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Background: In glioblastoma (GBM) therapy research, tumour treating fields by the company Novocure™, have shown promise for increasing patient overall survival. When used with the chemotherapeutic agent temozolomide, they extend median survival by five months. However, there is a space to design alternative systems that will be amenable for wider use in current research. Therefore, we sought to establish a custom-built alternating electric field device to investigate the effect of electrode design on the responsiveness of cancer cells to this therapy., Methods: A 96-well microtiter plate modified with an electrode array was fabricated to investigate its application as an in vitro alternating electric field device. This was initially performed with patient-derived GCE 31 and GIN 31 cell lines found in the core and invasive margin of the GBM tumour, respectively. We sought to establish the effect of the application of low-intensity (3 V/ cm) electric fields with an application duration of 4-48 h, using intermediate frequency (300 kHz) alternating currents (AC). To demonstrate that electric fields were entering the cell, GCE 31 and GIN 31 cells were treated with the inorganic, non-conductive zinc oxide (ZnO) nanoparticles (NP), previously demonstrated to enhance the efficacy of TTFs. After a 4-h exposure to NP, cells were then exposed to alternating electric fields or currents and their metabolic activity was assessed. To better understand how the position and morphology of cells can affect cell therapy responsiveness to alternating electric fields or currents, GBM results were compared to those from the semi-adherent brain tumour cell line, D425., Results: Contrary to previous findings, there was no significant difference between the GIN 31 and GCE 31 cells exposed to alternating electric fields or currents treated with or without NP compared to cells untreated and unstimulated. D425 cells exposed to alternating electric fields exhibited a pronounced metabolic increase (1.8-fold), while those exposed to alternating electric currents with or without ZnO had a reduced metabolism relative to the untreated control., Conclusions: The initial hypothesis for the lack of effect of electrical stimulation on the adherent cells was that, due to only a single pair of electrodes being used, the proportion of cells that were in the correct orientation for electric field effects was limited. However, the dramatic shift in cell behaviour of the semi-adherent cells shows that cell morphology plays an important role in the responsiveness of cancer cells to AC electric fields. This study highlights the lack of understanding of the complex mechanisms by which electric fields exert effects on cancer cells. We propose that, for the therapy to be enhanced for patients, research should first focus on the underlying mechanisms of action, specifically on how individual cancer cell types respond to this therapy., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Magnetic resonance imaging (MRI) of dry or solid materials in the gastrointestinal (GI) tract requires the use of contrast agents to enhance visualization of the dosage forms. In this study, we explore the novel use of manganese gluconate added to tablets. Manganese was released during tablet dissolution, generating a bright "halo" effect around the tablets, consistent with shortening of the longitudinal relaxation time of the bulk water surrounding the tablet. This is the first study to use MRI to directly image tablet dissolution in the fed stomach using a manganese gluconate contrast agent as dissolution marker.

