Imperial College London

Professor Molly Stevens FREng

Faculty of EngineeringDepartment of Materials

Prof of Biomedical Materials&Regenerative Medicine
 
 
 
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Contact

 

+44 (0)20 7594 6804m.stevens

 
 
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Location

 

208Royal School of MinesSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

348 results found

Sigmundsson K, Ojala JRM, Öhman MK, Österholm A-M, Moreno-Moral A, Domogatskaya A, Chong LY, Sun Y, Chai X, Steele JAM, George B, Patarroyo M, Nilsson A-S, Rodin S, Ghosh S, Stevens MM, Petretto E, Tryggvason Ket al., 2018, Culturing functional pancreatic islets on α5-laminins and curative transplantation to diabetic mice, Matrix Biology, Vol: 70, Pages: 5-19, ISSN: 0945-053X

The efficacy of islet transplantation for diabetes treatment suffers from lack of cadaver-derived islets, islet necrosis and long transfer times prior to transplantation. Here, we developed a method for culturing mouse and human islets in vitro on α5-laminins, which are natural components of islet basement membranes. Adhering islets spread to form layers of 1-3 cells in thickness and remained normoxic and functional for at least 7 days in culture. In contrast, spherical islets kept in suspension developed hypoxia and central necrosis within 16 h. Transplantation of 110-150 mouse islets cultured on α5-laminin-coated polydimethylsiloxane membranes for 3-7 days normalized blood glucose already within 3 days in mice with streptozotocin-induced diabetes. RNA-sequencing of isolated and cultured mouse islets provided further evidence for the adhesion and spreading achieved with α5-laminin. Our results suggest that use of such in vitro expanded islets may significantly enhance the efficacy of islet transplantation treatment for diabetes.

Journal article

Li C, Armstrong J, Pence I, Kit-Anan W, Puetzer J, Correia Carreira S, Stevens MMet al., 2018, Glycosylated superparamagnetic nanoparticle gradients for osteochondral tissue engineering, Biomaterials, Vol: 176, Pages: 24-33, ISSN: 0142-9612

In developmental biology, gradients of bioactive signals direct the formation of structural transitions in tissue that are key to physiological function. Failure to reproduce these native features in an in vitro setting can severely limit the success of bioengineered tissue constructs. In this report, we introduce a facile and rapid platform that uses magnetic field alignment of glycosylated superparamagnetic iron oxide nanoparticles, pre-loaded with growth factors, to pattern biochemical gradients into a range of biomaterial systems. Gradients of bone morphogenetic protein 2 in agarose hydrogels were used to spatially direct the osteogenesis of human mesenchymal stem cells and generate robust osteochondral tissue constructs exhibiting a clear mineral transition from bone to cartilage. Interestingly, the smooth gradients in growth factor concentration gave rise to biologically-relevant, emergent structural features, including a tidemark transition demarcating mineralized and non-mineralized tissue and an osteochondral interface rich in hypertrophic chondrocytes. This platform technology offers great versatility and provides an exciting new opportunity for overcoming a range of interfacial tissue engineering challenges.

Journal article

Wang Y, Howes P, Kim E, Spicer C, Thomas M, Lin Y, Crowder S, Pence I, Stevens MMet al., 2018, Duplex specific nuclease-amplified detection of microRNA using compact quantum dot-DNA conjugates, ACS Applied Materials and Interfaces, Vol: 10, Pages: 28290-28300, ISSN: 1944-8244

Advances in nanotechnology have provided new opportunities for the design of next-generation nucleic acid biosensors and diagnostics. Indeed, combining advances in functional nanoparticles, DNA nanotechnology, and nuclease-enzyme-based amplification can give rise to new assays with advantageous properties. In this work, we developed a microRNA (miRNA) assay using bright fluorescent quantum dots (QDs), simple DNA probes, and the enzyme duplex-specific nuclease. We employed an isothermal target-recycling mechanism, where a single miRNA target triggers the cleavage of many DNA signal probes. The incorporation of DNA-functionalized QDs enabled a quantitative fluorescent readout, mediated by Förster resonance energy transfer (FRET)-based interaction with the DNA signal probes. Our approach splits the reaction in two, performing the enzyme-mediated amplification and QD-based detection steps separately such that each reaction could be optimized for performance of the active components. Target recycling gave ca. 3 orders of magnitude amplification, yielding highly sensitive detection with a limit of 42 fM (or 1.2 amol) of miR-148, with excellent selectivity versus mismatched sequences and other miRNAs. Furthermore, we used an alternative target (miR-21) and FRET pair for direct and absolute quantification of miR-21 in RNA extracts from human cancer and normal cell lines.

