Imperial College London

Professor Molly Stevens

Faculty of EngineeringDepartment of Materials

Professor of Biomedical Materials and 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

390 results found

Higgins S, Lo Fiego A, Patrick I, Creamer A, Stevens Met al., 2020, Organic bioelectronics: using highly conjugated polymers to interface with biomolecules, cells and tissues in the human body, Advanced Materials Technologies, ISSN: 2365-709X

Conjugated polymers exhibit interesting material and optoelectronic properties that makethem well-suited to the development of biointerfaces. Their biologically relevant mechanicalcharacteristics, ability to be chemically modified, and mixed electronic and ionic chargetransport are captured within the diverse field of organic bioelectronics. Conjugated polymershave been used in wide range of device architectures, and cell and tissue scaffolds. Thesedevices enable biosensing of many biomolecules, such as metabolites, nucleic acids and more.Devices can be used to both stimulate and sense the behavior of cells and tissues. Similarly,tissue interfaces permit interaction with complex organs, aiding both fundamental biologicalunderstanding and providing new opportunities for stimulating regenerative behaviors andbioelectronic based therapeutics. Applications of these materials are broad, and muchcontinues to be uncovered about their fundamental properties. This report covers the currentunderstanding of the fundamentals of conjugated polymer biointerfaces and their interactionswith biomolecules, cells and tissues in the human body. An overview of current materials anddevices is presented, along with highlighted major in vivo and in vitro applications. Finally,open research questions and opportunities are discussed.

Journal article

Maynard S, Winter C, Cunnane E, Stevens Met al., Advancing cell instructive biomaterials through increased understanding of cell receptor spacing and material surface functionalization, Regenerative Engineering and Translational Medicine

Journal article

Hogset H, Horgan C, Bergholt M, Armstrong J, Torraca V, Chen Q, Keane TJ, Bugeon L, Dallman M, Mostowy S, Stevens Met al., In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy, Nature Communications, ISSN: 2041-1723

Journal article

J C, Najer A, Blakney A, McKay P, Bellahcene M, Winter C, Sintou A, Tang J, Keane TJ, Schneider M, Shattock R, Sattler S, Stevens Met al., Neutrophils enable local and non-invasive liposome delivery to inflamed skeletal muscle and ischemic heart, Advanced Materials, ISSN: 0935-9648

Journal article

Ouyang L, Armstrong J, Lin Y, Wojciechowski J, Lee-Reeves C, Hachim D, Zhou K, Burdick JA, Stevens Met al., 2020, Expanding and optimizing 3D bioprinting capabilities using complementary network bioinks, Science Advances, Vol: 6, Pages: 1-13, ISSN: 2375-2548

A major challenge in 3D bioprinting is the limited number of bioinks that fulfill the physiochemical requirements of printing, while also providing a desirable environment for encapsulated cells.Here, we address this limitation by temporarily stabilizing bioinks with a complementary thermo-reversible gelatin network. This strategy enablesthe effective printing of biomaterials that would typically not meet printing requirements, with instrument parameters and structural output largely independent of the base biomaterial. This approach is demonstrated across a library of photo-crosslinkable bioinks derived from natural and synthetic polymers, including gelatin, hyaluronic acid, chondroitin sulfate, dextran, alginate, chitosan, heparin,and poly(ethylene glycol). A range of complex and heterogeneous structures are printed, including soft hydrogel constructs supporting the 3D culture of astrocytes. This highly generalizable methodology expands the palette of available bioinks, allowing the biofabrication of constructs optimized to meet the biological requirements of cell culture and tissue engineering.