Resistance mechanisms in brain tumors, such as medulloblastoma and glioblastoma, frequently involve the entrapment of chemotherapeutic agents within endosomes and the extracellular expulsion of drugs. These barriers to effective treatment are exacerbated in nanotechnology-based drug delivery systems, where therapeutic nanoparticles often remain confined within endosomes, thus diminishing their therapeutic efficacy. Addressing this challenge necessitates the development of novel strategies to enhance the efficiency of cancer therapies. This study tests the hypothesis that external electrical stimuli can modulate intracellular trafficking of chemotherapeutic drugs in common malignant brain tumors in children (medulloblastoma) and adults (glioblastoma) by using gold nanoparticles (GNPs). In our experiments, alternating current (AC) stimulation ranging from 1 kHz to 5 MHz and at a strength of 1 V/cm significantly reduced cell viability in drug-resistant medulloblastoma and enhanced delivery of GNPs in glioblastoma. Low-frequency AC resulted in a 50% increase in apoptosis compared to controls and an 8-fold increase in cell death in cisplatin-resistant medulloblastoma cells, accompanied by a substantial reduction in EC 50 from 2.5 to 0.3 μM. Similarly, vincristine-resistant cells demonstrated a 4-fold enhancement in drug sensitivity. Furthermore, high-frequency AC facilitated a significant increase from 20 to 75% in the endosomal escape of GNPs in glioblastoma cells. These findings underscore the potential of AC to selectively disrupt cancer cell resistance mechanisms and bolster the efficacy of nanoparticle-based therapies. The results indicate the effectiveness of AC stimulation in circumventing the limitations inherent in current nanotechnology-based drug delivery systems but also illustrates its transformative potential for treating aggressive, drug-resistant brain tumors., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Drug delivery advances rely on using nano- and microsized carriers to transfer therapeutic molecules, although challenges persist in increasing the availability of new and even approved pharmaceutical products. Particle shape, a critical determinant in how these carriers distribute within the body after administration, raises opportunities of using, for instance, micrometer-sized nonspherical particles for vascular targeting and thereby creating new prospects for precise drug delivery to specific targeted areas. The versatility of polycrystalline silicon microfabrication allows for significant variation in the size and shape of microchips, and so, in the current work, photolithography was employed to create differently shaped polysilicon microchips, including cuboids, cubes, bars, and cylinders, to explore the influence of particle shape on cellular interactions. These microchips with different shapes and lateral dimensions, accounting for surface areas in the range of ca. 15 to 120 μm 2 and corresponding total volumes of 0.4 to 27 μm 3 , serve as ideal models for investigating their interactions with macrophages with diameters of ca. 20 μm. Side-scattering imaging flow cytometry was employed for studying the interaction of label-free prepared microchips with RAW 264.7 macrophages. Using a dose of 3 microchips per cell, results show that cuboids exhibit the highest cellular association (ca. 25%) and uptake (ca. 20%), suggesting their potential as efficient carriers for targeted drug delivery to macrophages. Conversely, similarly sized cylinders and bar-shaped microchips exhibit lower uptakes of about 8% and about 6%, respectively, indicating potential benefits in evading macrophage recognition. On average, 1-1.5 microchips were internalized, and ca. 1 microchip was surface-bound per cell, with cuboids showing the higher values overall. Macrophages respond to microchips by increasing their metabolic activity and releasing low levels of intracellular enzymes, indicating reduced toxicity. Interestingly, increasing the particle dose enhances macrophage metabolic activity without significantly affecting enzyme release.

Nano- and micro-carriers of therapeutic molecules offer numerous advantages for drug delivery, and the shape of these particles plays a vital role in their biodistribution and their interaction with cells. However, analysing how microparticles are taken up by cells presents methodological challenges. Qualitative methods like microscopy provide detailed imaging but are time-consuming, whereas quantitative methods such as flow cytometry enable high-throughput analysis but struggle to differentiate between internalised and surface-bound particles. Instead, imaging flow cytometry combines the best of both worlds, offering high-resolution imaging with the efficiency of flow cytometry, allowing for quantitative analysis at the single-cell level. This study focuses on fluorescently labelled silicon oxide microchips of various morphologies but related surface areas and volumes: rectangular cuboids and apex-truncated square pyramid microchips fabricated using photolithography techniques, offering a reliable basis for comparison with the more commonly studied spherical particles. Imaging flow cytometry was utilised to evaluate the effect of particle shape on cellular uptake using RAW 264.7 cells and revealed phagocytosis of particles with all shapes. Increasing the particle dose enhanced the uptake, while macrophage stimulation had minimal effect. Using a ratio particle:cell of 10:1 cuboids and spheres showed an uptake rate of approximately 50%, in terms of the percentage of cells with internalised particles, and the average number of particles taken up per cell ranging from about 1-1.5 particle/cell for all the different shapes. This study indicates how differently shaped micro-carriers offer insights into particle uptake variations, demonstrating the potential of non-spherical micro-carriers for precise drug delivery applications., (© 2024. The Author(s).)

Understanding the internalization of nanosized particles by mucosal epithelial cells is essential in a number of areas including viral entry at mucosal surfaces, nanoplastic pollution, as well as design and development of nanotechnology-type medicines. Here, we report our comparative study on pathways of cellular internalization in epithelial Caco-2 cells cultured in vitro as either a polarized, differentiated cell layer or as nonpolarized, nondifferentiated cells. The study reveals a number of differences in the extent that endocytic processes are used by cells, depending on their differentiation status and the nature of applied nanoparticles. In polarized cells , actin-driven and dynamin-independent macropinocytosis plays a prominent role in the internalization of both positively and negatively charged nanoparticles, contrary to its modest contribution in nonpolarized cells. Clathrin-mediated cellular entry plays a prominent role in the endocytosis of positive nanoparticles and cholesterol inhibition in negative nanoparticles. However, in nonpolarized cells, dynamin-dependent endocytosis is a major pathway in the internalization of both positive and negative nanoparticles. Cholesterol depletion affects both nonpolarized and polarized cells' internalization of positive and negative nanoparticles, which, in addition to the effect of cholesterol-binding inhibitors on the internalization of negative nanoparticles, indicates the importance of membrane cholesterol in endocytosis. The data collectively provide a new contribution to understanding endocytic pathways in epithelial cells, particularly pointing to the importance of the cell differentiation stage and the nature of the cargo.