Journal article

Holme MN, Rana S, Barriga H, Kauscher U, Brooks NJ, Stevens MMet al., 2018, A robust liposomal platform for direct colorimetric detection of sphingomyelinase enzyme and inhibitors, ACS Nano, Vol: 12, Pages: 8197-8207, ISSN: 1936-0851

The enzyme sphingomyelinase (SMase) is an important biomarker for several diseases such as Niemann Pick’s, atherosclerosis, multiple sclerosis, and HIV. We present a two-component colorimetric SMase activity assay that is more sensitive and much faster than currently available commercial assays. Herein, SMase-triggered release of cysteine from a sphingomyelin (SM)-based liposome formulation with 60 mol % cholesterol causes gold nanoparticle (AuNP) aggregation, enabling colorimetric detection of SMase activities as low as 0.02 mU/mL, corresponding to 1.4 pM concentration. While the lipid composition offers a stable, nonleaky liposome platform with minimal background signal, high specificity toward SMase avoids cross-reactivity of other similar phospholipases. Notably, use of an SM-based liposome formulation accurately mimics the natural in vivo substrate: the cell membrane. We studied the physical rearrangement process of the lipid membrane during SMase-mediated hydrolysis of SM to ceramide using small- and wide-angle X-ray scattering. A change in lipid phase from a liquid to gel state bilayer with increasing concentration of ceramide accounts for the observed increase in membrane permeability and consequent release of encapsulated cysteine. We further demonstrated the effectiveness of the sensor in colorimetric screening of small-molecule drug candidates, paving the way for the identification of novel SMase inhibitors in minutes. Taken together, the simplicity, speed, sensitivity, and naked-eye readout of this assay offer huge potential in point-of-care diagnostics and high-throughput drug screening.

Journal article

Amdursky N, Mazo M, Thomas MR, Humphrey E, Puetzer J, St-Pierre J, Skaalure S, Richardson RM, Terracciano C, Stevens MMet al., 2018, Elastic serum-albumin based hydrogels: mechanism of formation and application in cardiac tissue engineering, Journal of Materials Chemistry B, ISSN: 2050-750X

Hydrogels are promising materials for mimicking the extra-cellular environment. Here, we present a simple methodology for the formation of a free-standing viscoelastic hydrogel from the abundant and low cost protein serum albumin. We show that the mechanical properties of the hydrogel exhibit a complicated behaviour as a function of the weight fraction of the protein component. We further use X-ray scattering to shed light on the mechanism of gelation from the formation of a fibrillary network at low weight fractions to interconnected aggregates at higher fractions. Given the match between our hydrogel elasticity and that of the myocardium, we investigated its potential for supporting cardiac cells in vitro. Interestingly, the sehydrogels support the formation of several layers of myocytes and significantly promote the maintenance of a native-likegene expression profile compared to those cultured on glass. When confronted with a multicellular ventricular cell preparation, the hydrogels can support macroscopically contracting cardiac-like tissues with a distinct cell arrangement, and form mm-long vascular-like structures. We envisage that our simple approach for the formation of an elastic substrate from an abundant protein makes the hydrogel a compelling biomedical material candidate for a wide range of cell types.

Journal article

Holme MN, Rashid MH, Thomas MR, Barriga HMG, Herpoldt KL, Heenan RK, Dreiss CA, Banuelos JL, Xie HN, Yarovsky I, Stevens MMet al., 2018, Fate of liposomes in presence of phospholipase C and D: from atomic to supramolecular lipid arrangement, ACS Central Science, Vol: 4, Pages: 1023-1030, ISSN: 2374-7943

Understanding the origins of lipid membrane bilayer rearrangement in response to external stimuli is an essential component of cell biology and the bottom-up design of liposomes for biomedical applications. The enzymes phospholipase C and D (PLC and PLD) both cleave the phosphorus–oxygen bonds of phosphate esters in phosphatidylcholine (PC) lipids. The atomic position of this hydrolysis reaction has huge implications for the stability of PC-containing self-assembled structures, such as the cell wall and lipid-based vesicle drug delivery vectors. While PLC converts PC to diacylglycerol (DAG), the interaction of PC with PLD produces phosphatidic acid (PA). Here we present a combination of small-angle scattering data and all-atom molecular dynamics simulations, providing insights into the effects of atomic-scale reorganization on the supramolecular assembly of PC membrane bilayers upon enzyme-mediated incorporation of DAG or PA. We observed that PC liposomes completely disintegrate in the presence of PLC, as conversion of PC to DAG progresses. At lower concentrations, DAG molecules within fluid PC bilayers form hydrogen bonds with backbone carbonyl oxygens in neighboring PC molecules and burrow into the hydrophobic region. This leads initially to membrane thinning followed by a swelling of the lamellar phase with increased DAG. At higher DAG concentrations, localized membrane tension causes a change in lipid phase from lamellar to the hexagonal and micellar cubic phases. Molecular dynamics simulations show that this destabilization is also caused in part by the decreased ability of DAG-containing PC membranes to coordinate sodium ions. Conversely, PLD-treated PC liposomes remain stable up to extremely high conversions to PA. Here, the negatively charged PA headgroup attracts significant amounts of sodium ions from the bulk solution to the membrane surface, leading to a swelling of the coordinated water layer. These findings are a vital step toward a fundamental u

Journal article

Heeney MJ, Creamer A, Wood C, Howes P, Casey A, Cong S, Marsh A, Godin R, Panidi J, Anthopoulos T, Burgess C, Wu T, Fei Z, McLachlan M, Stevens Met al., 2018, Post-polymerisation functionalisation of conjugated polymer backbones and its application in multi-functional emissive nanoparticles, Nature Communications, Vol: 9, ISSN: 2041-1723

Backbone functionalisation of conjugated polymers is crucial to their performance in many applications, from electronic displays to nanoparticle biosensors, yet there are limited approaches to introduce functionality. To address this challenge we have developed a method for the direct modification of the aromatic backbone of a conjugated polymer, post-polymerisation. This is achieved via a quantitative nucleophilic aromatic substitution (SNAr) reaction on a range of fluorinated electron deficient comonomers. The method allows for facile tuning of the physical and optoelectronic properties within a batch of consistent molecular weight and dispersity. It also enables the introduction of multiple different functional groups onto the polymer backbone in a controlled manner. To demonstrate the versatility of this reaction, we designed and synthesised a range of emissive poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) based polymers for the creation of mono and multifunctional semiconducting polymer nanoparticles (SPNs) capable of two orthogonal bioconjugation reactions on the same surface.

