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.</p

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.</p

PEGylated nanomedicines: recent progress and remaining concerns

Introduction. Recent biopharma deals related to nanocarrier drug delivery technologieshighlight the emergence of nanomedicine. This is perhaps an expected culmination of manyyears of research demonstrating the potential of nanomedicine as the next generation oftherapeutics with improved performance. PEGylated nanocarriers play a key role within thisfield.Areas covered. The drug delivery advantages of nanomedicines in general are discussed,focusing on nanocarriers and PEGylated nanomedicines, including products under currentdevelopment/clinical evaluation. Well-established drug delivery benefits of PEGylation (e.g.prolonged circulation) are only briefly covered. Instead, attention is deliberately made to lesscommonly reported advantages of PEGylation, including mucosal delivery of nanomedicines.Finally, some of the issues related to the safety of PEGylated nanomedicines in clinic arediscussed.Expert opinion. The advent of nanomedicine providing therapeutic options of refinedperformance continues. PEGylation as a tool to improve the pharmacokinetics ofnanomedicines is well established and used clinically, but other benefits of ‘PEGnology’,including enhancement of physicochemical properties and/or biocompatibility of activesand/or drug carriers, as well as mucosal delivery, have attracted less attention. Concernsregarding the clinical use of PEGylated nanomedicines remain, but evidence suggests that atleast some safety issues may be controlled by adequate designs of nanosystems.</p

Insight into the relationship between the cell culture model, cell trafficking and siRNA silencing efficiency

Despite research efforts, cell uptake processes determining siRNA silencing efficiency remain unclear. Here, we examine the relationship between in vitro cell culture models, cellular trafficking and siRNA silencing efficiency to provide a mechanistic insight on siRNA delivery system design. Model siRNA-polyplexes, based on chitosan as a 'classical' condensing agent, were applied to a panel of lung epithelial cell lines, H1299, A549 and Calu-3 and cell internalization levels, trafficking pathways and gene silencing assessed on exposure to pharmacological inhibitors. The data reveal striking differences in the internalization behaviour and gene silencing efficiency in the tested cell lines, despite their common lung epithelial origins. The model system's silencing was lower where clathrin internalization pathway predominated in Calu-3, relative to silencing in H1299 cells where a non-clathrin internalization appears dominant. Increased silencing on endosomal disruption was apparent in Calu-3 cells, but absent when cellular internalization was not predominantly clathrin-mediated in A549 cells. This highlights that identifying cell trafficking pathways before incorporation of functional components to siRNA delivery systems (e.g. endosomolytic compounds) is crucial. The study hence stresses the importance of selection of appropriate cell culture model, relevant to in vivo target, to assess the gene silencing efficiency and decide which functionalities the 'stratified siRNA silencing vector' requires.</p

Insight into the relationship between the cell culture model, cell trafficking and siRNA silencing efficiency

Despite research efforts, cell uptake processes determining siRNA silencing efficiency remain unclear. Here, we examine the relationship between in vitro cell culture models, cellular trafficking and siRNA silencing efficiency to provide a mechanistic insight on siRNA delivery system design. Model siRNA-polyplexes, based on chitosan as a 'classical' condensing agent, were applied to a panel of lung epithelial cell lines, H1299, A549 and Calu-3 and cell internalization levels, trafficking pathways and gene silencing assessed on exposure to pharmacological inhibitors. The data reveal striking differences in the internalization behaviour and gene silencing efficiency in the tested cell lines, despite their common lung epithelial origins. The model system's silencing was lower where clathrin internalization pathway predominated in Calu-3, relative to silencing in H1299 cells where a non-clathrin internalization appears dominant. Increased silencing on endosomal disruption was apparent in Calu-3 cells, but absent when cellular internalization was not predominantly clathrin-mediated in A549 cells. This highlights that identifying cell trafficking pathways before incorporation of functional components to siRNA delivery systems (e.g. endosomolytic compounds) is crucial. The study hence stresses the importance of selection of appropriate cell culture model, relevant to in vivo target, to assess the gene silencing efficiency and decide which functionalities the 'stratified siRNA silencing vector' requires.</p

