Dissolving microneedles (MNs) hold promise as a versatile drug delivery platform, particularly suited to the delivery of complex molecules across the skin. Dissolving MNs are commonly manufactured using an accessible and reproducible two-step casting process. The selection of different polymers for both the needle and backing layer increases the adaptability of this platform. Previously, work has focused on the needle layer formulation and how the formulation will affect drug delivery. Less well understood is the role of the backing layer on insertion and, subsequently, drug delivery. Therefore, the aim of this work was to evaluate changes to the backing layer formulation on MN insertion and understand the relationships between material properties. The needle layer was formulated with polyvinylpyrrolidone-co-vinyl acetate, with and without insulin, a model protein therapeutic. A range of polymers was used to formulate the backing layer, including sodium carboxymethylcellulose (Na-CMC), poly(vinyl alcohol) (PVA), and polystyrene (PS). MNs manufactured with a PVA backing layer demonstrated an improved insertion profile (efficiency and depth). Permeation studies supported that the PVA backing layer offered an overall advantage in insulin delivery, with a cumulative recovery of 17.6% of the total insulin loading. This work demonstrates the importance of the backing layer formulation in MN arrays. Changing the backing layer formulation impacted both the insertion of MNs and subsequent drug delivery. Moving forward, the properties of polymers selected for use in MN backing layers should be thoroughly explored and rationally selected depending on the intended application.

Parkinson's disease is a complex neurodegenerative disease associated with the reduction of dopamine content in the brain. Dopamine, in the form of the precursor l-DOPA, is used as a replacement therapy, which provides temporary symptomatic relief. Long-term treatment with l-DOPA induces side effects such as involuntary movements (dyskinesia) due to the continuous and non-discriminatory exposure to dopamine. Here, we propose that a reduction in the dose and frequency of administration of l-DOPA might reduce such side effects. We hypothesise that by binding dopamine to a nanoparticle via a photoresponsive moiety, we can not only maintain dopamine's biological activity, but also modulate the availability of dopamine for receptor activation non-invasively through light. To test this hypothesis, we designed a nanoparticle surface functionalised with a photoresponsive spiropyran molecule that is conjugated to dopamine to stimulate activation of the dopamine D-1 receptor (D1R). The activity of the system was assessed using a cAMP assay on a Parkinson's disease modelled SH-SY5Y neuroblastoma cell line and DRD1/CRE transfected HEK293 cells. We found that cAMP concentration was elevated in treated cells, meaning that the biological activity of conjugated dopamine is maintained and that the dopaminergic receptor is activated on-demand by light stimulation in our model system.

Photo-responsive biomaterials are attractive because of the ability to non-invasively alter and control the material properties, thus allowing control over the cell response at the interface with the bioamaterial surface. While in silico mathematical models have been implemented for simulating single-cell force spectroscopy (SCFS) experiments on static and some dynamic biomaterials, these models have yet to be extended to light-responsive biointerfaces. So here, we develop a mathematical model that describes and predicts the strength of integrin-mediated cell adhesions to a photoswitchable biomaterial surface. The fluctuating biomaterial comprises photoswitchable azobenzene attached to a glass surface and terminated with peptide c(RGDfK). Upon irradiation with light at 530 nm, the azobenzenes rapidly fluctuate between an extended and contracted conformation, leading to a change in the length of the azobenzene that stimulates integrins bound to c(RGDfK). The mathematical model mimics the nascent adhesion and spatial fluctuations in the extra-cellular matrix (ECM) and is calibrated using single-cell force microscopy retraction curves. It relies on spring-based mechanics to describe the stretch and deformation of a cell and ensembles of integrins when applied to a fluctuating biomaterial. We use the model to simulate retraction curves and the proportion of bound integrins on the surface as the material fluctuates. Additionally, we use the model to predict SCFS retraction curves for varying experimental conditions. This includes the length of time the cell (attached to the tip of the atomic force microscopy cantilever) is kept in contact with the biomaterial before retraction, and for varying frequency of light-induced movement of the azobenzene conformations. These model outcomes provide an attractive route to integrate and rigorously control certain experimental variables, thus accelerating experimental design of the study of cell adhesion to light-responsive biomaterials. Furthermore the model complements experiments by providing estimates of variables that are experimentally inaccessible. Thus, the outcomes of the model provide a valuable resource to aid in the interpretation and design of light-responsive biointerface functionalities. ### Competing Interest Statement The authors have declared no competing interest. Engineering and Physical Sciences Research Council (EPSRC), EP/S023054/1