Journal article

Gray ER, Bain R, Varsaneux O, Peeling RW, Stevens MM, Mckendry RAet al., 2018, p24 revisited: a landscape review of antigen detection for early HIV diagnosis, AIDS, ISSN: 0269-9370

Despite major advances in HIV testing, early detection of infection at the point of care (PoC) remains a key challenge. While rapid antibody PoC and laboratory-based nucleic acid amplification tests dominate the diagnostics market, the viral capsid protein p24 is recognized as an alternative early virological biomarker of infection. However, the detection of ultra-low levels of p24 at the PoC has proven challenging. Here we review the landscape of p24-diagnostics to identify knowledge gaps and barriers and help shape future research agendas. 574 research papers to May 2018 that propose or evaluate diagnostic assays for p24 were identified and reviewed. We give a brief history of diagnostic development, and the utility of p24 as a biomarker in different populations such as infants, the newly infected, those on pre-exposure prophylaxis and self-testers. We review the performance of commercial p24 assays and consider elements such as immune complex disruption, resource-poor settings, prevalence, and assay antibodies. Emerging and ultrasensitive assays are reviewed and show a number of promising approaches but further translation has been limited. We summarize studies on the health economic benefits of using antigen testing. Finally, we speculate on the future uses of high performance p24 assays, particularly if available in self-test format.

Journal article

Spicer C, Pashuck E, Stevens MM, 2018, Achieving controlled biomolecule-biomaterial conjugation, Chemical Reviews, Vol: 118, Pages: 7702-7743, ISSN: 1520-6890

The conjugation of biomolecules can impart materials with the bioactivity necessary to modulate specific cell behaviors. While the biological roles of particular polypeptide, oligonucleotide, and glycan structures have been extensively reviewed, along with the influence of attachment on material structure and function, the key role played by the conjugation strategy in determining activity is often overlooked. In this review, we focus on the chemistry of biomolecule conjugation and provide a comprehensive overview of the key strategies for achieving controlled biomaterial functionalization. No universal method exists to provide optimal attachment, and here we will discuss both the relative advantages and disadvantages of each technique. In doing so, we highlight the importance of carefully considering the impact and suitability of a particular technique during biomaterial design.

Journal article

Armstrong JP, Stevens MM, 2018, Strategic design of extracellular vesicle drug delivery systems, Advanced Drug Delivery Reviews, ISSN: 0169-409X

Extracellular vesicles (EVs), sub-micron vectors used in intercellular communication, have demonstrated great promise as natural drug delivery systems. Recent reports have detailed impressive in vivo results from the administration of EVs pre-loaded with therapeutic cargo, including small molecules, nanoparticles, proteins and oligonucleotides. These results have sparked intensive research interest across a huge range of disease models. There are, however, enduring limitations that have restricted widespread clinical and pharmaceutical adoption. In this perspective, we discuss these practical and biological concerns, critically compare the relative merit of EVs and synthetic drug delivery systems, and highlight the need for a more comprehensive understanding of in vivo transport and delivery. Within this framework, we seek to establish key areas in which EVs can gain a competitive advantage in order to provide the tangible added value required for widespread translation.

Journal article

Barriga HMG, Holme MN, Stevens MM, 2018, Cubosomes: the next generation of smart lipid nanoparticles?, Angewandte Chemie, ISSN: 1521-3757

Cubosomes are highly stable nanoparticles formed from the lipid cubic phase and stabilized by a polymer based outer corona. Lipid cubic phases consist of a single lipid bilayer which forms a continuous periodic membrane lattice structure with pores formed by two interwoven water channels. Cubosome composition can be tuned to engineer pore sizes or include bioactive lipids, the polymer outer corona can be used for targeting and they are highly stable under physiological conditions. The structure provides a significantly higher membrane surface area for loading of membrane proteins and small drug molecules than liposomes. Due to recent advances, they can be engineered in vitro in both bulk and nanoparticle formats with applications including drug delivery, membrane bioreactors, artificial cells and biosensors. This review outlines recent advances in cubosome technology enabling their application and provides guidelines for the rational design of new systems for biomedical applications.

