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

457 results found

Ren Y, Autefage H, Jones JR, Stevens MM, Bagot PAJ, Moody MPet al., 2022, Developing atom probe tomography to characterize Sr-loaded bioactive glass for bone scaffolding, Microscopy and Microanalysis, Vol: 28, Pages: 1310-1320, ISSN: 1431-9276

In this study, atom probe tomography (APT) was used to investigate strontium-containing bioactive glass particles (BG-Sr10) and strontium-releasing bioactive glass-based scaffolds (pSrBG), both of which are attractive biomaterials with applications in critical bone damage repair. We outline the challenges and corresponding countermeasures of this nonconductive biomaterial for APT sample preparation and experiments, such as avoiding direct contact between focussed ion beam micromanipulators and the extracted cantilever to reduce damage during liftout. Using a low imaging voltage (≤3 kV) and current (≤500 pA) in the scanning electron microscope and a low acceleration voltage (≤2 kV) and current (≤200 pA) in the focussed ion beam prevents tip bending in the final stages of annular milling. To optimize the atom probe experiment, we considered five factors: total detected hits, multiple hits, the background level, the charge-state ratio, and the accuracy of the measured compositions, to explore the optimal laser pulse for BG-Sr10 bioactive glass. We show that a stage temperature of 30 K, 200–250 pJ laser pulse energy, 0.3% detection rate, and 200 kHz pulse rate are optimized experimental parameters for bioactive glass. The use of improved experimental preparation methods and optimized parameters resulted in a 90% successful yield of pSrBG samples by APT.

Journal article

Kim H, Yeow J, Najer A, Kit-Anan W, Wang R, Rifaie Graham O, Thanapongpibul C, Stevens Met al., 2022, Microliter scale synthesis of luciferase-encapsulated polymersomes as artificial organelles for optogenetic modulation of cardiomyocyte beating, Advanced Science, ISSN: 2198-3844

Constructing artificial systems that effectively replace or supplement natural biological machinery within cells is one of the fundamental challenges underpinning bioengineering. At the sub-cellular scale, artificial organelles (AOs) have significant potential as long-acting biomedical implants, mimicking native organelles by conducting intracellularly compartmentalized enzymatic actions. The potency of these AOs can be heightened when judiciously combined with genetic engineering, producing highly tailorable biohybrid cellular systems. Here, the authors present a cost-effective, microliter scale (10 µL) polymersome (PSome) synthesis based on polymerization-induced self-assembly for the in situ encapsulation of Gaussia luciferase (GLuc), as a model luminescent enzyme. These GLuc-loaded PSomes present ideal features of AOs including enhanced enzymatic resistance to thermal, proteolytic, and intracellular stresses. To demonstrate their biomodulation potential, the intracellular luminescence of GLuc-loaded PSomes is coupled to optogenetically engineered cardiomyocytes, allowing modulation of cardiac beating frequency through treatment with coelenterazine (CTZ) as the substrate for GLuc. The long-term intracellular stability of the luminescent AOs allows this cardiostimulatory phenomenon to be reinitiated with fresh CTZ even after 7 days in culture. This synergistic combination of organelle-mimicking synthetic materials with genetic engineering is therefore envisioned as a highly universal strategy for the generation of new biohybrid cellular systems displaying unique triggerable properties.

Journal article

Speidel A, Grigsby C, Stevens M, 2022, Ascendancy of semi-synthetic biomaterials from design towards democratization, Nature Materials, ISSN: 1476-1122

Semi-synthetic “goldilocks” material design integrates the advantageous tunable characteristics of synthetic materials and the refined complexity of natural components, and has opened doors for the progress of biomaterials across length scales. Through this multi-modal approach, accelerated translational success may be possible for more personalized and accessible products.

Journal article

Hsu CC, Serio A, Gopal S, Gelmi A, Chiappini C, Desai R, Stevens Met al., 2022, Biophysical regulations of epigenetic state and notch signalling in neural development using microgroove substrates, ACS Applied Materials and Interfaces, ISSN: 1944-8244

A number of studies have recently shown how surface topography can alter behaviour and differentiation patterns of different types of stem cells. Although the exact mechanisms and molecular pathways involved remain unclear, a consistent portion of the literature points to epigenetic changes induced by nuclear remodelling. In this study, we investigate the behaviour of clinically relevant neural populations derived from human pluripotent stem cells when cultured on polydimethylsiloxane microgrooves (3 μm- and 10 μm-depth grooves), to investigate what mechanisms are responsible for their differentiation capacity and functional behaviour. Our results show that microgrooves enhance cell alignment, modify nuclear geometry and significantly increase cellular stiffness, which we were able to measure at high resolution with a combination of light and electron microscopy, scanning ion conductance microscopy (SICM) and atomic force microscopy (AFM) coupled with quantitative image analysis. The microgrooves promoted significant changes in the epigenetic landscape, as revealed by the expression of key histone modification markers. The main behavioural change of neural stem cells on microgrooves was an increase of neuronal differentiation under basal conditions on the microgrooves. Through measurements of cleaved Notch1 levels, we found that microgrooves downregulate Notch signalling. We in fact propose that microgroovetopography affects the differentiation potential of neural stem cells by indirectly altering Notch signalling through geometric segregation and that this mechanism in parallel with topography-dependent epigenetic modulations acts in concert to enhance stem cell neuronal differentiation.