Liposomes are amongst the most promising and versatile nanomedicine products employed in recent years. In vitro release (IVR) tests are critical during development of new liposome-based products. The drug release characteristics of a formulation are affected by multiple factors related to the formulation itself and the IVR method used. While the effect of some of these parameters has been explored, their relative importance and contribution to the final drug release profile are not sufficiently understood to enable rational design choices. This prolongs the development and approval of new medicines. In this study, a machine learning workflow is developed which can be used to better understand patterns in liposome formulation properties, IVR methods, and the resulting drug release characteristics. A comprehensive database of liposome release profiles, including formulation properties, IVR method parameters, and drug release profiles is compiled from academic publications. A classification model is developed to predict the release profile type (kinetic class), with a significant increase in the balanced accuracy test score compared to a random baseline. The resulting machine learning approach enhances understanding of the complex liposome drug release dynamics and provides a predictive tool to accelerate the design of liposome IVR tests.

The process of programmed cell death, namely apoptosis, is a natural mechanism that regulates healthy tissue, multicellular structures, and homeostasis. An improved understanding of apoptosis can significantly enhance our knowledge of biological processes and systems. For instance, pathogens can manipulate the apoptotic process to either evade immune detection or to facilitate their spread. Furthermore of particular clinical interest is the ability of cancer cells to evade apoptosis, hence allowing them to survive and proliferate uncontrollably. Thus, in this work, we propose a phase-field model for simulating cellular apoptosis induced by a cytotoxin, including deriving the configurational mechanics underlying such phenomena. Along with exploring varying conditions needed to initiate or reduce apoptosis, this can serve as a starting point for computational therapeutic testing. To showcase model capabilities, we present simulations exhibiting different types of cellular dynamics produced when varying the mechanisms underlying apoptosis. The model is subsequently applied to probe different morphological transitions, such as cell shrinkage, membrane blebbing, cavity formation and fragmentation. Lastly, we compare the characteristics observed in our simulations to electron microscopy images, providing additional support for the model.

Machine learning and artificial intelligence (AI) is transforming the way pharmaceutical products are developed across drug discovery, process engineering, and pharmaceutics functions. AI for nanomedicine development is enabling faster and more accurate prediction of critical quality attributes (CQAs). However, the full potential of AI is limited by the quality and accessibility of data. Unlike adjacent fields such as the chemical sciences, the pharmaceutics domain lacks curated, open-access databases, particularly for nanomedicines. To address this, here we curate an open-access local database focused on liposomal formulations. The database includes formulation parameters, in vitro release (IVR) testing conditions, and digitised drug release data. By evaluating the entries in the database qualitatively and quantitatively, we identified challenges in current data reporting practices. This includes incomplete reporting of formulation and IVR testing conditions, as well as inconsistent quality of drug release plots and their data format. Based on our analysis, we propose a set of data standards and a database structure to support harmonisation for nanomedicine formulation and IVR data. Our open-access database aims to improve data accessibility and transparency to enable the development of robust AI models for IVR and CQA prediction, ultimately streamlining nanomedicine development.