Journal article

Tallia F, Russo L, Li S, Orrin A, Shi X, Chen S, Steele J, Meille S, Chevalier J, Lee P, Stevens M, Cipolla L, Jones JRet al., Bouncing and 3D printable hybrids with self-healing properties, Materials Horizons, ISSN: 2051-6355

Conventional composites often do not represent true synergy of their constituent materials. This is particularly evident in biomaterial applications where devices must interact with cells, resist cyclic loads and biodegrade safely. Here we propose a new hybrid system, with co-networks of organic and inorganic components, resulting in unprecedented mechanical properties, including “bouncy” elasticity and intrinsic ability to self-heal autonomously. They are also developed as new ‘inks’ that can be directly 3D printed. A hybrid is different from a nanocomposite because the components are indistinguishable from each other at the nanoscale and above. The properties are generated by a novel methodology that combines in situ cationic ring-opening polymerisation with sol–gel, creating silica/poly(tetrahydrofuran)/poly(ε-caprolactone) hybrids with molecular scale interactions and covalent links. Cartilage is notoriously difficult to repair and synthetic biomaterials have yet to mimic it closely. We show that 3D printed hybrid scaffolds with pore channels of ∼200 μm mimic the compressive behaviour of cartilage and provoke chondrocytes to produce markers integral to articular cartilage-like matrix. The synthesis method can be applied to different organic sources, leading to a new class of hybrid materials.

Journal article

Sang T, Li S, Ting HK, Stevens MM, Becer CR, Jones JRet al., 2018, Hybrids of silica/poly (caprolactone co-glycidoxypropyl trimethoxysilane) as biomaterials, Chemistry of Materials, Vol: 30, Pages: 3743-3751, ISSN: 0897-4756

Bioactive glasses stimulate bone regeneration but are brittle. Biomaterials are needed that share load with bone, promote bone regeneration and biodegrade at controlled rates. Sol-gel hybrids can achieve this through their intimate inorganic and organic co-networks, depending on the organic polymer used. Polycaprolactone degrades slowly but lacks functional groups for the critical step of covalent coupling to the silica co-network. Here, we synthesised a novel copolymer of caprolactone and glycidoxypropyl trimethoxysilane through one-pot ring opening polymerization (ROP). Hybrids with different organic content were fabricated using such a copolymer for the first time. The copolymer can directly bond to a silica network due its trimethoxysilane groups, which can hydrolyse, leaving silanol groups that undergo polycondensation with silanol groups of the silica network. Number of repeating units of caprolactone and glycidoxypropyl trimethoxysilane functional groups were controlled via ROP. The mechanical properties of the hybrids were tuned by weight percent and the number of repeating units of caprolactone independently, producing a homogeneous material with high strength (64 MPa) and strain to failure (20%) that deformed in a unique linear elastic manner until failure. MC3T3-E1 pre-osteoblast cells adhered to the hybrids. Introducing such a copolymer created a new way to fabricate covalently bonded polycaprolactone/silica hybrids for future bone repair.

Journal article

Elsharkawy S, Al-Jawad M, Pantano MF, Tejeda-Montes E, Mehta K, Jamal H, Agarwal S, Shuturminska K, Rice A, Tarakina NV, Wilson RM, Bushby AJ, Alonso M, Rodriguez-Cabello JC, Barbieri E, Del Rio Hernandez A, Stevens MM, Pugno NM, Anderson P, Mata Aet al., 2018, Protein disorder-order interplay to guide the growth of hierarchical mineralized structures, Nature Communications, Vol: 9, ISSN: 2041-1723

Journal article

Kapnisi M, Mansfield C, Marijon C, Guex AG, Perbellini F, Bardi I, Humphrey EJ, Puetzer J, Mawad D, Koutsogeorgis DC, Stuckey DJ, Terracciano CM, Harding SE, Stevens MMet al., 2018, Auxetic cardiac patches with tunable mechanical and conductive properties toward treating myocardial infarction, Advanced Functional Materials, Vol: 28, ISSN: 1616-301X

An auxetic conductive cardiac patch (AuxCP) for the treatment of myocardial infarction (MI) is introduced. The auxetic design gives the patch a negative Poisson's ratio, providing it with the ability to conform to the demanding mechanics of the heart. The conductivity allows the patch to interface with electroresponsive tissues such as the heart. Excimer laser microablation is used to micropattern a re‐entrant honeycomb (bow‐tie) design into a chitosan‐polyaniline composite. It is shown that the bow‐tie design can produce patches with a wide range in mechanical strength and anisotropy, which can be tuned to match native heart tissue. Further, the auxetic patches are conductive and cytocompatible with murine neonatal cardiomyocytes in vitro. Ex vivo studies demonstrate that the auxetic patches have no detrimental effect on the electrophysiology of both healthy and MI rat hearts and conform better to native heart movements than unpatterned patches of the same material. Finally, the AuxCP applied in a rat MI model results in no detrimental effect on cardiac function and negligible fibrotic response after two weeks in vivo. This approach represents a versatile and robust platform for cardiac biomaterial design and could therefore lead to a promising treatment for MI.

Journal article

Reznikov N, Matthew B, Leonardo L, Stevens MM, Kroger Ret al., 2018, Fractal-like hierarchical organization of bone begins at the nanoscale, Science, Vol: 360, ISSN: 0036-8075