Journal article

Armstrong JP, Pchelintseva E, Treumuth S, Campanella C, Meinert C, Klein TJ, Hutmacher DW, Drinkwater BW, Stevens MMet al., 2022, Tissue engineering cartilage with deep zone cytoarchitecture by high-resolution acoustic cell patterning, Advanced Healthcare Materials, ISSN: 2192-2640

The ultimate objective of tissue engineering is to fabricate artificial living constructs with a structural organization and function that faithfully resembles their native tissue counterparts. For example, the deep zone of articular cartilage possesses a distinctive anisotropic architecture with chondrocytes organized in aligned arrays ≈1–2 cells wide, features that are oriented parallel to surrounding extracellular matrix fibers and orthogonal to the underlying subchondral bone. Although there are major advances in fabricating custom tissue architectures, it remains a significant technical challenge to precisely recreate such fine cellular features in vitro. Here, it is shown that ultrasound standing waves can be used to remotely organize living chondrocytes into high-resolution anisotropic arrays, distributed throughout the full volume of agarose hydrogels. It is demonstrated that this cytoarchitecture is maintained throughout a five-week course of in vitro tissue engineering, producing hyaline cartilage with cellular and extracellular matrix organization analogous to the deep zone of native articular cartilage. It is anticipated that this acoustic cell patterning method will provide unprecedented opportunities to interrogate in vitro the contribution of chondrocyte organization to the development of aligned extracellular matrix fibers, and ultimately, the design of new mechanically anisotropic tissue grafts for articular cartilage regeneration.

Journal article

Najer A, Belessiotis Richards A, Kim H, Saunders C, Adrianus C, Fenaroli F, Che J, Tonkin R, Hogset H, Loercher S, Penna M, Higgins S, Meier W, Yarovsky I, Stevens MMet al., 2022, Block length-dependent protein fouling on Poly(2-oxazoline)-based polymersomes: influence on macrophage association and circulation behavior, Small, Vol: 18, ISSN: 1613-6810

Polymersomes are vesicular structures self-assembled from amphiphilic block copolymers and are considered an alternative to liposomes for applications in drug delivery, immunotherapy, biosensing, and as nanoreactors and artificial organelles. However, the limited availability of systematic stability, protein fouling (protein corona formation), and blood circulation studies hampers their clinical translation. Poly(2-oxazoline)s (POx) are valuable antifouling hydrophilic polymers that can replace the current gold-standard, poly(ethylene glycol) (PEG), yet investigations of POx functionality on nanoparticles are relatively sparse. Herein, a systematic study is reported of the structural, dynamic and antifouling properties of polymersomes made of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA). The study relates in vitro antifouling performance of the polymersomes to atomistic molecular dynamics simulations of polymersome membrane hydration behavior. These observations support the experimentally demonstrated benefit of maximizing the length of PMOXA (degree of polymerization (DP) > 6) while keeping PDMS at a minimal length that still provides sufficient membrane stability (DP > 19). In vitro macrophage association and in vivo blood circulation evaluation of polymersomes in zebrafish embryos corroborate these findings. They further suggest that single copolymer presentation on polymersomes is outperformed by blends of varied copolymer lengths. This study helps to rationalize design rules for stable and low-fouling polymersomes for future medical applications.

Journal article

Miller BS, Thomas MR, Banner M, Kim J, Chen Y, Wei Q, Tseng DK, Goeroecs ZS, Ozcan AM, Stevens MMA, McKendry RAet al., 2022, Sub-picomolar lateral flow antigen detection with two-wavelength imaging of composite nanoparticles, Biosensors and Bioelectronics, Vol: 207, Pages: 1-10, ISSN: 0956-5663

Lateral flow tests, commonly based on metal plasmonic nanoparticles, are rapid, robust, and low-cost. However, improvements in analytical sensitivity are required to allow detection of low-abundance biomarkers, for example detection of low antigen concentrations for earlier or asymptomatic diagnosis of infectious diseases. Efforts to improve sensitivity often require changes to the assay. Here, we developed optical methods to improve the sensitivity of absorption-based lateral flow tests, requiring no assay modifications to existing tests. We experimentally compared five different lock-in and subtraction-based methods, exploiting the narrow plasmonic peak of gold nanoparticles for background removal by imaging at different light wavelengths. A statistical framework and three fitting models were used to compare limits of detection, giving a 2.0–5.4-fold improvement. We then demonstrated the broad applicability of the method to an ultrasensitive assay, designing 530 nm composite nanoparticles to increase the particle volume, and therefore light absorption per particle, whilst retaining the plasmonic peak to allow background removal and without adding any assay steps. This multifaceted, modular approach gave a combined 58-fold improvement in the fundamental limit of detection using a biotin-avidin model over 50 nm gold nanoparticles with single-wavelength imaging. Applying to a sandwich assay for the detection of HIV capsid protein gave a limit of detection of 170 fM. Additionally, we developed an open-source software tool for performing the detection limit analysis used in this work.