Biocatalysis in ionic liquids enables novel routes for bioprocessing. Enzymes derived from extremophiles promise greater stability and activity under ionic liquid (IL) influence. Here, we probe the enzyme Alcohol Dehydrogenase 2 from the halophilic archaeon Haloferax volcanii in thirteen different ion combinations for relative specific activity and analyse the results against MD simulations of the same IL systems. We probe the ionic liquid property space based on ion polarizability and molecular electrostatic potential. Using radial distribution functions, survival probabilities and spatial distribution functions of ions we show that cooperative ion-ion interactions determine ion-protein interaction, specifically, strong ion-ion interactions equate to higher enzymatic activity if neither of the ions interact strongly with the protein surface. We further demonstrate a tendency for ions interacting with the protein surface to be least detrimental to enzymatic activity if they show a low polarizability and a small range of molecular electrostatic potential. We also find that the IL ion influence is not mitigated by the surplus of negatively charged residues of the halophilic enzyme. This is shown by free energy landscape analysis in root mean square deviation and distance variation plots of active site gating residues (Trp43 and His273) demonstrating no protection of specific structural elements relevant to preserving enzymatic activity. On the other hand, we observe a general effect across all IL systems that a tight binding of water at acidic residues is preferentially interrupted at these residues through the increased presence of potassium ions. Overall, this study demonstrates a co-ion interaction dependent influence on allosteric surface residues controlling the active/inactive conformation of halophilic Alcohol Dehydrogenase 2 and the necessity to engineer ionic liquid systems for enzymes that rely on the integrity of functional surface residues regardless of their halophilicity or thermophilicity for use in bioprocessing.

During the foreign body response (FBR), macrophages fuse to form foreign body giant cells (FBGCs). Modulation of FBGC formation can prevent biomaterial degradation and loss of therapeutic efficacy. However, the microenvironmental cues that dictate FBGC formation are poorly understood with conflicting reports. Here, we identified molecular and cellular factors involved in driving FBGC formation in vitro. Macrophages demonstrated distinct fusion competencies dependent on monocyte differentiation. The transition from a proinflammatory to a reparative microenvironment, characterised by specific cytokine and growth factor programmes, accompanied FBGC formation. Toll-like receptor signalling licensed the formation of FBGCs containing more than 10 nuclei but was not essential for cell-cell fusion to occur. Moreover, the fibroblast-macrophage crosstalk influenced FBGC development, with the fibroblast secretome inducing macrophages to secrete more PDGF, which enhanced large FBGC formation. These findings advance our understanding as to how a specific and timely combination of cellular and microenvironmental factors is required for an effective FBR, with monocyte differentiation and fibroblasts being key players.

Significance statementWe identified that PLGA‐PEG‐PLGA hydrogels, which have been used in human clinical trials and possess a demonstrable safety profile, induced significant cytotoxicity in conventional in vitro assays. This major contradiction may lead to inconsistent and misleading toxicology due to the limited biological representation of these assays. Cytotoxicity evaluation is a crucial element of screening the biological response to new biomaterials. However, as standard test methods do not recapitulate the in vivo environment, tailored adaptations may be required to reflect the true biological response elicited toward novel biomaterials.

Skeletal stem cells (SSCs, or mesenchymal stromal cells typically referred to as mesenchymal stem cells from the bone marrow) are a dynamic progenitor population that can enter quiescence, self-renew or differentiate depending on regenerative demand and cues from their niche environment. However, ex vivo, in culture, they are grown typically on hard polystyrene surfaces, and this leads to rapid loss of the SSC phenotype. While materials are being developed that can control SSC growth and differentiation, very few examples of dynamic interfaces that reflect the plastic nature of the stem cells have, to date, been developed. Achieving such interfaces is challenging because of competing needs: growing SSCs require lower cell adhesion and intracellular tension while differentiation to, for example, bone-forming osteoblasts requires increased adhesion and intracellular tension. We previously reported a dynamic interface where the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD) was presented to the cells upon activation by user-added elastase that cleaved a bulky blocking group hiding RGD from the cells. This allowed for a growth phase while the blocking group was in place and the cells could only form smaller adhesions, followed by an osteoblast differentiation phase that was induced after elastase was added which triggered exposure of RGD and subsequent cell adhesion and contraction. Here, we aimed to develop an autonomous system where the surface is activated according to the need of the cell by using matrix metalloprotease (MMP) cleavable peptide sequences to remove the blocking group with the hypothesis that the SSCs would produce higher levels of MMP as the cells reached confluence. The current studies demonstrate that SSCs produce active MMP-2 that can cleave functional groups on a surface. We also demonstrate that SSCs can grow on the uncleaved surface and, with time, produce osteogenic marker proteins on the MMP-responsive surface. These studies demonstrate the concept for cell-controlled surfaces that can modulate adhesion and phenotype with significant implications for stem cell phenotype modulation.

