In Situ Self-Assembling Liver Spheroids with Synthetic Nanoscaffolds for Preclinical Drug Screening Applications.

Drug-induced liver injury (DILI) is one of the most common reasons for acute liver failure and a major reason for the withdrawal of medications from the market. There is a growing need for advanced in vitro liver models that can effectively recapitulate hepatic function, offering a robust platform for preclinical drug screening applications. Here, we explore the potential of self-assembling liver spheroids in the presence of electrospun and cryomilled poly(caprolactone) (PCL) nanoscaffolds for use as a new preclinical drug screening tool. This study investigated the extent to which nanoscaffold concentration may have on spheroid size and viability and liver-specific biofunctionality. The efficacy of our model was further validated using a comprehensive dose-dependent acetaminophen toxicity protocol. Our findings show the strong potential of PCL-based nanoscaffolds to facilitate in situ self-assembly of liver spheroids with sizes under 350 µm. The presence of the PCL-based nanoscaffolds (0.005 and 0.01% w/v) improved spheroid viability and the secretion of critical liver-specific biomarkers, namely, albumin and urea. Liver spheroids with nanoscaffolds showed improved drug-metabolizing enzyme activity and greater sensitivity to acetaminophen compared to two-dimensional monolayer cultures and scaffold-free liver spheroids. These promising findings highlight the potential of our nanoscaffold-based liver spheroids as an in vitro liver model for drug-induced hepatotoxicity and drug screening.

Development of an in vitro co-culture model using Caco-2 and J774A.1 cells to mimic intestinal inflammation.

In vitro models that mimic the pathophysiology in vivo are important tools to study mechanisms of disease and assess the pharmacology and toxicity of drugs. In this work, we report the development of a novel model of intestinal inflammation. This model is based on the co-culture of intestinal epithelial Caco-2 cells and murine J774A.1 macrophages. The model is shown to mimic the intestinal barrier in both healthy and inflamed state. In the healthy state, without external stimulation, Caco-2 and J774A.1 cells were co-cultured in one system without affecting the barrier integrity of intestinal epithelial cells and without inducing release of cytokines from macrophages. To mimic the inflamed intestine, Caco-2 cells were primed with an optimised cytokine cocktail (TNF-⍺, IFN-γ and IL-1ß) and J774A.1 cells were pre-exposed to lipopolysaccharide (LPS) and IFN-γ for 24 h before combining the two cell lines into co-culture. In these conditions, a significant disruption of the epithelial barrier and an increase in pro-inflammatory cytokine (TNF-⍺ and IL-6) levels released from macrophages were detected. The data also show that inflammation in the co-culture model was temporary and reversible upon the removal of the inflammatory stimulus. This new in vitro model could be a valuable tool for investigating the safety and efficacy of drugs in the context of intestinal inflammation and provides advantages over other reported co-culture models of intestinal inflammation in terms of cost and simplicity.

Polymersomes for protein drug delivery across intestinal mucosa.

The oral administration is the route preferred by patients due to its multiple advantages. In the case of biopharmaceuticals, due to their low stability and absorption in the intestine, these molecules must be administered by injectable routes. To circumvent these problems, several strategies have been studied, among which the use of nanosystems, such as polymersomes, can be highlighted. In this work the potential of poloxamer 401 polymersomes as a system for oral delivery of antibodies was evaluated. IgG-FITC-loaded poloxamer 401 polymerosomes were initially used to assess whether it improves intestinal epithelial permeation in Caco-2 cell monolayers. Subsequently, epithelial/macrophage co-culture model was used to evaluate the ability of poloxamer 401 polymersomes containing adalimumab to reduce proinflammatory cytokine levels. The data showed that polymersome-encapsulated IgG increased the transport across intestinal Caco-2 monolayers 2.7-fold compared to the antibody in solution. Also, when comparing the groups of blank polymersomes with polymersomes containing adalimumab, decreases of 1.5-, 5.5-, and 2.4-fold in TNF-α concentrations were observed for the polymersomes containing 1.5, 3.75, and 15 µg/mL of adalimumab, respectively. This could indicate a possibility for the oral administration of biopharmaceuticals which would revolutionize many conditions that require the systemic administration such as in inflammatory bowel disease.