Journal article

Barriga HMG, Pence IJ, Holme M, Doutch J, Penders J, Nele V, Thomas M, Carroni M, Stevens Met al., 2022, Coupling lipid nanoparticle structure and automated single particle composition analysis to design phospholipase responsive nanocarriers, Advanced Materials, Vol: 34, Pages: 1-11, ISSN: 0935-9648

Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here we combine Single Particle Automated Raman Trapping Analysis (SPARTA®) with small angle scattering (SAXS / SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP - phospholipase D (PLD) interactions. Our analysis demonstrates that phospholipase D, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate was an order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme-responsive LNP solutions for stimuli-responsive delivery and diseases where PLD is dysregulated.

Journal article

Tallia F, Ting HK, Page S, Clark J, Li S, Sang T, Russo L, Stevens M, Hanna JV, Jones Jet al., 2022, Bioactive, degradable and tough hybrids through calcium and phosphate incorporation, Frontiers in Materials, Vol: 9, ISSN: 2296-8016

We report the first inorganic/organic hybrids that show outstanding mechanical properties (withstanding cyclic loading) and bone bioactivity. This new hybrid material may fulfil the unmet clinical need for bioactive synthetic bone grafts that can withstand cyclic loading. A SiO2/PTHF/PCL-diCOOH sol-gel hybrid system, that combined inorganic and organic co-networks at the molecular level, previously demonstrated unprecedented synergy of properties, with excellent flexibility and promoted formation of articular cartilage matrix in vitro. Here, for the first time, calcium and phosphate ions were incorporated into the inorganic component of the hybrid network, to impart osteogenic properties. Calcium methoxyethoxide and triethyl phosphate were the calcium and phosphate precursors because they allow for incorporation into the silicate network at low temperature. The hybrid network was characterised with ATR-FTIR, XRD and solid-state NMR,which proved calcium and phosphate incorporation and suggested the Ca2+ ions also interacted with PCL-diCOOH through ionic bonds. This resulted in an increased strength (17-64 MPa) and modulus of toughness (2.5-14 MPa) compared to the original SiO2/PTHF/PCL-diCOOH hybrid material (which showed strength of ̃3 MPa and modulus of toughness of ̃0.35 MPa), while also maintaining the ability to withstand cyclic loading. The presence of calcium and phosphates in the silicate network resulted in a more congruent dissolution of the inorganic and organic co-networks in TRIS buffer. This was shown by the presence of silicon, calcium and phosphate ions along with PCL in the TRIS buffer after 1 week, whereas Ca-free hybrids mainly released PCL with negligible Si dissolution. The presence of calcium and phosphates also enabled deposition of hydroxycarbonate apatite following immersion in simulated body fluid, which was not seen on Ca-free hybrid. All hybrids passed cell cytotoxicity tests and supported pre-osteoblast cell attachment. The phosphate-fr

Journal article

Tonkin R, Kloeckner A, Najer A, Simoes da Silva CJ, Echalier C, Dionne MS, Edwards AM, Stevens Met al., 2022, Bacterial toxin-triggered release of antibiotics from capsosomes protects a fly model from lethal methicillin-resistant Staphylococcus aureus (MRSA) infection, Advanced Healthcare Materials, Vol: 11, Pages: 1-14, ISSN: 2192-2640

Antibiotic resistance is a severe global health threat and hence demands rapid action to develop novel therapies, including microscale drug delivery systems. Herein, a hierarchical microparticle system is developed to achieve bacteria-activated single- and dual-antibiotic drug delivery for preventing methicillin-resistant Staphylococcus aureus (MRSA) bacterial infections. The designed system is based on a capsosome structure, which consists of a mesoporous silica microparticle coated in alternating layers of oppositely charged polymers and antibiotic-loaded liposomes. The capsosomes are engineered and shown to release their drug payloads in the presence of MRSA toxins controlled by the Agr quorum sensing system. MRSA-activated single drug delivery of vancomycin and synergistic dual delivery of vancomycin together with an antibacterial peptide successfully kills MRSA in vitro. The capability of capsosomes to selectively deliver their cargo in the presence of bacteria, producing a bactericidal effect to protect the host organism, is confirmed in vivo using a Drosophila melanogaster MRSA infection model. Thus, the capsosomes serve as a versatile multidrug, subcompartmentalized microparticle system for preventing antibiotic-resistant bacterial infections, with potential applications to protect wounds or medical device implants from infections.

Journal article

Steele JAM, Moore A, St-Pierre J, McCullen SD, Gormley A, Horgan CC, Black CRM, Meinert C, Klein T, Saifzadeh S, Steck R, Ren J, Woodruff M, Stevens Met al., 2022, In vitro and in vivo investigation of a zonal microstructured scaffold for osteochondral defect repair, Biomaterials, Vol: 286, Pages: 1-13, ISSN: 0142-9612