Interpretation of X‐ray photoelectron spectroscopy (XPS) spectra of complex material surfaces, such as those obtained after surface plasma treatment of polymers, is confined by the available references. The limited understanding of the chemical surface composition may impact the ability to determine suitable coupling chemistries used for surface decoration or assess surface‐related properties like biocompatibility. In this work, XPS is used to investigate the chemical composition of various ultra‐high‐molecular‐weight polyethylene (UHMWPE) surfaces. UHMWPE doped with α‐tocopherol or functionalised by active screen plasma nitriding (ASPN) was investigated as a model system. Subsequently, a more complex combined system obtained by ASPN treatment of α‐tocopherol doped UHMWPE was investigated. Through ab initio orbital calculations and by employing Koopmans' theorem, the core‐electron binding energies (CEBEs) were evaluated for a substantial number of possible chemical functionalities positioned on PE‐based model structures. The calculated ΔCEBEs showed to be in reasonable agreement with experimental reference data. The calculated ΔCEBEs were used to develop a material‐specific peak model suitable for the interpretation of merged high‐resolution C 1 s, N 1 s and O 1 s XPS spectra of PE‐based materials. In contrast to conventional peak fitting, the presented approach allowed the distinction of functionality positioning (i.e. centred or end‐chain) and evaluation of the long‐range effects of the chemical functionalities on the PE carbon backbone. Altogether, a more detailed interpretation of the modified UHMWPE surfaces was achieved whilst reducing the need for manual input and personal bias introduced by the spectral analyst.

Intraperitoneal (IP) drug delivery of chemotherapeutic agents, administered through hyperthermal intraperitoneal chemotherapy (HIPEC) and pressurized intraperitoneal aerosolized chemotherapy (PIPAC), is effective for the treatment of peritoneal malignancies. However, these therapeutic interventions are cumbersome in terms of surgical practice and are often associated with the formation of peritoneal adhesions, due to the catheters inserted into the peritoneal cavity during these procedures. Hence, there is a need for the development of drug delivery systems that can be administered into the peritoneal cavity. In this study, we have developed a nanocapsule (NCs)-loaded hydrogel for drug delivery in the peritoneal cavity. The hydrogel has been developed using poly(ethylene glycol) (PEG) and thiol-maleimide chemistry. NCs-loaded hydrogels were characterized by rheology and their resistance to dilution and drug release were determined in vitro. Using IVIS® to measure individual organ and recovered gel fluorescence intensity, an in vivo imaging study was performed and demonstrated that NCs incorporated in the PEG gel were retained in the IP cavity for 24 h after IP administration. NCs-loaded PEG gels could find potential applications as biodegradable, drug delivery systems that could be implanted in the IP cavity, for example at a the tumour resection site to prevent recurrence of microscopic tumours.

Peptide‐coated surfaces are widely employed in biomaterial design, but quantifiable correlation between surface composition and biological response is challenging due to, for example, instrumental limitations, a lack of suitable model surfaces or limitations in quantitatively correlating data from different surface analytical techniques. Here, we first establish a reference material that allows control over amino acid content. Reversible addition‐fragmentation chain‐transfer (RAFT) polymerisation is used to prepare a copolymer containing alkyne and furan units with well‐defined chain length and composition. Huisgen Cu(I)‐catalysed azide‐alkyne cycloaddition reaction is used to attach the model azido‐polyethyleneglycol‐amide‐modified pentafluoro‐l‐phenylalanine to the polymer. Different compositional ratios of the polymer provide a surface with varying amino acid content that is analysed by X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). Nitrogen‐related signals are compared with fluorine signals from both techniques. Fluorine and nitrogen signals from both techniques are found to be related to the copolymer compositions, but the homopolymer data deviate from this trend. The approach is then translated to a heparin‐binding peptide that supports cell adhesion. Human embryonic stem cells cultured on copolymer surfaces presenting different amounts of heparin‐binding peptide show strong cell growth while maintaining pluripotency after 72 h of culture. The early cell adhesion at 24 h can be correlated to the logarithm of the normalised CH4N+ ion intensity from ToF‐SIMS data, which is established as a suitable and generalisable marker ion for amino acids and peptides. This work contributes to the ability to use ToF‐SIMS in a more quantitative manner for the analysis of amino acid and peptide surfaces.