Journal article

Massi L, Najer A, Chapman R, Spicer C, Nele V, Che J, Booth MA, Doutch JJ, Stevens Met al., 2020, Tuneable peptide cross-linked nanogels for enzyme-triggered protein delivery, Journal of Materials Chemistry B, Vol: 8, Pages: 8894-8907, ISSN: 2050-750X

Many diseases are associated with the dysregulated activity of enzymes, such as matrixmetalloproteinases (MMPs). This dysregulation can be leveraged in drug delivery to achieve disease- orsite-specific cargo release. Self-assembled polymeric nanoparticles are versatile drug carrier materialsdue to the accessible diversity of polymer chemistry. However, efficient loading of sensitive cargo, suchas proteins, and introducing functional enzyme-responsive behaviour remain challenging. Herein,peptide-crosslinked, temperature-sensitive nanogels for protein delivery were designed to respond toMMP-7, which is overexpressed in many pathologies including cancer and inflammatory diseases. Theincorporation of N-cyclopropylacrylamide (NCPAM) into N-isopropylacrylamide (NIPAM)-basedcopolymers enabled us to tune the polymer lower critical solution temperature from 33 to 44 1C,allowing the encapsulation of protein cargo and nanogel-crosslinking at slightly elevated temperatures.This approach resulted in nanogels that were held together by MMP-sensitive peptides for enzymespecificprotein delivery. We employed a combination of cryogenic transmission electron microscopy(cryo-TEM), dynamic light scattering (DLS), small angle neutron scattering (SANS), and fluorescencecorrelation spectroscopy (FCS) to precisely decipher the morphology, self-assembly mechanism,enzyme-responsiveness, and model protein loading/release properties of our nanogel platform. Simplevariation of the peptide linker sequence and combining multiple different crosslinkers will enable us toadjust our platform to target specific diseases in the future.

Journal article

Whittaker T, Nagelkerke A, Nele V, Kauscher U, Stevens Met al., 2020, Experimental artefacts can lead to misattribution of bioactivity from soluble mesenchymal stem cell paracrine factors to extracellular vesicles, Journal of Extracellular Vesicles, Vol: 9, ISSN: 2001-3078

It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs.

Journal article

Li S, Tallia F, Mohammed AA, Stevens MM, Jones JRet al., 2020, Scaffold channel size influences stem cell differentiation pathway in 3-D printed silica hybrid scaffolds for cartilage regeneration, Biomaterials Science, Vol: 8, Pages: 4458-4466, ISSN: 2047-4830

We report that 3-D printed scaffold channel size can direct bone marrow derived stem cell differentiation. Treatment of articular cartilage trauma injuries, such as microfracture surgery, have limited success because durability is limited as fibrocartilage forms. A scaffold-assisted approach, combining microfracture with biomaterials has potential if the scaffold can promote articular cartilage production and share load with cartilage. Here, we investigated human bone marrow derived stromal cell (hBMSC) differentiation in vitro in 3-D printed silica/poly(tetrahydrofuran)/poly(ε-caprolactone) hybrid scaffolds with specific channel sizes. Channel widths of ∼230 μm (210 ± 22 μm mean strut size, 42.4 ± 3.9% porosity) provoked hBMSC differentiation down a chondrogenic path, with collagen Type II matrix prevalent, indicative of hyaline cartilage. When pores were larger (∼500 μm, 229 ± 29 μm mean strut size, 63.8 ± 1.6% porosity) collagen Type I was dominant, indicating fibrocartilage. There was less matrix and voids in smaller channels (∼100 μm, 218 ± 28 μm mean strut size, 31.2 ± 2.9% porosity). Our findings suggest that a 200–250 μm pore channel width, in combination with the surface chemistry and stiffness of the scaffold, is optimal for cell–cell interactions to promote chondrogenic differentiation and enable the chondrocytes to maintain their phenotype.

Journal article

Gurnani P, Blakney AK, Yeow J, Bouton CR, Shattock RJ, Stevens MM, Alexander Cet al., 2020, An improved synthesis of poly(amidoamine)s for complexation with self-amplifying RNA and effective transfection, Polymer Chemistry, Vol: 11, Pages: 5861-5869, ISSN: 1759-9954