Articular cartilage is comprised of zones that vary in architecture, extracellular matrix composition, and mechanical properties. Here, we designed and engineered a porous zonal microstructured scaffold from a single biocompatible polymer (poly [ϵ-caprolactone]) using multiple fabrication strategies: electrospinning, spherical porogen leaching, directional freezing, and melt electrowriting. With this approach we mimicked the zonal structure of articular cartilage and produced a stiffness gradient through the scaffold which aligns with the mechanics of the native tissue. Chondrocyte-seeded scaffolds accumulated extracellular matrix including glycosaminoglycans and collagen II over four weeks in vitro. This prompted us to further study the repair efficacy in a skeletally mature porcine model. Two osteochondral lesions were produced in the trochlear groove of 12 animals and repaired using four treatment conditions: (1) microstructured scaffold, (2) chondrocyte seeded microstructured scaffold, (3) MaioRegen™, and (4) empty defect. After 6 months the defect sites were harvested and analyzed using histology, micro computed tomography, and Raman microspectroscopy mapping. Overall, the scaffolds were retained in the defect space, repair quality was repeatable, and there was clear evidence of osteointegration. The repair quality of the microstructured scaffolds was not superior to the control based on histological scoring; however, the lower score was biased by the lack of histological staining due to the limited degradation of the implant at 6 months. Longer follow up studies (e.g., 1 yr) will be required to fully evaluate the efficacy of the microstructured scaffold. In conclusion, we found consistent scaffold retention, osteointegration, and prolonged degradation of the microstructured scaffold, which we propose may have beneficial effects for the long-term repair of osteochondral defects.

Journal article

Hachim D, Zhao J, Bhankharia J, Nuñez-Toldra R, Brito L, Seong H, Becce M, Ouyang L, Grigsby C, Higgins S, Terracciano C, Stevens Met al., 2022, Polysaccharide-polyplex nanofilm coatings enhance nanoneedle-based gene delivery and transfection efficiency, Small, ISSN: 1613-6810

Non-viral vectors represent versatile and immunologically safer alternatives fornucleic acid delivery, with polyplexes being a widely known option. Nanoneedles and highaspect ratio nanostructures are unconventional but interesting delivery systems, in which deliveryis mediated by surface interactions. Herein, we synergistically combined nanoneedles withpolysaccharide-polyplex nanofilms and observed enhanced transfection efficiency compared topolyplexes in suspension. Different polyplex-polyelectrolyte nanofilm combinations wereassessed and found that transfection efficiency was enhanced when using polysaccharide-basedpolyanions, rather than being only specific for hyaluronic acid, as suggested in previous studies.Moreover, results showed that enhanced transfection was not mediated by interactions with theCD44 receptor, hypothesized as a major mechanism mediating enhancement via hyaluronate. Incardiac tissue, nanoneedles were shown to increase the transfection efficiency of nanofilmscompared to flat substrates; while in vitro, high transfection efficiencies were observed innanostructures where cells presented large interfacing areas with the substrate. The results of thisstudy demonstrate that surface-mediated transfection using this system is efficient and safe,requiring amounts of nucleic acid with an order of magnitude lower than standard culturetransfection. These findings expand the spectrum of possible polyelectrolyte combinations thatcan be used for the development of suitable non-viral vectors for exploration in further clinicaltrials.

Journal article

Broto M, Kaminski MM, Adrianus C, Kim N, Greensmith R, Dissanayake-Perera S, Schubert AJ, Tan X, Kim H, Dighe AS, Collins JJ, Stevens Met al., 2022, Nanozyme-catalysed CRISPR assay for preamplification-free detection of non-coding RNAs, Nature Nanotechnology, ISSN: 1748-3387

CRISPR-based diagnostics enable specific sensing of DNA and RNA biomarkers associated withhuman diseases. This is achieved through the binding of guide RNAs to a complementarysequence which activates Cas enzymes to cleave reporter molecules. Currently, most CRISPRbased diagnostics rely on target preamplification to reach sufficient sensitivity for clinicalapplications. This limits quantification capability and adds complexity to the reaction chemistry.Here, we show the combination of a CRISPR/Cas-based reaction with a Nanozyme-LinkedImmunoSorbent Assay which allows for the quantitative and colorimetric readout of Cas13-mediated RNA detection through catalytic metallic nanoparticles at room temperature(CrisprZyme). We demonstrate CrisprZyme is easily adaptable to a lateral-flow-based readoutand different Cas enzymes, and enables the sensing of non-coding RNAs including microRNAs,long non-coding RNAs and circular RNAs. We utilise this platform to identify patients with acutemyocardial infarction and to monitor cellular differentiation in vitro and in tissue biopsies fromprostate cancer patients. We anticipate that CrisprZyme has significant potential as a universallyapplicable signal catalyst for CRISPR-based diagnostics which will expand the spectrum oftargets for preamplification-free, quantitative detection.

Journal article

Artzy-Schnirman A, Abu-Shah E, Chandrawati R, Altman E, Yusuf N, Wang S-T, Ramos J, Hansel C, Haus-Cohen M, Dahan R, Arif S, Dustin ML, Reiter Y, Stevens MMet al., 2022, Artificial antigen presenting cells for detection and desensitisation of auto-reactive T cells associated with Type 1 diabetes, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 22, Pages: 4376-4382, ISSN: 1530-6984

Autoimmune diseases and in particular type 1 diabetes rely heavily on treatments that target the symptoms rather than prevent the underlying disease. One of the barriers to better therapeutic strategies is the inability to detect and efficiently target rare autoreactive T-cell populations that are major drivers of these conditions. Here, we develop a unique artificial antigen-presenting cell (aAPC) system from biocompatible polymer particles that allows specific encapsulation of bioactive ingredients. Using our aAPC, we demonstrate that we are able to detect rare autoreactive CD4 populations in human patients, and using mouse models, we demonstrate that our particles are able to induce desensitization in the autoreactive population. This system provides a promising tool that can be used in the prevention of autoimmunity before disease onset.