Probing milk extracellular vesicles for intestinal delivery of RNA therapies.

BACKGROUND: Oral delivery remains unattainable for nucleic acid therapies. Many nanoparticle-based drug delivery systems have been investigated for this, but most suffer from poor gut stability, poor mucus diffusion and/or inefficient epithelial uptake. Extracellular vesicles from bovine milk (mEVs) possess desirable characteristics for oral delivery of nucleic acid therapies since they both survive digestion and traverse the intestinal mucosa. RESULTS: Using novel tools, we comprehensively examine the intestinal delivery of mEVs, probing whether they could be used as, or inform the design of, nanoparticles for oral nucleic acid therapies. We show that mEVs efficiently translocate across the Caco-2 intestinal model, which is not compromised by treatment with simulated intestinal fluids. For the first time, we also demonstrate transport of mEVs in novel 3D 'apical-out' and monolayer-based human intestinal epithelial organoids (IEOs). Importantly, mEVs loaded with small interfering RNA (siRNA) induced (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) gene silencing in macrophages. Using inflammatory bowel disease (IBD) as an example application, we show that administration of anti-tumour necrosis factor alpha (TNFα) siRNA-loaded mEVs reduced inflammation in a IBD rat model. CONCLUSIONS: Together, this work demonstrates that mEVs could either act as natural and safe systems for oral delivery or nucleic acid therapies, or inform the design of synthetic systems for such application.

Enhanced permeation by amphiphilic surfactant is spatially heterogenous at membrane and cell level.

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.

Exosome-based therapies for mucosal delivery.

Exosomes are membrane-bound extracellular nanovesicles secreted by most cells and found in multiple sources, including bodily fluids, plants, fruit, and bovine milk. They play an important role as mediators of intercellular communication, having a distinct ability to carry small molecules, proteins, and nucleic acids to recipient cells over large distances. Moreover, competency in crossing usually poorly permeable biological barriers has led to their promising use in diagnostics and in therapeutics, either as therapeutic entities on their own or as drug delivery vehicles, with superior stability, biocompatibility, circulation time and target specificity in comparison to conventional drug delivery systems. The aim of this review is to summarise and critically discuss the current literature on the use of exosomes in a therapeutic setting, with a particular focus on their use as drug delivery vehicles for mucosal drug delivery.

Non-Invasive Drug Delivery Systems.

Non-invasive drug delivery generally refers to painless drug administration methods involving drug delivery across the biological barriers of the mucosal surfaces or the skin [...].

Pulmonary Delivery of Biological Drugs.

In the last decade, biological drugs have rapidly proliferated and have now become an important therapeutic modality. This is because of their high potency, high specificity and desirable safety profile. The majority of biological drugs are peptide- and protein-based therapeutics with poor oral bioavailability. They are normally administered by parenteral injection (with a very few exceptions). Pulmonary delivery is an attractive non-invasive alternative route of administration for local and systemic delivery of biologics with immense potential to treat various diseases, including diabetes, cystic fibrosis, respiratory viral infection and asthma, etc. The massive surface area and extensive vascularisation in the lungs enable rapid absorption and fast onset of action. Despite the benefits of pulmonary delivery, development of inhalable biological drug is a challenging task. There are various anatomical, physiological and immunological barriers that affect the therapeutic efficacy of inhaled formulations. This review assesses the characteristics of biological drugs and the barriers to pulmonary drug delivery. The main challenges in the formulation and inhalation devices are discussed, together with the possible strategies that can be applied to address these challenges. Current clinical developments in inhaled biological drugs for both local and systemic applications are also discussed to provide an insight for further research.