Cationic polymers are widely used as materials to condense nucleic acids for gene-based therapies. These have been developed to mainly deliver DNA and RNA for cancer therapies but the ongoing COVID-19 pandemic has demonstrated an urgent need for new DNA and RNA vaccines. Given this, suitable manufacturing conditions for such cationic polymers which can protect the nucleic acid in the formulation and delivery stages but release the cargo in the correct cellular compartment effectively and safely are required. A number of polymers based on poly(amidoamine)s fit these criteria but their syntheses can be time-consuming, inefficient and poorly reproducible, precluding their adoption as manufacturable vaccine excipients. Here we report an improved synthesis of poly(cystamine bisacrylamide-co-4-amino-1-butanol), abbreviated as pABOL, via modifications in concentration, reaction time and reaction conditions. Optimisation of monomer contents and stoichiometries, solvents, diluents and temperature, combined with the application of microwaves, enabled the preparation of vaccine candidate pABOL materials in 4 h compared to 48 h reported for previous syntheses. These procedures were highly reproducible in multiple repeat syntheses. Transfection experiments with a model RNA showed that polymers of formulation with appropriate molar masses and mass distributions were as effective in model cell lines as polymers derived from the unoptimised syntheses which have been shown to have high efficacy as RNA vaccine formulation candidates.

Journal article

Nele V, Wojciechowski J, Armstrong J, Stevens Met al., 2020, Tailoring gelation mechanisms for advanced hydrogel applications, Advanced Functional Materials, ISSN: 1616-301X

Journal article

Ritzau-Reid K, Spicer C, Gelmi A, Grigsby CL, Ponder Jr J, Bemmer V, Creamer A, Vilar R, Serio A, Stevens Met al., 2020, An electroactive oligo-EDOT platform for neural tissue engineering, Advanced Functional Materials, ISSN: 1616-301X

The unique electrochemical properties of the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) make it an attractive material for use in neural tissue engineering applications. However, inadequate mechanical properties, and difficulties in processing and lack of biodegradability have hindered progress in this field. Here, we have improved the functionality of PEDOT:PSS for neural tissue engineering by incorporating 3,4-ethylenedioxythiophene (EDOT) oligomers, synthesised using a novel end-capping strategy, into block co-polymers. By exploiting end-functionalised oligoEDOT constructs as macroinitiators for the polymerization of poly(caprolactone) (PCL), we produce a block co-polymer that is electroactive, processable, and bio-compatible. By combining these properties, we were able to produce electroactive fibrous mats for neuronal culture via solution electrospinning and melt electrospinning writing (MEW). Importantly, we also show that neurite length and branching of neural stem cells can be enhanced on our materials under electrical stimulation, demonstrating the promise of these scaffolds for neural tissue engineering.

Journal article

Budd J, Miller BS, Manning EM, Lampos V, Zhuang M, Edelstein M, Rees G, Emery VC, Stevens MM, Keegan N, Short MJ, Pillay D, Manley E, Cox IJ, Heymann D, Johnson AM, McKendry RAet al., 2020, Digital technologies in the public-health response to COVID-19, NATURE MEDICINE, Vol: 26, Pages: 1183-1192, ISSN: 1078-8956

Journal article

Ahadian S, Finbloom JA, Mofidfar M, Emir Diltemiz S, Nasrollahi F, Davoodi E, Hosseini V, Mylonaki I, Sangabathuni S, Montazerian H, Fetah K, Nasiri R, Remzi Dokmeci M, Stevens M, Desai TA, Khademhosseini Aet al., 2020, Micro and nanoscale technologies in oral drug delivery, Advanced Drug Delivery Reviews, ISSN: 0169-409X

Oral administration is a pillar of the pharmaceutical industry and yet it remains challenging to administer hydrophilic therapeutics by the oral route. Smart and controlled oral drug delivery could bypass the physiological barriers that limit the oral delivery of these therapeutics. Micro- and nanoscale technologies, with an unprecedented ability to create, control, and measure micro- or nanoenvironments, have found tremendous applications in biology and medicine. In particular, significant advances have been made in using these technologies for oral drug delivery. In this review, we briefly describe biological barriers to oral drug delivery and micro and nanoscale fabrication technologies. Micro and nanoscale drug carriers fabricated using these technologies, including bioadhesives, microparticles, micropatches, and nanoparticles, are described. Other applications of micro and nanoscale technologies are discussed, including the fabrication of devices and tissue engineering models to precisely control or assess oral drug delivery in vivo and in vitro, respectively. Strategies to advance translation of micro and nanotechnologies into clinical trials for oral drug delivery are mentioned. Finally, challenges and future prospects on further integration of micro and nanoscale technologies with oral drug delivery systems are highlighted.