Journal article

Görgens A, Corso G, Hagey DW, Jawad Wiklander R, Gustafsson MO, Felldin U, Lee Y, Bostancioglu RB, Sork H, Liang X, Zheng W, Mohammad DK, van de Wakker SI, Vader P, Zickler AM, Mamand DR, Ma L, Holme MN, Stevens MM, Wiklander OPB, El Andaloussi Set al., 2022, Identification of storage conditions stabilizing extracellular vesicles pre, Journal of Extracellular Vesicles, Vol: 11, ISSN: 2001-3078

Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS-HAT). We report that PBS-HAT buffer facilitates clearly improved short-term and long-term EV preservation for samples stored at -80°C, stability throughout several freeze-thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.

Journal article

Mulvaney P, Buriak JM, Chen X, Hu T, Millstone JE, Stevens Met al., 2022, Nanoscience and Entrepreneurship, Publisher: AMER CHEMICAL SOC

Other

Dane EL, Belessiotis-Richards A, Backlund C, Wang J, Hidaka K, Milling LE, Bhagchandani S, Melo MB, Wu S, Li N, Donahue N, Ni K, Ma L, Okaniwa M, Stevens MM, Alexander-Katz A, Irvine DJet al., 2022, STING agonist delivery by tumour-penetrating PEG-lipid nanodiscs primes robust anticancer immunity, Nature Materials, Vol: 23/5/22, Pages: 710-720, ISSN: 1476-1122

Activation of the innate immune STimulator of INterferon Genes (STING) pathway potentiates antitumour immunity, but systemic delivery of STING agonists to tumours is challenging. We conjugated STING-activating cyclic dinucleotides (CDNs) to PEGylated lipids (CDN-PEG-lipids; PEG, polyethylene glycol) via a cleavable linker and incorporated them into lipid nanodiscs (LNDs), which are discoid nanoparticles formed by self-assembly. Compared to state-of-the-art liposomes, intravenously administered LNDs carrying CDN-PEG-lipid (LND-CDNs) exhibited more efficient penetration of tumours, exposing the majority of tumour cells to STING agonist. A single dose of LND-CDNs induced rejection of established tumours, coincident with immune memory against tumour rechallenge. Although CDNs were not directly tumoricidal, LND-CDN uptake by cancer cells correlated with robust T-cell activation by promoting CDN and tumour antigen co-localization in dendritic cells. LNDs thus appear promising as a vehicle for robust delivery of compounds throughout solid tumours, which can be exploited for enhanced immunotherapy.

Journal article

Najer A, Blight J, Ducker CB, Gasbarri M, Brown JC, Che J, Hogset H, Saunders C, Ojansivu M, Lu Z, Lin Y, Yeow J, Rifaie Graham O, Potter M, Tonkin R, Penders J, Doutch JJ, Georgiadou A, Barriga HMG, Holme MN, Cunnington AJ, Bugeon L, Dallman MJ, Barclay WS, Stellacci F, Baum J, Stevens MMet al., 2022, Potent virustatic polymer-lipid nanomimics block viral entry and inhibit malaria parasites in vivo, ACS Central Science, ISSN: 2374-7943

Infectious diseases continue to pose a substantial burden on global populations, requiring innovative broad-spectrum prophylactic and treatment alternatives. Here, we have designed modular synthetic polymer nanoparticles that mimic functional components of host cell membranes, yielding multivalent nanomimics that act by directly binding to varied pathogens. Nanomimic blood circulation time was prolonged by reformulating polymer–lipid hybrids. Femtomolar concentrations of the polymer nanomimics were sufficient to inhibit herpes simplex virus type 2 (HSV-2) entry into epithelial cells, while higher doses were needed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given their observed virustatic mode of action, the nanomimics were also tested with malaria parasite blood-stage merozoites, which lose their invasive capacity after a few minutes. Efficient inhibition of merozoite invasion of red blood cells was demonstrated both in vitro and in vivo using a preclinical rodent malaria model. We envision these nanomimics forming an adaptable platform for developing pathogen entry inhibitors and as immunomodulators, wherein nanomimic-inhibited pathogens can be secondarily targeted to sites of immune recognition.

Journal article

Ovrebo O, Perale G, Wojciechowski J, Echalier C, Jeffers J, Stevens M, Haugen H, Rossi Fet al., 2022, Design and clinical application of injectable hydrogels for musculoskeletal therapy, Bioengineering and Translational Medicine, Vol: 7, Pages: 1-21, ISSN: 2380-6761

Musculoskeletal defects are an enormous healthcare burden and source of pain and disability for individuals. With an ageing population, the proportion living with these medical indications will increase. Simultaneously, there is pressure on healthcare providers to source efficient solutions, which are cheaper and less invasive than conventional technology. This has led to an increased research focus on hydrogels as highly biocompatible biomaterials that can be delivered through minimally invasive procedures. This review will discuss how hydrogels can be designed for clinical translation, particularly in the context of the new European Medical Device Regulation (MDR). We will then do a deep dive into the clinically used hydrogel solutions that have been commercially approved or have undergone clinical trials in Europe or the US. We will discuss the therapeutic mechanism and limitations of these products. Due to the vast application areas of hydrogels, this work focuses only on treatments of cartilage, bone, and the nucleus pulposus. Lastly, the main steps towards clinical translation of hydrogels as medical devices are outlined. We suggest a framework for how academics can assist small and medium MedTech enterprises conducting the initial clinical investigation and Post-Market Clinical Follow-up (PMCF) required in the MDR. It is evident that the successful translation of hydrogels is governed by acquiring high-quality pre-clinical and clinical data confirming the device mechanism of action and safety.