INTRODUCTIONThe components of bone assemble hierarchically to provide stiffness and toughness. Deciphering the specific organization and relationship between bone’s principal components—mineral and collagen—requires answers to three main questions: whether the association of the mineral phase with collagen follows an intrafibrillar or extrafibrillar pattern, whether the morphology of the mineral building blocks is needle- or platelet-shaped, and how the mineral phase maintains continuity across an extensive network of cross-linked collagen fibrils. To address these questions, a nanoscale level of three-dimensional (3D) structural characterization is essential and has now been performed.RATIONALEBecause bone has multiple levels of 3D structural hierarchy, 2D imaging methods that do not detail the structural context of a sample are prone to interpretation bias. Site-specific focused ion beam preparation of lamellar bone with known orientation of the analyzed sample regions allowed us to obtain imaging data by 2D high-resolution transmission electron microscopy (HRTEM) and to identify individual crystal orientations. We studied higher-level bone mineral organization within the extracellular matrix by means of scanning TEM (STEM) tomography imaging and 3D reconstruction, as well as electron diffraction to determine crystal morphology and orientation patterns. Tomographic data allowed 3D visualization of the mineral phase as individual crystallites and/or aggregates that were correlated with atomic-resolution TEM images and corresponding diffraction patterns. Integration of STEM tomography with HRTEM and crystallographic data resulted in a model of 3D mineral morphology and its association with the organic matrix.RESULTSTo visualize and characterize the crystallites within the extracellular matrix, we recorded imaging data of the bone mineral in two orthogonal projections with respect to the arrays of mineralized collagen fibrils. Three motifs of minera

Journal article

Keane TJ, Horejs C, Stevens MM, 2018, Scarring vs. functional repair: matrix-based strategies to regulate tissue healing, Advanced Drug Delivery Reviews, Vol: 129, Pages: 407-419, ISSN: 0169-409X

All vertebrates possess mechanisms to restore damaged tissues with outcomes ranging from regeneration to scarring. Unfortunately, the mammalian response to tissue injury most often culminates in scar formation. Accounting for nearly 45% of deaths in the developed world, fibrosis is a process that stands diametrically opposed to functional tissue regeneration. Strategies to improve wound healing outcomes therefore require methods to limit fibrosis. Wound healing is guided by precise spatiotemporal deposition and remodelling of the extracellular matrix (ECM). The ECM, comprising the non-cellular component of tissues, is a signalling depot that is differentially regulated in scarring and regenerative healing. This Review focuses on the importance of the native matrix components during mammalian wound healing alongside a comparison to scar-free healing and then presents an overview of matrix-based strategies that attempt to exploit the role of the ECM to improve wound healing outcomes.

Journal article

Winther AK, Fejerskov B, ter Meer M, Jensen NBS, Dillion R, Schaffer JE, Chandrawati R, Schultze Kool LJ, Stevens MM, Simonsen U, Zelikin ANet al., 2018, Enzyme prodrug therapy achieves site-specific, personalized physiological responses to the locally produced nitric oxide, ACS Applied Materials and Interfaces, Vol: 10, Pages: 10741-10751, ISSN: 1944-8244

Nitric oxide (NO) is a highly potent but short-lived endogenous radical with a wide spectrum of physiological activities. In this work, we developed an enzymatic approach to the site-specific synthesis of NO mediated by biocatalytic surface coatings. Multilayered polyelectrolyte films were optimized as host compartments for the immobilized β-galactosidase (β-Gal) enzyme through a screen of eight polycations and eight polyanions. The lead composition was used to achieve localized production of NO through the addition of β-Gal–NONOate, a prodrug that releases NO following enzymatic bioconversion. The resulting coatings afforded physiologically relevant flux of NO matching that of the healthy human endothelium. The antiproliferative effect due to the synthesized NO in cell culture was site-specific: within a multiwell dish with freely shared media and nutrients, a 10-fold inhibition of cell growth was achieved on top of the biocatalytic coatings compared to the immediately adjacent enzyme-free microwells. The physiological effect of NO produced via the enzyme prodrug therapy was validated ex vivo in isolated arteries through the measurement of vasodilation. Biocatalytic coatings were deposited on wires produced using alloys used in clinical practice and successfully mediated a NONOate concentration-dependent vasodilation in the small arteries of rats. The results of this study present an exciting opportunity to manufacture implantable biomaterials with physiological responses controlled to the desired level for personalized treatment.

Journal article

Spicer C, Jumeaux C, Gupta B, Stevens MMet al., 2018, Peptide and protein nanoparticle conjugates: versatile platforms for biomedical applications, Chemical Society Reviews, Vol: 47, Pages: 3574-3620, ISSN: 1460-4744

Peptide– and protein–nanoparticle conjugates have emerged as powerful tools for biomedical applications, enabling the treatment, diagnosis, and prevention of disease. In this review, we focus on the key roles played by peptides and proteins in improving, controlling, and defining the performance of nanotechnologies. Within this framework, we provide a comprehensive overview of the key sequences and structures utilised to provide biological and physical stability to nano-constructs, direct particles to their target and influence their cellular and tissue distribution, induce and control biological responses, and form polypeptide self-assembled nanoparticles. In doing so, we highlight the great advances made by the field, as well as the challenges still faced in achieving the clinical translation of peptide- and protein-functionalised nano-drug delivery vehicles, imaging species, and active therapeutics.

Journal article

Fuhrmann G, Chandrawati R, Parmar P, Keane TJ, Maynard SA, Bertazzo S, Stevens MMet al., 2018, Engineering extracellular vesicles with the tools of enzyme prodrug therapy, Advanced Materials, Vol: 30, ISSN: 0935-9648

Extracellular vesicles (EVs) have been exploited as drug delivery vehicles but their therapeutic use may be limited due to off-target effects. To harness EVs’inherent properties and to couple them with site-specific drug delivery functions, EVs are incorporated into hydrogels and engineered with the tools of enzyme-prodrug therapy. Local sustained release of anti-inflammatory drugs is demonstrated in macrophage cell models.