Journal article

Li C, Ouyang L, Armstrong J, Stevens Met al., 2020, Advances in the fabrication of biomaterials for gradient tissue engineering, Trends in Biotechnology, ISSN: 0167-7799

Natural tissues and organs exhibit an array of spatial gradients, from the polar-ized neural tube during embryonic development to the osteochondral interfacepresent at articulating joints. The strong structure–function relationships inthese heterogeneous tissues have sparked intensive research into the develop-ment of methods that can replicate physiological gradients in engineered tis-sues. In this Review, we consider different gradients present in natural tissuesand discuss their critical importance in functional tissue engineering. Using thisbasis, we consolidate the existing fabrication methods into four categories: addi-tive manufacturing, component redistribution, controlled phase changes, andpostmodification. We have illustrated this with recent examples, highlightedprominent trends in thefield, and outlined a set of criteria and perspectives forgradient fabrication.

Journal article

Ouyang L, Armstrong J, Salmeron-Sanchez M, Stevens Met al., 2020, Assembly of living building blocks to engineer complex tissues, Advanced Functional Materials, Vol: 30, Pages: 1-22, ISSN: 1616-301X

The great demand for tissue and organ grafts, compounded by an aging demographic and a shortage of available donors, has driven the development of bioengineering approaches that can generate biomimetic tissues in vitro. Despite the considerable progress in conventional scaffold‐based tissue engineering, the recreation of physiological complexity has remained a challenge. Bottom‐up tissue engineering strategies have opened up a new avenue for the modular assembly of living building blocks into customized tissue architectures. This Progress Report overviews the recent progress and trends in the fabrication and assembly of living building blocks, with a key highlight on emerging bioprinting technologies that can be used for modular assembly and complexity in tissue engineering. By summarizing the work to date, providing new classifications of different living building blocks, highlighting state‐of‐the‐art research and trends, and offering personal perspectives on future opportunities, this Progress Report aims to aid and inspire other researchers working in the field of modular tissue engineering.

Journal article

Finbloom JA, Sousa F, Stevens MM, Desai TAet al., 2020, Engineering the drug carrier biointerface to overcome biological barriers to drug delivery, Advanced Drug Delivery Reviews, ISSN: 0169-409X

Micro and nanoscale drug carriers must navigate through a plethora of dynamic biological systems prior to reaching their tissue or disease targets. The biological obstacles to drug delivery come in many forms and include tissue barriers, mucus and bacterial biofilm hydrogels, the immune system, and cellular uptake and intracellular trafficking. The biointerface of drug carriers influences how these carriers navigate and overcome biological barriers for successful drug delivery. In this review, we examine how key material design parameters lead to dynamic biointerfaces and improved drug delivery across biological barriers. We provide a brief overview of approaches used to engineer key physicochemical properties of drug carriers, such as morphology, surface chemistry, and topography, as well as the development of dynamic responsive materials for barrier navigation. We then discuss essential biological barriers and how biointerface engineering can enable drug carriers to better navigate and overcome these barriers to drug delivery.

Journal article

Watts C, Hanham S, Armstrong J, Ahmad M, Stevens M, Klein Net al., 2020, Microwave dielectric sensing of free-flowing, single, living cells in aqueous suspension, IEEE Journal of Electromagnetics, RF and Microwaves in Medicine and Biology, Vol: 4, Pages: 97-208, ISSN: 2469-7249