Control over intracellular release of therapeutic compounds incorporated into nano-carriers will open new possibilities for targeted treatments of various diseases including cancer, and viral and bacterial infections. Here we report our study on mechanoresponsive nano-sized liposomes which, following internalization by cells, achieve intracellular delivery of encapsulated cargo on application of external ultrasound stimulus. This is demonstrated in a bespoke cell reporter system designed to assess free drug in cytoplasm. Biophysical analyses show that drug release is attributable to the action of a mechanoresponsive spiropyran-based compound embedded in the liposomal lipid membrane. Exposure to external ultrasound stimulus results in opening of the molecular structure of the embedded spiropyran, a consequent increase in liposomal lipid membrane fluidity, and size-dependent release of encapsulated model drugs, all pointing to lipid bilayer perturbation. The study hence illustrates feasibility of the proposed concept where intracellular drug release from mechanoresponsive liposomes can be triggered on demand by external ultrasound stimulus.

Polymeric, biodegradable, microspheres (MS) presenting a biomimetic surface of extracellular matrix (ECM) proteins are currently used for transporting cells and/or encapsulated proteins for regenerative medicine studies. They can be made of (lactic-co-glycolic acid) (PLGA) or of a more hydrophilic PLGA-P188 (Poloxamer188)-PLGA polymer allowing for the complete release of the therapeutic proteins. They promote stem cell adhesion, cell survival and differentiation after transplantation. Although the biological effectiveness of these microcarriers is established, a detailed understanding of the protein and cell interactions with the microcarrier surface remain unclear due to a lack of information of their surface properties. The aim of this study was to characterize the physicochemical properties of two polymeric MS systems and determine the effect of laminin and poly-d-lysine coated microcarriers on stem cell adhesion, survival and neuronal differentiation. The hydrophobicity and topography of PLGA MS promoted protein adsorption and the stem cells quickly adhered and spread on the surface of these microcarriers. In contrast less proteins adsorbed onto PLGA-P188-PLGA MS and although cells adhered to these microcarriers, they remained round and did not spread on their surface. Despite these early-stage differences, our results suggest that the nature of the MS does not strongly influence the long-term cell behavior. The cells exhibit the same cell number, differentiation profile and ability to secrete ECM molecules regardless of the type of microcarrier used. Likely the ECM molecules that form a microenvironment around both of these 3D microcarrier/cell constructs over time play a role in this converging cell behavior. We have thus furthered our understanding of the physicochemical properties of polymeric cell carriers affecting stem cell behavior to help tailor suitable microcarriers for neuroregenerative applications.

Controlling supramolecular self-assembly across multiple length scales to prepare gels with localised properties is challenging. Most strategies concentrate on fabricating gels with heterogeneous components, where localised properties are generated by the stimuli-responsive component. Here, as an alternative approach, we use a spiropyran-modified surface that can be patterned with light. We show that light-induced differences in surface chemistry can direct the bulk assembly of a low molecular weight gelator, 2-NapAV, meaning that mechanical gel properties can be controlled by the surface on which the gel is grown. Using grazing incidence X-ray diffraction and grazing incidence small angle X-ray scattering, we demonstrate that the origin of the different gel properties relates to differences in the architectures of the gels. This provides a new method to prepare a single domain (i.e., chemically homogeneous) hydrogel with locally controlled (i.e., mechanically heterogeneous) properties.