Oral dosage forms are the most widely and frequently used formulations to deliver active pharmaceutical ingredients (APIs), due to their ease of administration and noninvasiveness. Knowledge of intragastric release rates and gastric mixing is crucial for predicting the API release profile, especially for immediate release formulations. However, knowledge of the intragastric fate of oral dosage forms in vivo to date is limited, particularly for dosage forms administered when the stomach is in the fed state. An improved understanding of gastric food processing, dosage form location, disintegration times, and food effects is essential for greater understanding for effective API formulation design. In vitro standard and controlled modeling has played a significant role in predicting the behavior of dosage forms in vivo . However, discrepancies are reported between in vitro and in vivo disintegration times, with these discrepancies being greatest in the fed state. Studying the fate of a dosage form in vivo is a challenging process, usually requiring the use of invasive methods, such as intubation. Noninvasive, whole body imaging techniques can however provide unique insights into this process. A scoping review was performed systematically to identify and critically appraise published studies using MRI to visualize oral solid dosage forms in vivo in healthy human subjects. The review identifies that so far, an all-purpose robust contrast agent or dosage form type has not been established for dosage form visualization and disintegration studies in the gastrointestinal system. Opportunities have been identified for future studies, with particular focus on characterizing dosage form disintegration for development after the consumption food, as exemplified by the standard Food and Drug Administration (FDA) high fat meal.

The clinical use of protein and peptide biotherapeutics requires fabrication of stable products. This particularly concerns stability towards aggregation of proteins or peptides. Here, we tested a hypothesis that interactions between a synthetic peptide, which is an aggregation-prone region analogue, and its homologous sequence on a protein of interest, could be exploited to design excipients which stabilise the protein against aggregation. A peptide containing the analogue of lysozyme aggregation-prone region (GILQINSRW) was conjugated to a RAFT agent and used to initiate the polymerisation of N -hydroxyethyl acrylamide, generating a GILQINSRW-HEA 90 polymer, which profoundly reduced lysozyme aggregation. Substitution of tryptophan in GILQINSR W with glycine, to form GILQINSR G , revealed that tryptophan is a critical amino acid in the protein stabilisation by GILQINSR W -HEA 90 . Accordingly, polymeric peptide-mimetics of tryptophan, phenylalanine and isoleucine, which are often present in aggregation-prone regions, were synthesized. These were based on synthetic oligomers of acrylamide derivatives of indole-3 acetic acid (IND), phenylacetic acid (PHEN), or 2-methyl butyric acid (MBA), respectively, conjugated with hydrophilic poly( N -hydroxyethyl acrylamide) blocks to form amphiphilic copolymers denoted as IND m -, PHEN m - and MTB m - b -HEA n . These materials were tested as protein stabilisers and it was shown that solution properties and the abilities of these materials to stabilise insulin and the peptide IDR 1018 towards aggregation are dependent on the chemical nature of their side groups. These data suggest a structure-activity relationship, whereby the indole-based IND m - b -HEA n peptide-mimetic displays properties of a potential stabilising excipient for protein formulations.

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB., (© 2023. The Author(s).)

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

In the context of increased interest in permeability enhancement technologies to achieve mucosal delivery of drugs and biologics, we report our study on effects of the amphiphilic surfactant at cell membrane and cell population levels. Our results show that modulation in membrane order and fluidity initially occurs on insertion of individual surfactant molecules into the outer leaflet of membrane lipid bilayer; a process occurring at concentrations below surfactant's critical micellar concentration. The surfactant insertion, and consequent increase in membrane fluidity, are observed to be spatially heterogenous, i.e. manifested as 'patches' of increased membrane fluidity. At the cell population level, spatially heterogeneous activity of surfactant is also manifested, with certain cells displaying high permeability amongst a 'background' population. We propose that this heterogeneity is further manifested in a broad profile of intracellular and nuclear exposure levels to a model drug (doxorubicin) observed in cell population. The study points to heterogeneous nature of surfactant effects at cell membrane and cells in population levels., (Copyright © 2022. Published by Elsevier B.V.)