Temperature-Responsive Methylcellulose-Hyaluronic Hydrogel as a 3D Cell Culture Matrix.

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.

Exploiting disease-induced changes for targeted oral delivery of biologics and nanomedicines in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic and progressive disorder with destructive inflammation in the gastrointestinal tract (GIT). Biologics have changed the management of IBD, but have serious limitations, which is associated with their systemic administration via injection. Oral administration is the most accepted route of drug administration. However, the physiological barriers of the GIT pose significant challenges for oral administration of biologics, making this route of administration currently unavailable. The status of tissue barriers to oral drug delivery is altered in IBD. This may bring more challenges, but also present opportunities for oral delivery of biologics. This article provides an overview of disease-induced alterations of GIT barriers in IBD and discusses challenges, opportunities and commonly-utilised strategies for oral delivery of complex therapeutics, including biologics and nanomedicines.

Use of Engineered Nanoparticles (ENPs) for the Study of High-Affinity IgE FcεRI Receptor Engagement and Rat Basophilic Leukemia (RBL) Cell Degranulation.

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. ß-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 ß-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.

Hypromellose - A traditional pharmaceutical excipient with modern applications in oral and oromucosal drug delivery.

Hydroxypropylmethylcellulose (HPMC), also known as Hypromellose, is a traditional pharmaceutical excipient widely exploited in oral sustained drug release matrix systems. The choice of numerous viscosity grades and molecular weights available from different manufacturers provides a great variability in its physical-chemical properties and is a basis for its broad successful application in pharmaceutical research, development, and manufacturing. The excellent mucoadhesive properties of HPMC predetermine its use in oromucosal delivery systems including mucoadhesive tablets and films. HPMC also possesses desirable properties for formulating amorphous solid dispersions increasing the oral bioavailability of poorly soluble drugs. Printability and electrospinnability of HPMC are promising features for its application in 3D printed drug products and nanofiber-based drug delivery systems. Nanoparticle-based formulations are extensively explored as antigen and protein carriers for the formulation of oral vaccines, and oral delivery of biologicals including insulin, respectively. HPMC, being a traditional pharmaceutical excipient, has an irreplaceable role in the development of new pharmaceutical technologies, and new drug products leading to continuous manufacturing processes, and personalized medicine. This review firstly provides information on the physical-chemical properties of HPMC and a comprehensive overview of its application in traditional oral drug formulations. Secondly, this review focuses on the application of HPMC in modern pharmaceutical technologies including spray drying, hot-melt extrusion, 3D printing, nanoprecipitation and electrospinning leading to the formulation of printlets, nanoparticle-, microparticle-, and nanofiber-based delivery systems for oral and oromucosal application. Hypromellose is an excellent excipient for formulation of classical dosage forms and advanced drug delivery systems. New methods of hypromellose processing include spray draying, hot-melt extrusion, 3D printing, and electrospinning.

Cow Milk and Intestinal Epithelial Cell-derived Extracellular Vesicles as Systems for Enhancing Oral Drug Delivery.

Ingestion is the preferred way for drug administration. However, many drugs have poor oral bioavailability, warranting the use of injections. Extracellular vesicles (EVs) from cow milk have shown potential utility in improving oral drug bioavailability. However, EVs produced by intestinal epithelial cells have not been investigated for this application. We compared the capacity of cow milk EVs and intestinal epithelial cell-derived counterparts to enhance oral drug bioavailability. EVs were isolated, fluorescently labelled, and loaded with curcumin (CUR) as a model poorly absorbable drug. These were then characterised before testing in an intestinal model (Caco-2). Epithelial cell-derived EVs showed notably higher cell uptake compared to cow milk EVs. Cell uptake was significantly higher in differentiated compared to undifferentiated cells for both types of EVs. While both milk- and cell-derived EVs improved the cell uptake and intestinal permeability of CUR (confirming oral drug bioavailability enhancement potential), epithelial cell EVs demonstrated a superior effect.