Dielectric measurements offer the possibility of highly sensitive detection of physical cell properties, and are of interest for clinical applications due to their non-destructive nature and the lack of need for cell labelling. Here we report sensitive measurements on single, living, free-flowing cells (not electrostatically or dielectrophoretically trapped, cultured or fixed directly on sensing elements) in aqueous medium at ~9.8 GHz taken using a coupled dielectric-split ring resonator assembly. Inductive coupling between the two resonators enabled separation of microfluidic chips from RF connectors and allowed for time-resolved continuous-wave measurements on flowing single cells via the coaxial ports of a dielectric-loaded microwave cavity. Analysis via an equivalent circuit model showed that the novel resonator assembly maintained the permittivity-dependent sensitivity of a split ring resonator while operating at quality factors >1000 with lossy aqueous media (typically ~1900). Using a microfluidic channel with a 300 x 300 μm cross section, at a water-loaded resonant amplitude of ~-22 dB at 0 dBm input power level, shifts in amplitude due to individual cells passing through the sensing region of up to -0.0015 dB were observed. Correlations between averaged amplitude shifts and cell size as well as material properties demonstrate the diagnostic potential of this technique.

Journal article

Horgan C, Nagelkerke A, Whittaker TE, Nele V, Massi L, Kauscher U, Bergholt M, Hood SR, Stevens Met al., 2020, Molecular imaging of extracellular vesicles in vitro via Raman metabolic labelling, Journal of Materials Chemistry B, Vol: 8, Pages: 4447-4459, ISSN: 2050-750X

Extracellular vesicles (EVs) are biologically-derived nanovectors important for intercellular communication and trafficking. As such, EVs show great promise as disease biomarkers and therapeutic drug delivery vehicles. However, despite the rapidly growing interest in EVs, understanding of the biological mechanisms that govern their biogenesis, secretion, and uptake remains poor. Advances in this field have been hampered by both the complex biological origins of EVs, which make them difficult to isolate and identify, and a lack of suitable imaging techniques to properly study their diverse biological roles. Here, we present a new strategy for simultaneous quantitative in vitro imaging and molecular characterisation of EVs in 2D and 3D based on Raman spectroscopy and metabolic labelling. Deuterium, in the form of deuterium oxide (D2O), deuterated choline chloride (d-Chol), or deuterated D-glucose (d-Gluc), is metabolically incorporated into EVs through the growth of parent cells on medium containing one of these compounds. Isolated EVs are thus labelled with deuterium, which acts as a bio-orthogonal Raman-active tag for direct Raman identification of EVs when introduced to unlabelled cell cultures. Metabolic deuterium incorporation demonstrates no apparent adverse effects on EV secretion, marker expression, morphology, or global composition, indicating its capacity for minimally obstructive EV labelling. As such, our metabolic labelling strategy could provide integral insights into EV biocomposition and trafficking. This approach has the potential to enable a deeper understanding of many of the biological mechanisms underpinning EVs, with profound implications for the design of EVs as therapeutic delivery vectors and applications as disease biomarkers.

Journal article

Moroz-Omori EV, Satyapertiwi D, Ramel MC, Hogset H, Sunyovszki I, Liu Z, Wojciechowski J, Zhang Y, Grigsby CL, Casimiro Brito L, Bugeon L, Dallman M, Stevens Met al., 2020, Photoswitchable gRNAs for spatiotemporally controlled CRISPR-Cas-based genomic regulation, ACS Central Science, Vol: 6, Pages: 695-703, ISSN: 2374-7943

The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a light-responsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.

Journal article

Blakney AK, Zhu Y, McKay PF, Bouton CR, Yeow J, Tang J, Hu K, Samnuan K, Grigsby CL, Shattock RJ, Stevens MMet al., 2020, Big is beautiful: enhanced saRNA delivery and immunogenicity by a higher molecular weight, bioreducible, cationic polymer, ACS Nano, Vol: 14, Pages: 5711-5727, ISSN: 1936-0851

Self-amplifying RNA (saRNA) vaccines are highly advantageous, as they result in enhanced protein expression compared to mRNA (mRNA), thus minimizing the required dose. However, previous delivery strategies were optimized for siRNA or mRNA and do not necessarily deliver saRNA efficiently due to structural differences of these RNAs, thus motivating the development of saRNA delivery platforms. Here, we engineer a bioreducible, linear, cationic polymer called “pABOL” for saRNA delivery and show that increasing its molecular weight enhances delivery both in vitro and in vivo. We demonstrate that pABOL enhances protein expression and cellular uptake via both intramuscular and intradermal injection compared to commercially available polymers in vivo and that intramuscular injection confers complete protection against influenza challenge. Due to the scalability of polymer synthesis and ease of formulation preparation, we anticipate that this polymer is highly clinically translatable as a delivery vehicle for saRNA for both vaccines and therapeutics.