Oral specially coated formulations have the potential to improve treatment outcomes of a range of diseases in distal intestinal tract whilst limiting systemic drug absorption and adverse effects. Their development is challenging, partly because of limited knowledge of the physiological and pathological distal gastrointestinal factors, including colonic chyme fluid distribution and motor function. Recently, non-invasive techniques such as magnetic resonance imaging (MRI) have started to provide novel important insights. In this feasibility study, we formulated a coated capsule consisting of a hydroxypropyl methylcellulose (HPMC) shell, coated with a synthetic polymer based on polymethacrylate-based copolymer (Eudragit ® ) that can withstand the upper gastrointestinal tract conditions. The capsule was filled with olive oil as MRI-visible marker fluid. This allowed us to test the ability of MRI to track such a coated capsule in the gastrointestinal tract and to assess whether it is possible to image its loss of integrity by exploiting the ability of MRI to image fat and water separately and in combination. Ten healthy participants were administered capsules with varying amounts of coating and underwent MRI imaging of the gastrointestinal tract at 45 min intervals. The results indicate that it is feasible to track the capsules present in the gastrointestinal tract at different locations, as they were detected in all 10 participants. By the 360 min endpoint of the study, in nine participants the capsules were imaged in the small bowel, in eight participants in the terminal ileum, and in four in the colon. Loss of capsule integrity was observed in eight participants, occurring predominantly in distal intestinal regions. The data indicate that the described approach could be applied to assess performance of oral formulations in undisturbed distal gastrointestinal regions, without the need for ionizing radiation or contrast agents.

The temporary silencing of disease-associated genes utilising short interfering RNA (siRNA) is a potent and selective route for addressing a wide range of life limiting disorders. However, the few clinically approved siRNA therapies rely on lipid based formulations, which although potent, provide limited chemical space to tune the stability, efficacy and tissue selectivity. In this study, we investigated the role of molar mass and histidinylation for poly(lysine) based non-viral vectors, synthesised through a fully aqueous thermal condensation polymerisation. Formulation and in vitro studies revealed that higher molar mass derivatives yielded smaller polyplexes attributed to a greater affinity for siRNA at lower N/P ratios yielding greater transfection efficiency, albeit with some cytotoxicity. Histidinylation had a negligible effect on formulation size, yet imparted a moderate improvement in biocompatibility, but did not provide any meaningful improvement over silencing efficiency compared to non-histidinylated derivatives. This was attributed to a greater degree of cellular internalisation for non-histidinylated analogues, which was enhanced with the higher molar mass material.

Dietary lipids and some pharmaceutical lipid excipients can facilitate the targeted delivery of drugs to the intestinal lymphatics. Here, the feasibility of magnetic resonance imaging (MRI) for imaging lipid uptake into the intestinal lymphatics was assessed, shedding light on which lymph nodes can be targeted using this approach. Three healthy male volunteers were scanned at 3.0 T at baseline, 120, 180, 240, and 300 min post high-fat meal. A sagittal multi-slice image was acquired using a diffusion-weighted whole-body imaging sequence with background suppression (DWIBS) (pre inversion TI = 260 ms). Changes in area, major, and minor axis length were compared at each time point. Apparent diffusion coefficient (ADC) was calculated (b = 0 and 600 s/mm 2 ) across eight slices. An average of 22 nodes could be visualised across all time points. ADC increased at 120 and 180 min compared to the baseline in all three participants by an average of 9.2% and 6.8%, respectively. In two participants, mean node area and major axis lengths increased at 120 and 180 min relative to the baseline. In conclusion, the method described shows potential for repeated lymph node measurements and the tracking of lipid uptake into the lymphatics. Further studies should focus on methodology optimisation in a larger cohort.