Journal article

Belessiotis-Richards A, Larsen AH, Higgins SG, Stevens MM, Alexander-Katz Aet al., 2022, Coarse-grained simulations suggest potential competing roles of phosphoinositides and amphipathic helix structures in membrane curvature sensing of the AP180 N-terminal homology domain, The Journal of Physical Chemistry B: Biophysical Chemistry, Biomaterials, Liquids, and Soft Matter, Vol: 126, Pages: 2789-2797, ISSN: 1520-5207

The generation and sensing of membrane curvature by proteins has become of increasing interest to researchers with multiple mechanisms, from hydrophobic insertion to protein crowding, being identified. However, the role of charged lipids in the membrane curvature-sensing process is still far from understood. Many proteins involved in endocytosis bind phosphatidylinositol 4,5-bisphosphate (PIP2) lipids, allowing these proteins to accumulate at regions of local curvature. Here, using coarse-grained molecular dynamics simulations, we study the curvature-sensing behavior of the ANTH domain, a protein crucial for endocytosis. We selected three ANTH crystal structures containing either an intact, split, or truncated terminal amphipathic helix. On neutral membranes, the ANTH domain has innate curvature-sensing ability. In the presence of PIP2, however, only the domain with an intact helix senses curvature. Our work sheds light on the role of PIP2 and its modulation of membrane curvature sensing by proteins.

Journal article

Utterström J, Barriga HMG, Holme MN, Selegård R, Stevens MM, Aili Det al., 2022, Peptide-folding triggered phase separation and lipid membrane destabilization in cholesterol-rich lipid vesicles., Bioconjugate Chemistry, Vol: 33, Pages: 736-746, ISSN: 1043-1802

Liposome-based drug delivery systems are widely used to improve drug pharmacokinetics but can suffer from slow and unspecific release of encapsulated drugs. Membrane-active peptides, based on sequences derived or inspired from antimicrobial peptides (AMPs), could offer means to trigger and control the release. Cholesterol is used in most liposomal drug delivery systems (DDS) to improve the stability of the formulation, but the activity of AMPs on cholesterol-rich membranes tends to be very low, complicating peptide-triggered release strategies. Here, we show a de novo designed AMP-mimetic peptide that efficiently triggers content release from cholesterol-containing lipid vesicles when covalently conjugated to headgroup-functionalized lipids. Binding to vesicles induces peptide folding and triggers a lipid phase separation, which in the presence of cholesterol results in high local peptide concentrations at the lipid bilayer surface and rapid content release. We anticipate that these results will facilitate the development of peptide-based strategies for controlling and triggering drug release from liposomal drug delivery systems.

Journal article

Minelli C, Wywijas M, Bartczak D, Cuello-Nuñez S, Infante HG, Deumer J, Gollwitzer C, Krumrey M, Murphy KE, Johnson ME, Montoro Bustos AR, Strenge IH, Faure B, Høghøj P, Tong V, Burr L, Norling K, Höök F, Roesslein M, Kocic J, Hendriks L, Kestens V, Ramaye Y, Contreras Lopez MC, Auclair G, Mehn D, Gilliland D, Potthoff A, Oelschlägel K, Tentschert J, Jungnickel H, Krause BC, Hachenberger YU, Reichardt P, Luch A, Whittaker TE, Stevens MM, Gupta S, Singh A, Lin F-H, Liu Y-H, Costa AL, Baldisserri C, Jawad R, Andaloussi SEL, Holme MN, Lee TG, Kwak M, Kim J, Ziebel J, Guignard C, Cambier S, Contal S, Gutleb AC, Kuba Tatarkiewicz J, Jankiewicz BJ, Bartosewicz B, Wu X, Fagan JA, Elje E, Rundén-Pran E, Dusinska M, Kaur IP, Price D, Nesbitt I, O Reilly S, Peters RJB, Bucher G, Coleman D, Harrison AJ, Ghanem A, Gering A, McCarron E, Fitzgerald N, Cornelis G, Tuoriniemi J, Sakai M, Tsuchida H, Maguire C, Prina-Mello A, Lawlor AJ, Adams J, Schultz CL, Constantin D, Thanh NTK, Tung LD, Panariello L, Damilos S, Gavriilidis A, Lynch I, Fryer B, Carrazco Quevedo A, Guggenheim E, Briffa S, Valsami-Jones E, Huang Y, Keller AA, Kinnunen V-T, Perämäki S, Krpetic Z, Greenwood M, Shard AGet al., 2022, Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles, Nanoscale, Vol: 14, Pages: 4690-4704, ISSN: 2040-3364

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.

