Particle characterisation in drug delivery

The use of materials in nano-scale dimensions is proving to be a promising approach to overcome drug delivery challenges. ‘Nanomedicine’ technologies are gradually achieving commercial success and reaching the clinic. Sub-micron nanocarriers have the potential to ferry the therapeutic to its site of action and in this process overcome the biological barriers and achieve targeted drug delivery, controlled or stimuli-responsive delivery and protect the therapeutic from biological milieus. Many different types of nanocarriers have been described, including polymeric nanoparticles (NPs), liposomes, solid lipid NPs, micelles, dendrimers and metal NPs among other systems (the terms ‘nanomedicine’, ‘NP’ and ‘nanocarriers’ are used herein to describe all nanosystems). Of particular interest are nanocarriers with the ability to act selectively and target cell internalisation processes, guiding the therapeutic into subcellular regions. NP features important in dictating their drug delivery performance, including targeted delivery and cellular trafficking, are their size, shape and surface characteristics such as surface charge, chemistry and the distribution of ligands

In vitro investigation into strategies for mucosal delivery of proteins

Mucosal surfaces offer a potential for non-invasive delivery of proteins. The role of these surfaces, however, is to limit the movement of material from the external environment (mucosal lumen) into systemic circulation. Mucosal absorption of protein therapeutics is constrained through several physiological barriers such as mucus and mucociliary clearance, protease enzymes, epithelial tight junctions (TJs) and cellular membranes. This work explores different strategies with the view to improving the transport of macromolecules (proteins and protein drug models) across polarised epithelial cell layers in vitro, which could potentially be a reflection of improved mucosal absorption and bioavailability in vivo. The Calu-3 cell line was used in this work to produce such layers, serving as an in vitro model of the airway epithelium. Following growth of Calu-3 cells on filters under air-interface culture conditions polarised layers of closely packed cells were formed. The cell layers exhibited a TEER ≥500 Ωcm2 and cells showed structural features similar to the native epithelium, including the TJs, the microvilli and the secretory granules. Cell layers presented a barrier to the permeability of FITC-dextrans (FDs, paracellular markers) and nanoparticles (NPs). The first class of tested compounds, namely alkylglycoside (AG) surfactants, exhibited severe toxicity at concentrations considerably lower than those used in the literature. Data indicated that the cellular toxicity of AGs possibly results from a membrane effect. Investigation of calcium depletion as a proposed strategy to improve mucosal absorption of protein therapeutics, revealed that calcium depletion on the apical side produced limited TJ opening, as demonstrated by reversible decrease in transepithelial electrical resistance (TEER) and modest enhancement of permeability of macromolecules. Although combined apical and basolateral calcium exhaustion produced significant effects on TJs, this scenario becomes irrelevant in an in vivo situation. Application of chitosan in the form of solution and NPs to Calu-3 layers demonstrated that chitosan NPs formulated by the ionic gelation method, exhibit a similar TJ-opening effect to solution. This was shown by similarities in measurable indicators of TJ opening such as reduction in TEER and enhancement of dextran permeability across the cell layers. Furthermore, chitosan NP and solution exhibited similar effects on the TJ protein, Zonula Occludens-1. These results therefore indicated that chitosan NPs could potentially be used to carry and protect fragile therapeutic proteins to the mucosal surface(s) of interest and at the same time promote their absorption through TJ opening. TJ opening as a strategy to improve mucosal absorption of macromolecular therapeutics is rather inefficient for larger proteins such as antibodies, or for nano- sized drug carriers following their mucosal administration; this led to investigation of the IgG/neonatal Fc receptor (FcRn) transcytotic pathway. Immunostaining data demonstrated that Calu-3 cells express FcRn. IgG was shown to traverse the Calu-3 layers and studies characterizing this transport indicated FcRn involvement in this process. Confocal microscopy revealed that IgG- or Fc-adsorbed NPs were taken up by Calu-3 cells. Adsorption of Fc on the surface of NPs was seen to promote their cellular uptake and transport across the cell layers. Characterisation of cell uptake and transport of Fc-NPs revealed data that strongly suggested FcRn involvement in these processes