PEGylation affects the self-assembling behaviour of amphiphilic octapeptides.

Surfactant-like peptides are a class of amphiphilic macromolecules, which are able to self-assemble in water forming different supramolecular structures. Among them, octapeptides composed of six hydrophobic and two hydrophilic residues have attracted interest since they have a length similar to those of natural phospholipids. Supramolecular structures of different amphiphilic octapeptides have been widely reported, but no study has been performed aimed at investigating the effect of PEGylation on their self-assembling behaviour. The aim of the present work was to synthesize and characterise the self-assembling behaviour of PEGylated alanine- or valine based amphiphilic octapeptides (mPEG1.9kDa-DDAAAAAA and mPEG1.9kDa-DDVVVVVV) in comparison to the non-PEGylated ones (DDAAAAAA and DDVVVVVV). The self-aggregation process in ultrapure water was investigated by fluorescence spectroscopy, small angle neutron scattering (SANS), dynamic light scattering (DLS), while the secondary structure was assessed by circular dichroism. PEGylation markedly affects the self-assembling behaviour of these amphiphilic octapeptides in terms of both critical aggregation concentration (CAC) and shape of the formed supramolecular aggregates. Indeed, PEGylation increases CAC and prevents the self-aggregation into fibrillary supramolecular aggregates (as observed for non-PEGylated peptides), by promoting the formation of micelle-like structures (as demonstrated for valine-based octapeptide). On the other side, the secondary structure of peptides seems not to be affected by PEGylation. Overall, these results suggest that self-assembling behaviour of amphiphilic octapeptides can be modified by PEGylation, with a great potential impact for the future applications of these nanomaterials.

Delivery of Nanoparticles across the Intestinal Epithelium via the Transferrin Transport Pathway.

The aim of this study was to probe whether the transferrin (Tf) transport pathway can be exploited for intestinal delivery of nanoparticles. Tf was adsorbed on 100 nm model polystyrene nanoparticles (NP), followed by size characterisation of these systems. Cell uptake of Tf and Tf-adsorbed NP was investigated in intestinal epithelial Caco-2 cells cultured on multi-well plates and as differentiated polarised monolayers. Tf-NP demonstrated a remarkably higher cell uptake compared to unmodified NP in both non-polarised (5-fold) and polarised cell monolayers (16-fold difference). Application of soluble Tf significantly attenuated the uptake of Tf-NP. Notably, Tf-NP displayed remarkably higher rate (23-fold) of epithelial transport across Caco-2 monolayers compared to unmodified NP. This study therefore strongly suggests that the Tf transport pathway should be considered as a candidate biological transport route for orally-administered nanomedicines and drugs with poor oral bioavailability.

Quaternary Ammonium Leucine-Based Surfactants: The Effect of a Benzyl Group on Physicochemical Properties and Antimicrobial Activity.

Quaternary ammonium amphiphiles are a class of compounds with a wide range of commercial and industrial uses. In the pharmaceutical field, the most common quaternary ammonium surfactant is benzalkonium chloride (BAC), which is employed as a preservative in several topical formulations for ocular, skin, or nasal application. Despite the broad antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as fungi and small enveloped viruses, safety concerns regarding its irritant and cytotoxic effect on epithelial cells still remain. In this work, quaternary ammonium derivatives of leucine esters (C10, C12 and C14) were synthesised as BAC analogues. These cationic surfactants were characterised in terms of critical micelle concentration (CMC, by tensiometry), cytotoxicity (MTS and LDH assays on the Caco-2 and Calu-3 cell lines) and antimicrobial activity on the bacterial species Staphylococcus aureus and Enterococcus faecalis among the Gram-positives, Escherichia coli and Pseudomonas aeruginosa among the Gram-negatives and the yeast Candida albicans. They showed satisfactory surface-active properties, and a cytotoxic effect that was dependent on the length of the hydrophobic chain. Lower minimum inhibiting concentration (MIC) values were calculated for C14-derivatives, which were comparable to those calculated for BAC toward Gram-positive bacteria and slightly higher for Gram-negative bacteria and C. albicans. Thus, the synthesised leucine-based quaternary ammonium cationic surfactants can potentially find application as promising surface-active compounds with antimicrobial activity.