Journal article

Seong H, Higgins SG, Penders J, Armstrong JPK, Crowder SW, Moore AC, Sero JE, Becce M, Stevens MMet al., 2020, Size-tunable nanoneedle arrays for influencing stem cell morphology, gene expression and nuclear membrane curvature, ACS Nano, Vol: 14, Pages: 5371-5381, ISSN: 1936-0851

High-aspect-ratio nanostructures have emerged as versatile platforms for intracellular sensing and biomolecule delivery. Here, we present a microfabrication approach in which a combination of reactive ion etching protocols was used to produce high-aspect-ratio, nondegradable silicon nanoneedle arrays with tip diameters that can be finely tuned between 20 and 700 nm. We used these arrays to guide the long-term culture of human mesenchymal stem cells (hMSCs). Notably, we used the nanoneedle tip diameter to control the morphology, nuclear size and F-actin alignment of interfaced hMSCs, and to regulate the expression of nuclear lamina genes, Yes-associated protein (YAP) target genes and focal adhesion genes. These topography-driven changes were attributed to signaling by Rho-family GTPase pathways, differences in the effective stiffness of the nanoneedle arrays and the degree of nuclear membrane impingement, with the latter clearly visualized using focused-ion beam scanning electron microscopy (FIB-SEM). Our approach to design high-aspect-ratio nanostructures will be broadly applicable to design biomaterials and biomedical devices used for long-term cell stimulation and monitoring.

Journal article

Kauscher U, Pender J, Nagelkerke A, Holme M, Nele V, Massi L, Gopal S, Whittaker T, Stevens Met al., 2020, Gold nanocluster extracellular vesicle supraparticles: Self-assembled nanostructures for 3D uptake visualization, Langmuir: the ACS journal of surfaces and colloids, Vol: 36, Pages: 3912-3923, ISSN: 0743-7463

Extracellular vesicles (EVs) are secreted by the vast majority of cells and are being intensively studied due to their emerging involvement in a variety of cellular communication processes. However, the study of their cellular uptake and fate has been hampered by difficulty in imaging EVs against the cellular background. Here, we show that EVs combined with hydrophobic gold nanoclusters (AuNCs) can self-assemble into supraparticles, offering an excellent labeling strategy for high-resolution electron microscopic imaging in vitro. We have tracked and visualized the reuptake of breast cancer cell-derived EV AuNC supraparticles into their parent cells, from early endocytosis to lysosomal degradation, using focused ion beam-scanning electron microscopy (FIB-SEM). The presence of gold within the EVs and lysosomes was confirmed via DF-STEM EDX analysis of lift-out sections. The demonstrated formation of AuNC EV supraparticles will facilitate future applications in EV imaging as well as the EV-assisted cellular delivery of AuNCs.

Journal article

Liu H, Du Y, St-Pierre J-P, Bergholt MS, Autefage H, Wang J, Cai M, Yang G, Stevens M, Shengmin Zet al., 2020, Bioenergetic-active materials enhance tissue regeneration by modulating cellular metabolic state, Science Advances, Vol: 6, Pages: 1-15, ISSN: 2375-2548

Cellular bioenergetics (CBE) plays a critical role in tissue regeneration. Physiologically, an enhanced metabolic state facilitates anabolic biosynthesis and mitosis to accelerate regeneration. However, the development of approaches to reprogram CBE, towards the treatment of substantial tissue injuries, hasbeen limited thus far. Here, we show that induced repair in a rabbit model of weight-bearing bone defects is greatly enhanced using a bioenergetic-active material (BAM) scaffold, compared to commercialized poly (lactic acid) and calcium phosphate ceramic scaffolds. This material was composed of energy-active units that can be released in a sustained degradation-mediated fashion once implanted. By establishing an intramitochondrial metabolic bypass, the internalized energy-active units significantly elevatemitochondria membrane potential (ΔΨm) to supply increased bioenergetic levels and accelerate bone formation. The ready-to-use material developed here represents a highly efficient and easy-to-implement therapeutic approach toward tissue regeneration, withpromise for bench-to-bedside translation.