Bioresponsive nanoparticles (NPs) are of interest for anticancer nanomedicines, owing to the possibility to 'design in' selective modulation of drug release at target sites. Here we describe the double emulsion formulation of redox-responsive NPs based on modified polyethylene glycol (PEG)-co-poly(lactic-co-glycolic acid) (PLGA) block copolymers and oligo (β-aminoesters) (OBAE), both of which contained disulfide linkages, for the co-delivery of a cytotoxic small molecule drug and a nucleic acid. In particular, we focused our attention on docetaxel (DTX) and a siRNA against TUBB3, a gene that encodes for βIII-tubulin, in order to have a synergistic effect in the treatment of lung cancer. Spherical NPs of around 150 nm with negative zeta potential and high loading efficiencies of both drugs were obtained. Stability and release studies showed "on demand" drug release under reducing conditions. Unloaded NPs containing PEG-disulfide-PLGA and OBAE were well-tolerated by lung cancer cells, thus masking the intrinsic cytotoxicity of OBAE, while for intracellular siRNA delivery, redox responsive NPs demonstrated a higher cell internalization with a preferential cytoplasmic accumulation of siRNA, with a subsequent fast gene-silencing efficiency. The viability of cells treated with combined DTX/TUBB3-siRNA NPs significantly decreased as compared to NPs loaded only with DTX, thus showing an efficient combined anticancer effect, due to a substantial reduction of β-tubulin expression. Finally, in an in vivo feasibility study employing an orthotopic lung cancer model, NPs formulated with an anti-luciferase siRNA distributed throughout the lungs following oro-tracheal administration, and demonstrated effective gene knockdown and no apparent cytotoxicity. Taken together, these results show that the double emulsion formulated redox responsive PEG-PLGA and OBAE systems represent a promising new therapeutic approach for the local combined chemo- and gene-therapy of lung cancer.

Medulloblastoma (MB) is the most common malignant brain tumour in children and is subdivided into four subgroups: WNT, SHH, Group 3, and Group 4. These molecular subgroups differ in their metastasis patterns and related prognosis rates. Conventional 2D cell culture methods fail to recapitulate these clinical differences. Realistic 3D models of the cerebellum are therefore necessary to investigate subgroup-specific functional differences and their role in metastasis and chemoresistance. A major component of the brain extracellular matrix (ECM) is the glycosaminoglycan hyaluronan. MB cell lines encapsulated in hyaluronan hydrogels grew as tumour nodules, with Group 3 and Group 4 cell lines displaying clinically characteristic laminar metastatic patterns and levels of chemoresistance. The glycoproteins, laminin and vitronectin, were identified as subgroup-specific, tumour-secreted ECM factors. Gels of higher complexity, formed by incorporation of laminin or vitronectin, revealed subgroup-specific adhesion and growth patterns closely mimicking clinical phenotypes. ECM subtypes, defined by relative levels of laminin and vitronectin expression in patient tissue microarrays and gene expression data sets, were able to identify novel high-risk MB patient subgroups and predict overall survival. Our hyaluronan model system has therefore allowed us to functionally characterize the interaction between different MB subtypes and their environment. It highlights the prognostic and pathological role of specific ECM factors and enables preclinical development of subgroup-specific therapies. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland., (© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.)

Helicobacter pylori ( H. pylori ) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcβ1-4GlcNAc, occurring in the gastric mucosa. LabA 47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 ​Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20-49%), LabA shares an L-shaped fold with SabA and BabA. The 'head' region contains a 4 ​+ ​3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain ('crown' region), the 'J99-type' (insertion ~31 ​amino acids), and the H. pylori '26695 type' (insertion ~46 ​amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcβ1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

This study investigated the application of a temperature-responsive methylcellulose-hyaluronic acid (MC-HA) hydrogel to support 3D cell growth in vitro . Initial work focused on the preparation of hydrogels for 3D culture, followed by investigations of the biological compatibility of hydrogel components and optimization of the cell culture environment. Evaluation of viability and proliferation of HCT116 cells cultured in the MC-HA hydrogel was used to adjust the blend composition to design a hydrogel with optimal properties to support cell growth. Two important aspects in terms of the application of the proposed polymeric matrix in 3D cell culture were demonstrated: (i) 3D cultured cell aggregates can be released/recovered from the matrix via a gentle procedure that will preserve cell viability and (ii) the hydrogel matrix is amenable to application in a 96-well plate format as a standard approach employed in in vitro tissue culture tests. The work therefore shows that MC-HA hydrogels demonstrate potential for in vitro 3D cell culture as inexpensive and well-defined alternatives to animal-derived or complex synthetic systems.