Evaluation of a Methylcellulose and Hyaluronic Acid Hydrogel as a Vehicle for Rectal Delivery of Biologics.

Biologics have changed the management of Inflammatory Bowel Disease (IBD), but there are concerns regarding unexpected systemic toxicity and loss of therapeutic response following administration by injection. Local delivery of biologics directly to the inflamed mucosa via rectal enema administration addresses the problems associated with systemic administration. Hydrogels are potentially useful delivery vehicles enabling rectal administration of biologics. Here, we prepared a hydrogel system based on methylcellulose (MC) and hyaluronic acid (HA), which possesses mucosal healing properties, incorporating a model macromolecular drug, namely (fluorescently-labeled) bovine serum albumin (BSA). The BSA-loaded MCHA hydrogel showed temperature-dependent gelation (liquid-like at 20 °C and gel-like at 37 °C) and shear thinning behavior, with these being important and desirable characteristics for rectal application (enabling easy application and retention). BSA release from the MCHA system at 37 °C was linear, with 50% of the loaded drug released within 2 h. The system demonstrated acceptable toxicity towards intestinal (colon) Caco-2 epithelial cells, even at high concentrations. Importantly, application of the BSA-loaded MCHA hydrogel to polarized Caco-2 monolayers, with or without an exemplar absorption enhancer, resulted in transintestinal permeability of BSA. The study therefore indicates that the MCHA hydrogel shows potential for topical (rectal) delivery of biologics in IBD.

Nanoparticle modification in biological media: implications for oral nanomedicines.

Nanomedicine has shown potential in enabling oral administration of poorly absorbable drugs, such as biologics. As part of the process related to optimisation of the safety and efficacy of nanomedicines, it is imperative that the interaction of nanoparticles with the biological systems - including the gut - is fully characterised. In this article, we provide an overview of the major mechanisms by which nanoparticles may transform upon introduction in biological media. Specifically, the phenomena of association, dissolution and biomolecule adsorption are discussed, together with factors which influence the occurrence of each phenomenon. The implications of these phenomena within the context of therapeutic action of nanomedicines, which includes reduced targeting efficiency, are also explored. Finally, we will comment on nanoparticle modification within the gut environment, including the currently available gastrointestinal models for the study of nano-bio interactions, with implications in the area of nanomedicines for oral administration.

Role of the Basement Membrane as an Intestinal Barrier to Absorption of Macromolecules and Nanoparticles.

Full understanding of the barrier property of mucosal tissues is imperative for development of successful mucosal drug delivery strategies, particularly for biologics and nanomedicines. The contribution of the mucosal basement membrane (BM) to this barrier is currently not fully appreciated. This work examined the role of the BM as a barrier to intestinal absorption of model macromolecules (5 and 10 kDa dextrans) and 100 nm polystyrene nanoparticles. Dextrans and nanoparticles were applied either directly to BM-coated inserts or to an intestinal model, namely, differentiated intestinal epithelial monolayers (Caco-2) cultured on BM-modified inserts. The work shows that the BM per se does not impact the diffusion of dextran macromolecules but severely hinders the movement of nanoparticles. However, importantly, Caco-2 monolayers cultured on BM-coated inserts, which show a remarkably different morphology, display a significantly larger barrier to the translocation of one dextran, as well as nanoparticle systems compared to cells cultured on unmodified inserts. Therefore, this work shows that, in addition to presenting a direct physical barrier to the movement of nanoparticles, the BM also exerts an indirect barrier effect, likely due to its influence on epithelial cell physiology. This work is important as it highlights the currently unmet need to consider and further study the barrier properties of the BM in mucosal delivery of biologics and nanomedicines.