Journal article

von Erlach T, Bertazzo S, Wozniak MA, Horejs C, Maynard SA, Attwood S, Robinson BK, Autefage H, Kallepitis C, Del Rio Hernandez A, Chen CS, Goldoni S, Stevens MMet al., 2018, Cell geometry dependent changes in plasma membrane order direct stem cell signalling and fate, Nature Materials, Vol: 17, Pages: 237-242, ISSN: 1476-1122

Cell size and shape affect cellular processes such as cell survival, growth and differentiation1,2,3,4, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

Journal article

Albro M, Bergholt M, St-Pierre JP, Vinals Guitart A, Zlotnick HM, Evita EG, Stevens MMet al., 2018, Raman Spectroscopic Imaging for Quantification of Depth-Dependent and Local Heterogeneities in Native and Engineered Cartilage, npj Regenerative Medicine, Vol: 3, ISSN: 2057-3995

Articular cartilage possesses a remarkable, mechanically-robust extracellular matrix (ECM) that is organized and distributed throughout the tissue to resist physiologic strains and provide low friction during articulation. The ability to characterize the make-up and distribution of the cartilage ECM is critical to both understand the process by which articular cartilage undergoes disease-related degeneration and to develop novel tissue repair strategies to restore tissue functionality. However, the ability to quantitatively measure the spatial distribution of cartilage ECM constituents throughout the tissue has remained a major challenge. In this experimental investigation, we assessed the analytical ability of Raman micro-spectroscopic imaging to semi-quantitatively measure the distribution of the major ECM constituents in cartilage tissues. Raman spectroscopic images were acquired of two distinct cartilage tissue types that possess large spatial ECM gradients throughout their depth: native articular cartilage explants and large engineered cartilage tissue constructs. Spectral acquisitions were processed via multivariate curve resolution to decompose the “fingerprint” range spectra (800–1800 cm−1) to the component spectra of GAG, collagen, and water, giving rise to the depth dependent concentration profile of each constituent throughout the tissues. These Raman spectroscopic acquired-profiles exhibited strong agreement with profiles independently acquired via direct biochemical assaying of spatial tissue sections. Further, we harness this spectroscopic technique to evaluate local heterogeneities through the depth of cartilage. This work represents a powerful analytical validation of the accuracy of Raman spectroscopic imaging measurements of the spatial distribution of biochemical components in a biological tissue and shows that it can be used as a valuable tool for quantitatively measuring the distribution and organization of ECM con

Journal article

Hsu C-C, Serio A, Amdursky N, Besnard C, Stevens MMet al., 2018, Fabrication of Hemin-Doped Serum Albumin-Based Fibrous Scaffolds for Neural Tissue Engineering Applications, ACS Applied Materials and Interfaces, Vol: 10, Pages: 5305-5317, ISSN: 1944-8244

Neural tissue engineering (TE) represents a promising new avenue of therapy to support nerve recovery and regeneration. To recreate the complex environment in which neurons develop and mature, the ideal biomaterials for neural TE require a number of properties and capabilities including the appropriate biochemical and physical cues to adsorb and release specific growth factors. Here, we present neural TE constructs based on electrospun serum albumin (SA) fibrous scaffolds. We doped our SA scaffolds with an iron-containing porphyrin, hemin, to confer conductivity, and then functionalized them with different recombinant proteins and growth factors to ensure cell attachment and proliferation. We demonstrated the potential for these constructs combining topographical, biochemical, and electrical stimuli by testing them with clinically relevant neural populations derived from human induced pluripotent stem cells (hiPSCs). Our scaffolds could support the attachment, proliferation, and neuronal differentiation of hiPSC-derived neural stem cells (NSCs), and were also able to incorporate active growth factors and release them over time, which modified the behavior of cultured cells and substituted the need for growth factor supplementation by media change. Electrical stimulation on the doped SA scaffold positively influenced the maturation of neuronal populations, with neurons exhibiting more branched neurites compared to controls. Through promotion of cell proliferation, differentiation, and neurite branching of hiPSC-derived NSCs, these conductive SA fibrous scaffolds are of broad application in nerve regeneration strategies.

Journal article

Jumeaux C, Wahlsten O, Block S, Kim E, Chandrawati R, Howes PD, Hook P, Stevens MMet al., 2018, MicroRNA detection by DNA-mediated liposome fusion, ChemBioChem, Vol: 19, Pages: 434-438, ISSN: 1439-4227

Membrane fusion is a process of fundamental importance in biological systems that involves highly selective recognition mechanisms for the trafficking of molecular and ionic cargos. Mimicking natural membrane fusion mechanisms for the purpose of biosensor development holds great potential for amplified detection because relatively few highly discriminating targets lead to fusion and an accompanied engagement of a large payload of signal-generating molecules. In this work, sequence-specific DNA-mediated liposome fusion is used for the highly selective detection of microRNA. The detection of miR-29a, a known flu biomarker, is demonstrated down to 18 nm within 30 min with high specificity by using a standard laboratory microplate reader. Furthermore, one order of magnitude improvement in the limit of detection is demonstrated by using a novel imaging technique combined with an intensity fluctuation analysis, which is coined two-color fluorescence correlation microscopy.