Doxorubicin (DOX), an anthracycline, is widely used in cancer treatment by interfering RNA and DNA synthesis. Its broad antitumour spectrum makes it an effective therapy for a wide array of cancers. However, the prevailing drug-resistant cancer has proven to be a significant drawback to the success of the conventional chemotherapy regime and DOX has been identified as a major hurdle. Furthermore, the clinical application of DOX has been limited by rapid breakdown, increased toxicity, and decreased half-time life, highlighting an urgent need for more innovative delivery methods. Although advancements have been made, achieving a complete cure for cancer remains elusive. The development of nanoparticles offers a promising avenue for the precise delivery of DOX into the tumour microenvironment, aiming to increase the drug concentration at the target site while reducing side effects. Despite the good aspects of this technology, the classical nanoparticles struggle with issues such as premature drug leakage, low bioavailability, and insufficient penetration into tumours due to an inadequate enhanced permeability and retention (EPR) effect. Recent advancements have focused on creating stimuli-responsive nanoparticles and employing various chemosensitisers, including natural compounds and nucleic acids, fortifying the efficacy of DOX against resistant cancers. The efforts to refine nanoparticle targeting precision to improve DOX delivery are reviewed. This includes using receptor-mediated endocytosis systems to maximise the internalisation of drugs. The potential benefits and drawbacks of these novel techniques constitute significant areas of ongoing study, pointing to a promising path forward in addressing the challenges posed by drug-resistant cancers., Competing Interests: Declarations. Conflict of interest: The authors have no competing interests to declare that are relevant to the content of this article. Ethical approval: This review does not contain any studies with human participants or animals performed by any authors. Consent to participate: Informed consent is not applicable. Publication consent: All the authors have given their consent and permission to the publisher in the publication of this work upon successfully passing through peer-review process., (© 2025. Controlled Release Society.)

Mucus is the first biological component inhaled drugs encounter on their journey towards their pharmacological target in the upper airways. Yet, how mucus may influence drug disposition and efficacy in the lungs has been essentially overlooked. In this study, a simple in vitro system was developed to investigate the factors promoting drug interactions with airway mucus in physiologically relevant conditions. Thin layers of porcine tracheal mucus were prepared in Transwell ® inserts and initially, the diffusion of various fluorescent dyes across those layers was monitored over time. A deposition system featuring a MicroSprayer ® aerosolizer was optimized to reproducibly deliver liquid aerosols to multiple air-facing layers and then exploited to compare the impact of airway mucus on the transport of inhaled bronchodilators. Both the dyes and drugs tested were distinctly hindered by mucus with high logP compounds being the most affected. The diffusion rate of the bronchodilators across the layers was in the order: ipratropium glycopyronnium > formoterol > salbutamol > indacaterol, suggesting hydrophobicity plays an important role in their binding to mucus but is not the unique parameter involved. Testing of larger series of compounds would nevertheless be necessary to better understand the interactions of inhaled drugs with airway mucus., Competing Interests: The authors declare no conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Degranulation of mast cells and basophils occurs after the cross-linking of FcεRI receptor-bound IgE by multivalent allergens, resulting in the release of a range of de novo synthesized and preformed mediators of the allergic response. β-Hexosaminidase release is usually measured as a simple readout for degranulation. Furthermore, the rat basophilic leukemia (RBL)-2H3 cell line is commonly used for measuring degranulation, monitoring β-hexosaminidase release. Here, we describe surface-engineered and modified nanoparticles with specific ligands in order to study the signaling and cellular responses of the RBL-2H3 cell line.