Drug delivery: epithelial cell models for drug transport and toxicology studies

The development of medicines during the 20th Century was initially based on oral delivery of drugs via the gastrointestinal tract. To enhance understanding of rate of uptake of different drugs and formulations and reduce the need for animal testing, in vitro models based on gut epithelial cell models were developed in the 1980s and 1990s. With the advent of biotechnology, an increasing number of drugs based on proteins and other biomolecules are being produced, which currently require parenteral administration (by injection). To avoid the need for injection, alternative routes of delivery are being sought for these molecules, including mucosal routes of the gastrointestinal tract and the lung. In parallel with this, the field of ‘nanotechnology’ began to develop. Nanotechnology offers both solutions and problems. ‘Nanomedicines’ over a range of nano sizes appear to offer some solutions for delivery, provided that they could cross epithelial barriers. In contrast, there remains considerable concern that the many different types of nanoparticles in development for electronics and new materials may be taken up into the body and cause harm. There are therefore clear needs for epithelial models which allow us to not only screen conventional drugs for absorption, but also assess potential non-invasive delivery of biologics and nanomedicines, as well as screen easily and reliably for nanotoxicology. As it is the same barrier involved in all of these cases, we need a single epithelial model that can adequately reflect and give accurate answers for all of these different barrier problems. In this article, we assess the properties needed for an epithelial cellular model, the current state of the art, and some recent work developing a more accurate and comprehensive model.</p

Basement membrane influences intestinal epithelial cell growth and presents a barrier to the movement of macromolecules

This work examines the potential drug delivery barrier of the basement membrane (BM) by assessing the permeability of select macromolecules and nanoparticles. The study further extends to probing the effect of BM on intestinal epithelial cell attachment and monolayer characteristics, including cell morphology. Serum-free cultured Caco-2 cells were grown on BM-containing porous supports, which were obtained by prior culture of airway epithelial cells (Calu-3), shown to assemble and deposit a BM on the growth substrate, followed by decellularisation. Data overall show that the attachment capacity of Caco-2 cells, which is completely lost in serum-free culture, is fully restored when the cells are grown on BM-coated substrates, with cells forming intact monolayers with high electrical resistance and low permeability to macromolecules. Caco-2 cells cultured on BM-coated substrates displayed strikingly different morphological characteristics, suggestive of a higher level of differentiation and closer resemblance to the native intestinal epithelium. BM was found to notably hinder the diffusion of macromolecules and nanoparticles in a size dependent manner. This suggests that the specialised network of extracellular matrix proteins may have a significant impact on transmucosal delivery of certain therapeutics or drug delivery systems.</p

The mechanisms of nanoparticle internalization and transport across an intestinal epithelial cell model: effect of size and surface charge

This study investigated the effect of nanoparticle size and surface charge on their interaction with Caco-2 monolayers as a model of the intestinal epithelium, including cell internalization pathways and the level of transepithelial transport. Initially, toxicity assays showed that cell viability and cell membrane integrity were dependent on the surface charge and applied mass, number and total surface area of nanoparticles, as tested in two epithelial cell lines, colon carcinoma Caco-2 and airway Calu-3. This also identified suitable nanoparticle concentrations for subsequent cell uptake experiments. Nanoparticle application at doses below EC50 revealed that the transport efficiency (ratio of transport to cell uptake) across Caco-2 cell monolayers is significantly higher for negatively charged nanoparticles compared to their positively charged counterparts (of similar size), despite the higher level of internalization of positively charged systems. Cell internalization pathways were hence probed using a panel of pharmacological inhibitors aiming to establish whether the discrepancy in transport efficiency is due to different uptake and transport pathways. Vesicular trans-monolayer transport for both positively and negatively charged nanoparticles was confirmed via inhibition of dynamin (by dynasore) and microtubule network (via nocodazole), which significantly reduced the transport of both nanoparticle systems. For positively charged nanoparticles a significant decrease in internalization and transport (46% and 37%, respectively) occurred in the presence of a clathrin pathway inhibitor (chlorpromazine), macropinocytosis inhibition (42%; achieved by 5-(N-ethyl-N-isopropyi)-amiloride) and under cholesterol depletion (38%; via methyl-ß-cyclodextrin), but remained unaffected by the inhibition of lipid raft associated uptake (caveolae) by genistein. On the contrary, the most prominent reduction in internalization and transport of negatively charged nanoparticles (51% and 48%, respectively) followed the inhibition of lipid raft-associated pathway (caveolae inhibition by genistein), but was not significantly affected by the inhibition of clathrin pathway.</p