Protein-coated polymer-based microparticles are attractive supports for cell delivery, but the interplay between microparticle properties, protein coating, and cell response is poorly understood. The interest in alternative microparticle formulations increases the need for a better understanding of how functional protein coatings form on different microparticles. In this work, microparticle formulations based on biodegradable polymers [poly (lactic-co-glycolic acid) (PLGA) and the triblock copolymer PLGA-poloxamer-PLGA] were prepared via an emulsion-based process. To explore the impact that the use of a surfactant has on the properties of the microparticles, the emulsion was stabilized by using either a surfactant, poly(vinyl alcohol), or an organic solvent, propylene glycol. Four different types of microparticles were prepared through combinations of the two types of polymers and the two types of stabilizers. The coating of microparticles with proteins/polypeptides such as fibronectin and poly-d-lysine has been demonstrated before and is an integral step for their application as microcarriers, e.g., for cell delivery; however, the impact of the microparticles’ surface chemical properties on the formation (prevalence and distribution) of the mixed polypeptide coatings and the influence on subsequent cell attachment remain to be elucidated. Using a colocalization analysis approach on ToF-SIMS images of protein-coated microparticles, we show that the use of propyleneglycol over PVA as well as the substitution of PLGA by the triblock copolymer resulted in enhanced protein adsorption. Furthermore, if propyleneglycol is used, the substitution of PLGA with the triblock copolymer leads to increased stem cell adhesion.

Background: The standard treatment for ovarian cancer (OC) is cytoreductive surgery, performed either in between or followed by intravenous. The difficulty in excising all the disease leads to recurrence in many patients. Hence, there is an urgent need for treatment options that can remove microscopic or macroscopic disease effectively. Poor tolerability limits the usage of many therapies for OC. However, loco-regional delivery is being used for the therapeutic index improvement. Nevertheless, intraperitoneal (IP) chemotherapy has not yet been widely adopted for OC treatment due to the risk of local toxicity, catheterassociated infections, bowel perforations and obstructions, and lack of products specifically approved. Nanoparticles (NPs) have been investigated for IP delivery of chemotherapeutics because of their potential to offer higher tumour penetration. However, in general, NPs are rapidly cleared from the peritoneal cavity. Hence, to maximise this NPs loco-regional effect, we have proposed the incorporation of nanocapsules (NCs) within a hydrogel for IP delivery and to provide a more sustained release profile. The first part of this work involves the NCs formulation optimization and incorporation of paclitaxel (PCX), a first-line drug for OC and the focus herein. The second part will be the development and evaluation of a cross-linked polyethylene glycol (PEG) NCs loaded hydrogel for IP drug delivery, for use at the point of surgical resection, and will be the focus of future studies.

The surface of a medical implant is required to interact favourably with ions, biomolecules and cells in vivo, commonly resulting in the formation of the extracellular matrix. Medical grade Ti6Al4V alloy is widely used in orthopaedic and dental applications for bone replacement due to its advantageous mechanical properties and biocompatibility, which enhances the adhesion between native tissue and the implanted material. In this study, chemical and thermal modification of a medical-grade Ti6Al4V alloy were performed to enhance electrostatic interactions at the alloy surface with a synthetic peptide, suitable for conferring drug release capabilities and antimicrobial properties. The modified surfaces exhibited a range of topographies and chemical compositions depending primarily on the treatment temperature. The surface wetting behaviour was found to be pH-dependent, as were the adhesive properties, evidenced by chemical force titration atomic force microscopy.

Among the diversity of existing supramolecular hydrogels, nucleic acid-based hydrogels are of particular interest for potential drug delivery and tissue engineering applications because of their inherent biocompatibility. Hydrogel performance is directly related to the nanostructure and the self-assembly mechanism of the material, an aspect that is not well-understood for nucleic acid-based hydrogels in general and has not yet been explored for cytosine-based hydrogels in particular. Herein, we use a broad range of experimental characterization techniques along with molecular dynamics (MD) simulation to demonstrate the complementarity and applicability of both approaches for nucleic acid-based gelators in general and propose the self-assembly mechanism for a novel supramolecular gelator, N4-octanoyl-2′-deoxycytidine. The experimental data and the MD simulation are in complete agreement with each other and demonstrate the formation of a hydrophobic core within the fibrillar structures of these mainly water-containing materials. The characterization of the distinct duality of environments in this cytidine-based gel will form the basis for further encapsulation of both small hydrophobic drugs and biopharmaceuticals (proteins and nucleic acids) for drug delivery and tissue engineering applications.