Helicobacter pylori is a pathogenic microorganism infecting approximately 50% of the global population, and establishes life-long colonization despite the hostile stomach environment. H. pylori employs a wide range of outer membrane proteins (adhesins) for epithelial attachment, which specifically bind to glycans or non-carbohydrate structures expressed on the gastric epithelium. A recently described adhesin from H. pylori is LabA, named after its ability to bind to a disaccharide present in gastric mucus (LacdiNAc-specific adhesin). Here, we describe the recombinant expression of LabA from H. pylori strains J99 and 26695 in E. coli. High yields of recombinant LabA were obtained using periplasmic expression. We found that the addition of a C-terminal hexalysine (6K) tag enhanced the thermal stability of LabA without affecting its secondary structure, using differential scanning fluorimetry and circular dichroism spectroscopy. In contrast to our previous report for another H. pylori adhesin (BabA), the 6K tag did not enhance recombinant protein yield or solubility. Both versions of LabA, with or without the 6K tag, were expressed and isolated from the periplasmic space of Escherichia coli, with a surprisingly high yield of at least 40 mg/L for each independent preparation, following a two-step purification protocol. The proteins were analyzed with mass spectrometry (MS). Unlike its reported effect on stability of BabA, the 6K tag did not appear to protect the N-term of recombinant LabA from partial periplasmic degradation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Although the mucus layer is the first biological barrier encountered by inhaled drugs upon their deposition in the upper airways, its potential impact on drug dissolution and absorption in the lung has hardly been investigated. Bio-relevant in vitro models were therefore used to assess the role of airway mucus in the fate of drug particles at the air-epithelium interface. Salbutamol and indomethacin were used as model Biopharmaceutics Classification System (BCS) class III and class II drugs, respectively. Dry powders were reproducibly aerosolised using a PennCentury™ Dry Powder Insufflator onto multiple air-liquid interfaced layers of the broncho-epithelial cell line Calu-3 or thin layers of porcine tracheal mucus mounted onto Transwells ® inserts, as well as on empty Transwells ® . Comparison of the permeation profiles of the two drugs indicated that mucus acted as a barrier for salbutamol transport but increased that of indomethacin, suggesting it facilitates the dissolution of poorly soluble drugs. In presence of Calu-3 layers, the permeability of salbutamol was even more restricted while indomethacin transport was enhanced further. This study demonstrates mucus distinctly affects the absorption characteristics of drugs with different physico-chemical properties. Hence, drug-mucus interactions should be considered during the development of inhaled drugs., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Trans-plasma membrane electron transfer (tMPET) is a process by which reducing equivalents, either electrons or reductants like ascorbic acid, are exported to the extracellular environment by the cell. TPMET is involved in a number of physiological process and has been hypothesised to play a role in the redox regulation of cancer metabolism. Here, we use a new electrochemical assay to elucidate the 'preference' of cancer cells for different trans tPMET systems. This aids in proving a biochemical framework for the understanding of tPMET role, and for the development of novel tPMET-targeting therapeutics. We have delineated the mechanism of tPMET in 3 lung cancer cell models to show that the external electron transfer is orchestrated by ascorbate mediated shuttling via tPMET. In addition, the cells employ a different, non-shuttling-based mechanism based on direct electron transfer via Dcytb. Results from our investigations indicate that tPMETs are used differently, depending on the cell type. The data generated indicates that tPMETs may play a fundamental role in facilitation of energy reprogramming in malignant cells, whereby tPMETs are utilised to supply the necessary energy requirement when mitochondrial stress occurs. Our findings instruct a deeper understanding of tPMET systems, and show how different cancer cells may preferentially use distinguishable tPMET systems for cellular electron transfer processes., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Animal models are effective for assessing tumor localization of nanosystems but difficult to use for studying penetration beyond the vasculature. Here, we have used well-characterized HCT116 colorectal cancer spheroids to study the effect of nanoparticle (NP) physicochemical properties on penetration and uptake. Incubation of spheroids with Hoechst 33342 resulted in a dye gradient, which facilitated discrimination between the populations of cells in the core and at the periphery of spheroids by flow cytometry. This approach was used to compare doxorubicin and liposomal doxorubicin (Caelyx) and a range of model poly(styrene) nanoparticles of different sizes (30 nm, 50 nm, 100 nm) and with different surface chemistries (50 nm uniform plain, carboxylated, aminated and a range of NPs and polyethylene glycol modified NPs prepared from a promising new functionalized biodegradable polymer (poly(glycerol-adipate), PGA). Unmodified poly(styrene) nanoparticles (30 nm/50 nm) were able to penetrate to the core of HCT116 spheroids more efficiently than larger poly(styrene) nanoparticles (100 nm). Surprisingly, penetration of 30 and 50 nm particles was as good as clinically relevant doxorubicin concentrations. However, penetration was reduced with higher surface charge. PGA NPs of 100 nm showed similar penetration into spheroids as 50 nm poly(styrene) nanoparticles, which may be related to polymer flexibility. PEG surface modification of polymeric particles significantly improved penetration into the spheroid core. The new model combining the use of spheroids Hoechst staining and flow cytometry was a useful model for assessing NP penetration and gives useful insights into the effects of NPs' physical properties when designing nanomedicines.

A model cancer cell line was used to initiate polymerisation of pyrrole to form the conducting material polypyrrole. The polymerisation was shown to occur through the action of cytosolic exudates rather than that of the membrane redox sites that normally control the oxidation state of iron as ferricyanide or ferrocyanide. The data demonstrate for the first time that mammalian cells can be used to initiate synthesis of conducting polymers and suggest a possible route to detection of cell damage and/or transcellular processes through in situ and amplifiable signal generation., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)