The mechanisms of nanoparticle internalization and transport across an intestinal epithelial cell model: effect of size and surface charge

This study investigated the effect of nanoparticle size and surface charge on their interaction with Caco-2 monolayers as a model of the intestinal epithelium, including cell internalization pathways and the level of transepithelial transport. Initially, toxicity assays showed that cell viability and cell membrane integrity were dependent on the surface charge and applied mass, number and total surface area of nanoparticles, as tested in two epithelial cell lines, colon carcinoma Caco-2 and airway Calu-3. This also identified suitable nanoparticle concentrations for subsequent cell uptake experiments. Nanoparticle application at doses below EC50 revealed that the transport efficiency (ratio of transport to cell uptake) across Caco-2 cell monolayers is significantly higher for negatively charged nanoparticles compared to their positively charged counterparts (of similar size), despite the higher level of internalization of positively charged systems. Cell internalization pathways were hence probed using a panel of pharmacological inhibitors aiming to establish whether the discrepancy in transport efficiency is due to different uptake and transport pathways. Vesicular trans-monolayer transport for both positively and negatively charged nanoparticles was confirmed via inhibition of dynamin (by dynasore) and microtubule network (via nocodazole), which significantly reduced the transport of both nanoparticle systems. For positively charged nanoparticles a significant decrease in internalization and transport (46% and 37%, respectively) occurred in the presence of a clathrin pathway inhibitor (chlorpromazine), macropinocytosis inhibition (42%; achieved by 5-(N-ethyl-N-isopropyi)-amiloride) and under cholesterol depletion (38%; via methyl-ß-cyclodextrin), but remained unaffected by the inhibition of lipid raft associated uptake (caveolae) by genistein. On the contrary, the most prominent reduction in internalization and transport of negatively charged nanoparticles (51% and 48%, respectively) followed the inhibition of lipid raft-associated pathway (caveolae inhibition by genistein), but was not significantly affected by the inhibition of clathrin pathway.</p

Ligand density and clustering effects on endocytosis of folate modified nanoparticles

This study investigates the effects of surface ligand distribution pattern (ligand clustering and density) on the internalisation of nanoparticles by a bronchial epithelial in vitro model (Calu-3 cells cultured as polarised layers). Control of ligand clustering and its surface density was achieved through the use of ovalbumin as an intermediate species to anchor the ligand to the nanoparticle surface. The model particulate system consisted of polystyrene nanoparticles surface-decorated via the adsorption of ovalbumin with conjugated folate ligand. The density of the displayed ligand was manipulated by controlling the conjugation level of folate to ovalbumin, while ligand clustering was achieved by co-adsorption of varying mixtures of folate-ovalbumin conjugate (at different ligand density levels) and unconjugated ovalbumin. Increasing overall ligand density on the nanoparticle surface resulted in increased internalisation of modified nanoparticles by the cells, up to a saturation level. Surface ligand density also affected the cellular uptake pathway; from predominantly clathrin to predominantly caveolae-mediated as the ligand density is increased. We further demonstrate that surface clustering of the folate ligand enhances cellular internalisation of nanoparticles, relative to its dispersed surface distribution. Our work suggests a simple way to prepare a model system where surface manipulations of ligand density and its distribution are possible and which can be used to study nanoparticle-cell interaction processes.</p