Journal article

Belessiotis-Richards A, Larsen AH, Higgins SG, Alexander-Katz A, Stevens Met al., 2022, Coarse-grained simulations suggest phosphoinositides and amphipathic helix structure play opposing roles in membrane curvature sensing of the AP180 N-terminal homology domain, The Journal of Physical Chemistry B: Biophysical Chemistry, Biomaterials, Liquids, and Soft Matter, ISSN: 1520-5207

The generation and sensing of membrane curvature by proteins has become of increasinginterest to researchers with multiple mechanisms, from hydrophobic insertion to protein crowding, beingidentified. However, the role of charged lipids in the membrane curvature sensing process is still far fromunderstood. Many proteins involved in endocytosis bind phosphatidylinositol 4,5-bisphosphate (PIP2)lipids, allowing these proteins to accumulate at regions of local curvature. Here, using coarse-grainedmolecular dynamics simulations, we study the curvature sensing behavior of the ANTH domain, a proteincrucial for endocytosis. We selected three ANTH crystal structures containing either an intact, split, ortruncated terminal amphipathic helix. On neutral membranes, the ANTH domain has innate curvaturesensing ability. In the presence of PIP2, however, only the domain with an intact helix senses curvature.Our work sheds light on the role of PIP2 and its modulation of membrane curvature sensing by proteins.

Journal article

Kotov NA, Akinwande D, Brinker CJ, Buriak JM, Chan WCW, Chen X, Chhowalla M, Chueh W, Glotzer SC, Gogotsi Y, Hersam MC, Ho D, Hu T, Javey A, Kagan CR, Kataoka K, Kim I-D, Lee S-T, Lee YH, Liz-Marzan LM, Millstone JE, Mulvaney P, Nel AE, Nordlander P, Parak WJ, Penner RM, Rogach AL, Salanne M, Schaak RE, Sood AK, Stevens M, Tsukruk V, Wee ATS, Voets I, Weil T, Weiss PSet al., 2022, Tanks and Truth, Publisher: AMER CHEMICAL SOC

Other

Lachowski D, Matellan C, Gopal S, Cortes E, Robinson BK, Saiani A, Miller AF, Stevens MM, Del Río Hernández AEet al., 2022, Substrate stiffness-driven membrane tension modulates vesicular trafficking via caveolin-1., ACS Nano, Vol: 16, Pages: 4322-4337, ISSN: 1936-0851

Liver fibrosis, a condition characterized by extensive deposition and cross-linking of extracellular matrix (ECM) proteins, is idiosyncratic in cases of chronic liver injury. The dysregulation of ECM remodeling by hepatic stellate cells (HSCs), the main mediators of fibrosis, results in an elevated ECM stiffness that drives the development of chronic liver disease such as cirrhosis and hepatocellular carcinoma. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a key element in the regulation of ECM remodeling, which modulates the degradation and turnover of ECM components. We have previously reported that a rigid, fibrotic-like substrate can impact TIMP-1 expression at the protein level in HSCs without altering its mRNA expression. While HSCs are known to be highly susceptible to mechanical stimuli, the mechanisms through which mechanical cues regulate TIMP-1 at the post-translational level remain unclear. Here, we show a mechanism of regulation of plasma membrane tension by matrix stiffness. We found that this effect is orchestrated by the β1 integrin/RhoA axis and results in elevated exocytosis and secretion of TIMP-1 in a caveolin-1- and dynamin-2-dependent manner. We then show that TIMP-1 and caveolin-1 expression increases in cirrhosis and hepatocellular carcinoma. These conditions are associated with fibrosis, and this effect can be recapitulated in 3D fibrosis models consisting of hepatic stellate cells encapsulated in a self-assembling polypeptide hydrogel. This work positions stiffness-dependent membrane tension as a key regulator of enzyme secretion and function and a potential target for therapeutic strategies that aim at modulating ECM remodeling in chronic liver disease.

Journal article

Bost JP, Ojansivu M, Munson MJ, Wesén E, Gallud A, Gupta D, Gustafsson O, Saher O, Rädler J, Higgins SG, Lehto T, Holme MN, Dahlén A, Engkvist O, Strömstedt P-E, Andersson S, Edvard Smith CI, Stevens MM, Esbjörner EK, Collén A, El Andaloussi Set al., 2022, Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides, Communications Biology, Vol: 5, ISSN: 2399-3642

The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.

Journal article

Hu K, McKay PF, Samnuan K, Najer A, Blakney AK, Che J, O'Driscoll G, Cihova M, Stevens MM, Shattock RJet al., 2022, Presentation of antigen on extracellular vesicles using transmembrane domains from viral glycoproteins for enhanced immunogenicity, Journal of Extracellular Vesicles, Vol: 11, ISSN: 2001-3078

A vaccine antigen, when launched as DNA or RNA, can be presented in various forms, including intracellular, secreted, membrane-bound, or on extracellular vesicles (EVs). Whether an antigen in one or more of these forms is superior in immune induction remains unclear. In this study, we used GFP as a model antigen and first compared the EV-loading efficiency of transmembrane domains (TMs) from various viral glycoproteins, and then investigated whether EV-bound GFP (EV-GFP) would enhance immune induction. Our data showed that GFP fused to viral TMs was successfully loaded onto the surface of EVs. In addition, GFP-bound EVs were predominantly associated with the exosome marker CD81. Immunogenicity study with EV-GFP-producing plasmids in mice demonstrated that antigen-specific IgG and IgA were significantly increased in EV-GFP groups, compared to soluble and intracellular GFP groups. Similarly, GFP-specific T cell response-related cytokines produced by antigen-stimulated splenocytes were also enhanced in mice immunized with EV-GFP constructs. Immunogenicity study with purified soluble GFP and GFP EVs further confirmed the immune enhancement property of EV-GFP in mice. In vitro uptake assays indicated that EV-GFP was more efficiently taken up than soluble GFP by mouse splenocytes and such uptake was B cell preferential. Taken together, our data indicate that viral TMs can efficiently load antigens onto the EV surface, and that EV-bound antigen enhances both humoral and cell-mediated antigen-specific responses.