Journal article

Wang ST, Gray MA, Xuan S, Lin Y, Byrnes J, Nguyen AI, Todorova N, Stevens M, Bertozzi CR, Zuckermann RN, Gang Oet al., 2020, DNA origami protection and molecular interfacing through engineered sequence-defined peptoids, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 6339-6348, ISSN: 0027-8424

DNA nanotechnology has established approaches for designing programmable and precisely controlled nanoscale architectures through specific Watson−Crick base-pairing, molecular plasticity, and intermolecular connectivity. In particular, superior control over DNA origami structures could be beneficial for biomedical applications, including biosensing, in vivo imaging, and drug and gene delivery. However, protecting DNA origami structures in complex biological fluids while preserving their structural characteristics remains a major challenge for enabling these applications. Here, we developed a class of structurally well-defined peptoids to protect DNA origamis in ionic and bioactive conditions and systematically explored the effects of peptoid architecture and sequence dependency on DNA origami stability. The applicability of this approach for drug delivery, bioimaging, and cell targeting was also demonstrated. A series of peptoids (PE1–9) with two types of architectures, termed as “brush” and “block,” were built from positively charged monomers and neutral oligo-ethyleneoxy monomers, where certain designs were found to greatly enhance the stability of DNA origami. Through experimental and molecular dynamics studies, we demonstrated the role of sequence-dependent electrostatic interactions of peptoids with the DNA backbone. We showed that octahedral DNA origamis coated with peptoid (PE2) can be used as carriers for anticancer drug and protein, where the peptoid modulated the rate of drug release and prolonged protein stability against proteolytic hydrolysis. Finally, we synthesized two alkyne-modified peptoids (PE8 and PE9), conjugated with fluorophore and antibody, to make stable DNA origamis with imaging and cell-targeting capabilities. Our results demonstrate an approach toward functional and physiologically stable DNA origami for biomedical applications.

Journal article

Armstrong JPK, Keane TJ, Roques AC, Stephen Patrick P, Mooney CM, Kuan W-L, Pisupati V, Oreffo ROC, Stuckey D, Watt FM, Forbes SJ, Barker RA, Stevens Met al., A blueprint for translational regenerative medicine, Science Translational Medicine, ISSN: 1946-6234

The last few decades have produced a large number of proof-of-concept studies in regenerative medicine.However, the route to clinical adoption is fraught with technical and translational obstacles that frequentlyconsign promising academic solutions to the so-called “valley of death.” This review is intended to serve as ablueprint for translational regenerative medicine: we suggest principles to help guide cell and materialselection, present key in vivo imaging modalities and argue that the host immune response should beconsidered throughout therapeutic development. Finally, we suggest a pathway to navigate the oftencomplex regulatory and manufacturing landscape of translational regenerative medicine.

Journal article

Higgins S, Becce M, Belessiotis Richards A, Seong H, Sero J, Stevens Met al., 2020, High-aspect-ratio nanostructured surfaces as biological metamaterials, Advanced Materials, Vol: 32, Pages: 1-44, ISSN: 0935-9648

Materials patterned with high-aspect-ratio nanostructures have features on similar lengthscales to cellular components. These surfaces are an extreme topography on the cellular leveland have become useful tools for perturbing and sensing the cellular environment. Motivationcomes from the ability of high-aspect-ratio nanostructures to deliver cargoes into cells andtissues, access the intracellular environment, and control cell behavior. These structuresdirectly perturb cells’ ability to sense and respond to external forces, influencing cell fate andenabling new mechanistic studies. Through careful design of their nanoscale structure, thesesystems act as biological metamaterials, eliciting unusual biological responses. Whilepredominantly used to interface eukaryotic cells, there is growing interest in non-animal andprokaryotic cell interfacing. Both experimental and theoretical studies have attempted todevelop a mechanistic understanding for the observed behaviors, predominantly focusing onthe cell – nanostructure interface. Here, we consider how high-aspect-ratio nanostructuredsurfaces are used to both stimulate and sense biological systems and discuss remainingresearch questions.