Journal article

Rizzo R, Alvaro M, Danz N, Napione L, Descrovi E, Schmieder S, Sinibaldi A, Rana S, Chandrawati R, Munzert P, Schubert T, Maillart E, Anopchenko A, Rivolo P, Mascioletti A, Förster E, Sonntag F, Stevens MM, Bussolino F, Michelotti Fet al., 2018, Bloch surface wave enhanced biosensor for the direct detection of angiopoietin-2 tumor biomarker in human plasma, Biomedical Optics Express, Vol: 9, Pages: 529-542, ISSN: 2156-7085

Quantitative detection of angiogenic biomarkers provides a powerful tool to diagnose cancers in early stages and to follow its progression during therapy. Conventional tests require trained personnel, dedicated laboratory equipment and are generally time-consuming. Herein, we propose our developed biosensing platform as a useful tool for a rapid determination of Angiopoietin-2 biomarker directly from patient plasma within 30 minutes, without any sample preparation or dilution. Bloch surface waves supported by one dimensional photonic crystal are exploited to enhance and redirect the fluorescence arising from a sandwich immunoassay that involves Angiopoietin-2. The sensing units consist of disposable and low-cost plastic biochips coated with the photonic crystal. The biosensing platform is demonstrated to detect Angiopoietin-2 in plasma samples at the clinically relevant concentration of 6 ng/mL, with an estimated limit of detection of approximately 1 ng/mL. This is the first Bloch surface wave based assay capable of detecting relevant concentrations of an angiogenic factor in plasma samples. The results obtained by the developed biosensing platform are in close agreement with enzyme-linked immunosorbent assays, demonstrating a good accuracy, and their repeatability showed acceptable relative variations.

Journal article

Brangel P, Sobarzo A, Parolo C, Miller BS, Howes PD, Gelkop S, Lutwama JJ, Dye JM, McKendry RA, Lobel L, Stevens MMet al., 2018, A Serological Point-of-Care Test for the Detection of IgG Antibodies against Ebola Virus in Human Survivors, ACS Nano, Vol: 12, Pages: 63-73, ISSN: 1936-0851

Ebola virus disease causes widespread and highly fatal epidemics in human populations. Today, there is still great need for point-of-care tests for diagnosis, patient management and surveillance, both during and post outbreaks. We present a point-of-care test comprising an immunochromatographic strip and a smartphone reader, which detects and semiquantifies Ebola-specific antibodies in human survivors. We developed a Sudan virus glycoprotein monoplex platform and validated it using sera from 90 human survivors and 31 local noninfected controls. The performance of the glycoprotein monoplex was 100% sensitivity and 98% specificity compared to standard whole antigen enzyme-linked immunosorbent assay (ELISA), and it was validated with freshly collected patient samples in Uganda. Moreover, we constructed a multiplex test for simultaneous detection of antibodies against three recombinant Sudan virus proteins. A pilot study comprising 15 survivors and 5 noninfected controls demonstrated sensitivity and specificity of 100% compared to standard ELISA. Finally, we developed a second multiplex subtype assay for the identification of exposure to three related EVD species: Sudan virus, Bundibugyo virus and Ebola virus (formerly Zaire) using recombinant viral glycoprotein. This multiplex test could distinguish between the host’s immunity to specific viral species and identify cross-reactive immunity. These developed serological platforms consisted of capture ligands with high specificity and sensitivity, in-house developed strips and a compatible smartphone application. These platforms enabled rapid and portable testing, data storage and sharing as well as geographical tagging of the tested individuals in Uganda. This platform holds great potential as a field tool for diagnosis, vaccine development, and therapeutic evaluation.

Journal article

Bergholt MS, Serio A, McKenzie JS, Boyd A, Soares RF, Tillner J, Chiappini C, Wu V, Dannhorn A, Takats Z, Williams A, Stevens MMet al., 2017, Correlated heterospectral lipidomics for biomolecular profiling of remyelination in multiple sclerosis, ACS Central Science, Vol: 4, Pages: 39-51, ISSN: 2374-7943

Analyzing lipid composition and distribution within the brain is important to study white matter pathologies that present focal demyelination lesions, such as multiple sclerosis. Some lesions can endogenously re-form myelin sheaths. Therapies aim to enhance this repair process in order to reduce neurodegeneration and disability progression in patients. In this context, a lipidomic analysis providing both precise molecular classification and well-defined localization is crucial to detect changes in myelin lipid content. Here we develop a correlated heterospectral lipidomic (HSL) approach based on coregistered Raman spectroscopy, desorption electrospray ionization mass spectrometry (DESI-MS), and immunofluorescence imaging. We employ HSL to study the structural and compositional lipid profile of demyelination and remyelination in an induced focal demyelination mouse model and in multiple sclerosis lesions from patients ex vivo. Pixelwise coregistration of Raman spectroscopy and DESI-MS imaging generated a heterospectral map used to interrelate biomolecular structure and composition of myelin. Multivariate regression analysis enabled Raman-based assessment of highly specific lipid subtypes in complex tissue for the first time. This method revealed the temporal dynamics of remyelination and provided the first indication that newly formed myelin has a different lipid composition compared to normal myelin. HSL enables detailed molecular myelin characterization that can substantially improve upon the current understanding of remyelination in multiple sclerosis and provides a strategy to assess remyelination treatments in animal models.