In vitro investigation into strategies for mucosal delivery of proteins

Mucosal surfaces offer a potential for non-invasive delivery of proteins. The role of these surfaces, however, is to limit the movement of material from the external environment (mucosal lumen) into systemic circulation. Mucosal absorption of protein therapeutics is constrained through several physiological barriers such as mucus and mucociliary clearance, protease enzymes, epithelial tight junctions (TJs) and cellular membranes. This work explores different strategies with the view to improving the transport of macromolecules (proteins and protein drug models) across polarised epithelial cell layers in vitro, which could potentially be a reflection of improved mucosal absorption and bioavailability in vivo. The Calu-3 cell line was used in this work to produce such layers, serving as an in vitro model of the airway epithelium. Following growth of Calu-3 cells on filters under air-interface culture conditions polarised layers of closely packed cells were formed. The cell layers exhibited a TEER ≥500 Ωcm2 and cells showed structural features similar to the native epithelium, including the TJs, the microvilli and the secretory granules. Cell layers presented a barrier to the permeability of FITC-dextrans (FDs, paracellular markers) and nanoparticles (NPs). The first class of tested compounds, namely alkylglycoside (AG) surfactants, exhibited severe toxicity at concentrations considerably lower than those used in the literature. Data indicated that the cellular toxicity of AGs possibly results from a membrane effect. Investigation of calcium depletion as a proposed strategy to improve mucosal absorption of protein therapeutics, revealed that calcium depletion on the apical side produced limited TJ opening, as demonstrated by reversible decrease in transepithelial electrical resistance (TEER) and modest enhancement of permeability of macromolecules. Although combined apical and basolateral calcium exhaustion produced significant effects on TJs, this scenario becomes irrelevant in an in vivo situation. Application of chitosan in the form of solution and NPs to Calu-3 layers demonstrated that chitosan NPs formulated by the ionic gelation method, exhibit a similar TJ-opening effect to solution. This was shown by similarities in measurable indicators of TJ opening such as reduction in TEER and enhancement of dextran permeability across the cell layers. Furthermore, chitosan NP and solution exhibited similar effects on the TJ protein, Zonula Occludens-1. These results therefore indicated that chitosan NPs could potentially be used to carry and protect fragile therapeutic proteins to the mucosal surface(s) of interest and at the same time promote their absorption through TJ opening. TJ opening as a strategy to improve mucosal absorption of macromolecular therapeutics is rather inefficient for larger proteins such as antibodies, or for nano- sized drug carriers following their mucosal administration; this led to investigation of the IgG/neonatal Fc receptor (FcRn) transcytotic pathway. Immunostaining data demonstrated that Calu-3 cells express FcRn. IgG was shown to traverse the Calu-3 layers and studies characterizing this transport indicated FcRn involvement in this process. Confocal microscopy revealed that IgG- or Fc-adsorbed NPs were taken up by Calu-3 cells. Adsorption of Fc on the surface of NPs was seen to promote their cellular uptake and transport across the cell layers. Characterisation of cell uptake and transport of Fc-NPs revealed data that strongly suggested FcRn involvement in these processes

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

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

Live imaging of cellular internalization of single colloidal particle by combined label-free and fluorescence total internal reflection microscopy

In this work we utilise the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labelled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently-tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF co-imaging enables live visualization of the process of colloidal particle interaction with the labelled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm model polystyrene colloid associates with clathrin, prior to and during its cellular internalisation. This association is not apparent with larger, 1 μm colloid, indicating an upper particle size limit for clathrin-mediated endocytosis

Nanoparticle transport in epithelial cells: pathway switching through bioconjugation