Journal article

Roberts DA, Pilgrim BS, Dell TN, Stevens MMet al., 2020, Dynamic pH responsivity of triazole-based self-immolative linkers, Chemical Science, Vol: 11, Pages: 3713-3718, ISSN: 2041-6520

Gating the release of chemical payloads in response to transient signals is an important feature of ‘smart’ delivery systems. Herein, we report a triazole-based self-immolative linker that can be reversibly paused or slowed and restarted throughout its elimination cascade in response to pH changes in both organic and organic-aqueous solvents. The linker is conveniently prepared using the alkyne–azide cycloaddition reaction, which introduces a 1,4-triazole ring that expresses a pH-sensitive intermediate during its elimination sequence. Using a series of model compounds, we demonstrate that this intermediate can be switched between active and dormant states depending on the presence of acid or base, cleanly gating the release of payload in response to a fluctuating external stimulus.

Journal article

Armstrong J, Stevens M, 2020, Using remote fields for complex tissue engineering, Trends in Biotechnology, Vol: 38, Pages: 254-263, ISSN: 0167-7799

Great strides have been taken towards the in vitro engineering of clinically-relevant tissue constructsusing the classic triad of cells, materials and biochemical factors. In this perspective, we highlight ways in which these elements can be manipulated or stimulated using a fourth component: the application of remote fields.This arena has gained great momentum over the last few years, with a recent surge of interest in using magnetic, optical and acoustic fields to guide the organization of cells, materials and growth factors. We summarize recent developments and trends in this arena and then lay out a series of challenges that we believe, if met, could enable the widespread adoption of remote fields in mainstream tissue engineering.

Journal article

Spicer CD, Pujari-Palmer M, Autefage H, Insley G, Procter P, Engqvist H, Stevens M, Stevens Met al., 2020, Synthesis of phospho-amino acid analogues as tissue adhesive cement additives, ACS Central Science, Vol: 6, Pages: 226-231, ISSN: 2374-7943

In this paper we report the synthesis of a library of phospho-amino acid analogues, via a novel single-step allyl-phosphoester protection/Pd-mediated deprotection strategy. These phosphoserine and phosphotyrosine analogues were then applied as additives to create adhesive calcium phosphate cements, allowing us to probe the chemical origins of the increased surface binding strength. We demonstrate the importance of multiple calcium binding motifs in mediating adhesion, as well as highlighting the crucial role played by substrate hydrophobicity and orientation in controlling binding strength.

Journal article

Nele V, Schutt CE, Wojciechowski J, Kit-Anan W, Doutch JJ, Armstrong J, Stevens Met al., 2020, Ultrasound-triggered enzymatic gelation, Advanced Materials, Vol: 32, Pages: 1-8, ISSN: 0935-9648

Hydrogels are formed using various triggers, including light irradiation, pH adjustment, heating,cooling or chemical addition. In this report, a new method for forming hydrogels is introduced:ultrasound-triggered enzymatic gelation. Specifically, ultrasound is used as a stimulus to liberateliposomal calcium ions, which then trigger the enzymatic activity of transglutaminase. Theactivated enzyme catalyzes the formation of fibrinogen hydrogels through covalent intermolecularcrosslinking. The catalysis and gelation processes are monitored in real time and both the enzymekinetics and final hydrogel properties are controlled by varying the initial ultrasound exposure time.This technology is extended to microbubble-liposome conjugates, which exhibit a stronger responseto the applied acoustic field and are also used for ultrasound-triggered enzymatic hydrogelation. Tothe best of our knowledge, these results are the first instance in which ultrasound has been used as atrigger for either enzyme catalysis or enzymatic hydrogelation. This approach is highly versatile and Peer reviewed version of the manuscript published in final form at Advanced Materials (2020)2could be readily applied to different ion-dependent enzymes or gelation systems. Moreover, thiswork paves the way for the use of ultrasound as a remote trigger for in vivo hydrogelation.

Journal article

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