Journal article

Loynachan C, Thomas MR, Gray ER, Richards DA, Kim J, MIller BS, Brookes JC, Chudasama V, McKendry RA, Stevens MMet al., 2017, Platinum Nanocatalyst Amplification: Redefining the Gold Standard for Lateral Flow Immunoassays with Ultra-broad Dynamic Range, ACS Nano, Vol: 12, Pages: 279-288, ISSN: 1936-0851

Paper-based lateral flow immunoassays (LFIAs) are one of the most widely used point-of-care (PoC) devices; however, their application in early disease diagnostics is often limited due to insufficient sensitivity for the requisite sample sizes and the short time frames of PoC testing. To address this, we developed a serum-stable, nanoparticle catalyst-labeled LFIA with a sensitivity surpassing that of both current commercial and published sensitivities for paper-based detection of p24, one of the earliest and most conserved biomarkers of HIV. We report the synthesis and characterization of porous platinum core–shell nanocatalysts (PtNCs), which show high catalytic activity when exposed to complex human blood serum samples. We explored the application of antibody-functionalized PtNCs with strategically and orthogonally modified nanobodies with high affinity and specificity toward p24 and established the key larger nanoparticle size regimes needed for efficient amplification and performance in LFIA. Harnessing the catalytic amplification of PtNCs enabled naked-eye detection of p24 spiked into sera in the low femtomolar range (ca. 0.8 pg·mL–1) and the detection of acute-phase HIV in clinical human plasma samples in under 20 min. This provides a versatile absorbance-based and rapid LFIA with sensitivity capable of significantly reducing the HIV acute phase detection window. This diagnostic may be readily adapted for detection of other biomolecules as an ultrasensitive screening tool for infectious and noncommunicable diseases and can be capitalized upon in PoC settings for early disease detection.

Journal article

You AYF, Bergholt MS, St-Pierre JP, Chester AH, Yacoub MH, Bertazzo S, Stevens MMet al., 2017, Raman spectroscopy imaging reveals interplay between atherosclerosis and medial calcification in human aorta, Science Advances, Vol: 3, ISSN: 2375-2548

Medial calcification in the human aorta accumulates during aging and is known to be aggravated in several diseases. Atherosclerosis, another major cause of cardiovascular calcification, shares some common aggravators. However, the mechanisms of cardiovascular calcification remain poorly understood. To elucidate the relationship between medial aortic calcification and atherosclerosis, we characterized the cross-sectional distributions of the predominant minerals in aortic tissue, apatite and whitlockite, and the associated extracellular matrix. We also compared the cellular changes between atherosclerotic and nonatherosclerotic human aortic tissues. This was achieved through the development of Raman spectroscopy imaging methods that adapted algorithms to distinguish between the major biomolecules present within these tissues. We present a relationship between apatite, cholesterol, and triglyceride in atherosclerosis, with the relative amount of all molecules concurrently increased in the atherosclerotic plaque. Further, the increase in apatite was disproportionately large in relation to whitlockite in the aortic media directly underlying a plaque, indicating that apatite is more pathologically significant in atherosclerosis-aggravated medial calcification. We also discovered a reduction of β-carotene in the whole aortic intima, including a plaque in atherosclerotic aortic tissues compared to nonatherosclerotic tissues. This unprecedented biomolecular characterization of the aortic tissue furthers our understanding of pathological and physiological cardiovascular calcification events in humans.

Journal article

Milner P, Parkes M, Puetzer J, Chapman R, Cann P, Stevens M, Jeffers Jet al., 2017, A Low Friction, Biphasic and Boundary Lubricating Hydrogel for Cartilage Replacement, Acta Biomaterialia, Vol: 65, Pages: 102-111, ISSN: 1742-7061

Partial joint repair is a surgical procedure where an artificial material is used to replace localised chondral damage. These artificial bearing surfaces must articulate against cartilage, but current materials do not replicate both the biphasic and boundary lubrication mechanisms of cartilage. A research challenge therefore exists to provide a material that mimics both boundary and biphasic lubrication mechanisms of cartilage.In this work a polymeric network of a biomimetic boundary lubricant, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), was incorporated into an ultra-tough double network (DN) biphasic (water phase + polymer phase) gel, to form a PMPC triple network (PMPC TN) hydrogel with boundary and biphasic lubrication capability. The presence of this third network of MPC was confirmed using ATR-FTIR. The PMPC TN hydrogel had a yield stress of 26 MPa, which is an order of magnitude higher than the peak stresses found in the native human knee. A preliminary pin on plate tribology study was performed where both the DN and PMPC TN hydrogels experienced a reduction in friction with increasing sliding speed which is consistent with biphasic lubrication. In the physiological sliding speed range, the PMPC TN hydrogel halved the friction compared to the DN hydrogel indicating the boundary lubricating PMPC network was working.A biocompatible, tough, strong and chondral lubrication imitating PMPC TN hydrogel was synthesised in this work. By complementing the biphasic and boundary lubrication mechanisms of cartilage, PMPC TN hydrogel could reduce the reported incidence of chondral damage opposite partial joint repair implants, and therefore increase the clinical efficacy of partial joint repair.Statement of SignificanceThis paper presents the synthesis, characterisation and preliminary tribological testing of a new biomaterial that aims to recreate the primary chondral lubrication mechanisms: boundary and biphasic lubrication. This work has demonstrated that the

Journal article

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