The understanding and control of nanoparticle transport into and through cellular compartments is central to biomedical applications of nanotechnology. Here, it is shown that the transport pathway of 50 nm polystyrene nanoparticles decorated with vitamin B12 in epithelial cells is different compared to both soluble B12 ligand and unmodified nanoparticles, and this is not attributable to B12 recognition alone. Importantly, the study indicates that vitamin B12-conjugated nanoparticles circumnavigate the lysosomal compartment, the destination of soluble vitamin B12 ligand. Whereas cellular trafficking of soluble B12 is confirmed to occur via the clathrin-mediated pathway, transport of B12-conjugated nanoparticles appears to predominantly take place by a route that is perturbed by caveolae-specific inhibitors. This data suggests that, following its conjugation to nanoparticles, in addition to dramatically increasing the cellular uptake of nanoparticles, the normal cell trafficking of B12 is switched to an alternative pathway, omitting the lysosomal stage: a result with important implications for oral delivery of nanoparticulate diagnostics and therapeutics

Ultrasound and Microbubbles Promote the Retention of Fluorescent Compounds in the Small Intestine

Focused ultrasound (US) is a novel means to increase the passage of medication through the wall of the small intestine. The purpose of this study was to determine whether US and microbubbles (MBs) can facilitate delivery of macromolecular therapeutic agents across the intestinal epithelium in vitro and in vivo. In vitro experiments involved delivery of compounds across a cell monolayer, namely Caco-2 cells cultured on ThinCert filters. The cells were cultured for a minimum of 3 weeks to mimic the polarised intestinal epithelium. A suspension of dextran with or without MBs, prepared in growth medium, was introduced into the apical chamber of the ThinCert with a syringe pump through a channel in the centre of a miniature focused US transducer (4 MHz, 1 MPa PNP). Each in vivo experiment involved a tethered endoscopic capsule with an US transducer and a delivery channel inserted into the small intestine of a terminally anaesthetised pig via a surgical stoma. The amount of fluorescent dextran delivered across the Caco-2 monolayer when employing US, MBs and dextran was higher than the amount delivered with dextran alone. With this approach, fluorescent marking of the wall of the small intestine was achieved in vivo by applying US and MBs. Our work indicates that US has potential for application in targeted treatment of gastrointestinal disease and oral drug delivery

Unsaturated fatty acids lactose esters: cytotoxicity, permeability enhancement and antimicrobial activity

Sugar based surfactants conjugated with fatty acid chains are an emerging broad group of highly biocompatible and biodegradable compounds with established and potential future applications in the pharmaceutical, cosmetic and food industries. In this work, we investigated absorption enhancing and antimicrobial properties of disaccharide lactose, mono-esterified with unsaturated fatty acids through an enzymatic synthetic approach. After chemical and cytotoxicity characterizations, their permeability enhancing activity was demonstrated using intestinal Caco-2 monolayers through transepithelial electrical resistance (TEER) and permeability studies. The synthesised compounds, namely lactose palmitoleate (URB1076) and lactose nervonate (URB1077), were shown to exhibit antimicrobial activity versus eight pathogenic species belonging to Gram-positive, Gram-negative microorganisms and fungi

Water-soluble substituted chitosan derivatives as technology platform for inhalation delivery of siRNA

Despite research efforts full potential of siRNA-based therapeutics has not yet been fully realized due to a need for suitable, effective delivery formulations. Here, we examine a potential of a new class of water-soluble chitosans as siRNA platform for pulmonary delivery. The system is based on piperazine-substituted chitosans, a material designed to integrate established, safe application of chitosan for mucosal administration with novel properties: the piperazine-substituted chitosans are freely water-soluble at physiological pH, possess low cytotoxicity (no significant reduction in cell viability up to 0.1 mg/ml), and provide efficient incorporation of siRNA into sub-300 nm colloidal complexes (at relatively low polymer/siRNA ratio of 5:1). In vitro, the complexes achieved silencing of a model gene at a level of 40–80%, when tested in a panel of lung epithelial cells. Considering the formulation ‘developability’, there were no significant changes in the complexes’ size and integrity on aerosolisation by microsprayer (PenCenturyTM) device. Following intratracheal aerolisation, the complexes deposited throughout the lung, although relatively inhomogeneously, as judged from IVIS imaging of the isolated mouse lung (visualizing DY647-siRNA). In vivo data illustrate absence of adverse effects on repeated administration of complexes and significant tumor reduction in atopical lung cancer model in mice. Altogether, the data illustrates potential of substituted chitosan derivatives to be utilized as a safe system for inhalation delivery of siRNA