Journal article

Geng H, Pedersen S, Ma Y, Haghighi T, Dai H, Howes PD, Stevens Met al., 2022, Noble metal nanoparticle biosensors: from fundamental studies towards point-of-care diagnostics, Accounts of Chemical Research, Vol: 55, Pages: 593-604, ISSN: 0001-4842

Noble metal nanoparticles (NMNPs) have become firmly established as effective agents to detect various biomolecules with extremely high sensitivity. This ability stems from the collective oscillation of free electrons and extremely large electric field enhancement under exposure to light, leading to various light–matter interactions such as localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering. A remarkable feature of NMNPs is their customizability by mechanisms such as particle etching, growth, and aggregation/dispersion, yielding distinct color changes and excellent opportunities for colorimetric biosensing in user-friendly assays and devices. They are readily functionalized with a large variety of capping agents and biomolecules, with resultant bioconjugates often possessing excellent biocompatibility, which can be used to quantitatively detect analytes from physiological fluids. Furthermore, they can possess excellent catalytic properties that can achieve significant signal amplification through mechanisms such as the catalytic transformation of colorless substrates to colored reporters. The various excellent attributes of NMNP biosensors have put them in the spotlight for developing high-performance in vitro diagnostic (IVD) devices that are particularly well-suited to mitigate the societal threat that infectious diseases pose. This threat continues to dominate the global health care landscape, claiming millions of lives annually. NMNP IVDs possess the potential to sensitively detect infections even at very early stages with affordable and field-deployable devices, which will be key to strengthening infectious disease management. This has been the major focal point of current research, with a view to new avenues for early multiplexed detection of infectious diseases with portable devices such as smartphones, especially in resource-limited settings.

Journal article

Constantinou AP, Nele V, Doutch JJ, S Correia J, Moiseev RV, Cihova M, Gaboriau DCA, Krell J, Khutoryanskiy VV, Stevens MM, Georgiou TKet al., 2022, Investigation of the thermogelation of a promising biocompatible ABC triblock terpolymer and its comparison with pluronic F127, Macromolecules, Vol: 55, Pages: 1783-1799, ISSN: 0024-9297

Thermoresponsive polymers with the appropriate structure form physical networks upon changes in temperature, and they find utility in formulation science, tissue engineering, and drug delivery. Here, we report a cost-effective biocompatible alternative, namely OEGMA30015-b-BuMA26-b-DEGMA13, which forms gels at low concentrations (as low as 2% w/w); OEGMA300, BuMA, and DEGMA stand for oligo(ethylene glycol) methyl ether methacrylate (MM = 300 g mol–1), n-butyl methacrylate, and di(ethylene glycol) methyl ether methacrylate, respectively. This polymer is investigated in depth and is compared to its commercially available counterpart, Poloxamer P407 (Pluronic F127). To elucidate the differences in their macroscale gelling behavior, we investigate their nanoscale self-assembly by means of small-angle neutron scattering and simultaneously recording their rheological properties. Two different gelation mechanisms are revealed. The triblock copolymer inherently forms elongated micelles, whose length increases by temperature to form worm-like micelles, thus promoting gelation. In contrast, Pluronic F127’s micellization is temperature-driven, and its gelation is attributed to the close packing of the micelles. The gel structure is analyzed through cryogenic scanning and transmission electron microscopy. Ex vivo gelation study upon intracameral injections demonstrates excellent potential for its application to improve drug residence in the eye.

Journal article

Ouyang L, Wojciechowski A, Tang J, Guo Y, Stevens Met al., 2022, Tunable microgel-templated porogel (MTP) bioink for 3D bioprinting applications, Advanced Healthcare Materials, Vol: 11, ISSN: 2192-2640

Micropores are essential for tissue engineering to ensure adequate mass transportation for embedded cells. Despite the considerable progress made by advanced 3D bioprinting technologies, it remains challenging to engineer micropores of 100 µm or smaller in cell-laden constructs. Here, a microgel-templated porogel (MTP) bioink platform is reported to introduce controlled microporosity in 3D bioprinted hydrogels in the presence of living cells. Templated gelatin microgels are fabricated with varied sizes (≈10, ≈45, and ≈100 µm) and mixed with photo-crosslinkable formulations to make composite MTP bioinks. The addition of microgels significantly enhances the shear-thinning and self-healing viscoelastic properties and thus the printability of bioinks with cell densities up to 1 × 108 mL−1 in matrix. Consistent printability is achieved for a series of MTP bioinks based on different component ratios and matrix materials. After photo-crosslinking the matrix phase, the templated microgels dissociated and diffused under physiological conditions, resulting in corresponding micropores in situ. When embedding osteoblast-like cells in the matrix phase, the MTP bioinks support higher metabolic activity and more uniform mineral formation than bulk gel controls. The approach provides a facile strategy to engineer precise micropores in 3D printed structures to compensate for the limited resolution of current bioprinting approaches.

Journal article

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