Caitriona M. O'Driscoll

Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories

Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.

PEGylated gold nanoparticles: polymer quantification as a function of PEG lengths and nanoparticle dimensions

Au nanoparticles with diameters ranging between 15 and 170 nm have been synthesised in aqueous solution using a seed-mediated growth method, employing hydroxylamine hydrochloride as a reducing agent. Thiolated polyethylene glycol (mPEG-SH) polymers, with molecular weights ranging from 2100 to 51000 g mol−1, were used as efficient particle stabilising ligands. Dynamic light scattering and zeta potential measurements confirmed that the overall mean diameter and zeta potential of the capped nanoparticles increased in a non-linear way with increasing molecular weight of the mPEG-SH ligand. Electron microscopy and thermal gravimetric analysis of the polymer-capped nanoparticles, with a mean gold core diameter of 15 nm, revealed that the grafting density of the mPEG-SH ligands decreased from 3.93 to 0.31 PEG nm−2 as the molecular weight of the ligands increased from 2100 to 51400 g mol−1 respectively, due to increased steric hindrance and polymer conformational entropy with increase in the PEG chain length. Additionally, the number of bound mPEG-SH ligands, with a molecular weight of 10800 g mol−1, was found to increase in a non-linear way from 278 (σ = 42) to approximately 12960 PEG (σ = 1227) when the mean Au core diameter increased from 15 to 115 nm respectively. However, the grafting density of mPEG10000-SH ligands was higher on 15 nm Au nanoparticles and decreased slightly from 1.57 to 0.8 PEG nm−2 when the diameter increased; this effect can be attributed to the fact that smaller particles offer higher surface curvature, therefore allowing increased polymer loading per nm2. Au nanoparticles were also shown to interact with CT-26 cells without causing noticeable toxicity.

Therapeutic targeting in the silent era: advances in non-viral siRNA delivery

Gene silencing using RNA-interference, first described in mammalian systems almost a decade ago, is revolutionizing therapeutic target validation efforts both in vitro and in vivo. Moreover, the potential for using short interfering RNA (siRNA) as a therapy in its own right is also progressing at a significant pace. However, the widespread use of such approaches is contingent on having appropriate systems to achieve clinically appropriate, safe, and efficient delivery of siRNA. There are many physicochemical and biological barriers to such delivery, and a growing emphasis on the design and characterisation of non-viral technologies that will overcome these barriers and expedite targeted delivery. This review discusses the considerations and challenges associated with use of siRNA-based therapeutics, including stability and off-target effects. Speculation is made on the properties of an ideal delivery system and the non-viral delivery approaches used to date, both in vitro and in vivo, are classified and discussed. Moreover, the ability of cyclodextrin-based delivery vectors to fulfil many of the criteria of an ideal delivery construct is also elaborated.

The Effects of Pluronic® Block Copolymers and Cremophor® EL on Intestinal Lipoprotein Processing and the Potential Link with P-Glycoprotein in Caco-2 Cells

AbstractPurpose. This investigation was performed to study the effects of Pluronic® block copolymers and Cremophor® EL on intestinal lipoprotein processing and to investigate a potential link between lipoprotein processing and P-glycoprotein. Methods. Caco-2 cells were used to monitor changes in lipoprotein production and secretion following exposure to excipients. Effects on P-glycoprotein were monitored using cyclosporin A as a model substrate. Results. A range of surfactants commonly used as pharmaceutical excipients in lipid-based oral drug delivery systems, including Pluronic® block copolymers L81, P85, and F68 and Cremophor® EL, inhibited intestinal lipoprotein secretion. The effects were concentration dependent and reversible. The mechanism of inhibition appears to be related to the assembly and secretion of lipoproteins rather than to initial intracellular triglyceride synthesis. A strong correlation was found between excipient-mediated inhibition of lipoprotein secretion and inhibition of P-glycoprotein efflux, implying a link between the two biochemical processes. Conclusions. The ability of such bioactive excipients to simultaneously manipulate different cellular processes must be considered in selecting excipients for oral drug delivery systems. Such information is particularly relevant when the drug is lipophilic, a candidate for P-glycoprotein efflux, and where intestinal lymphatic targeting via chylomicron stimulation is desirable.

A comparison of the permeation enhancement potential of simple bile salt and mixed bile salt:fatty acid micellar systems using the CaCo-2 cell culture model

The aim of this study was to compare the permeation enhancing potential and toxicity of simple bile salt and bile salt:fatty acid mixed micellar systems using the CaCo-2 cell culture model. The effects of micellar systems of sodium cholate, (NaC), and sodium taurocholate, (NaTC), on the permeability of the hydrophilic markers, mannitol (182) and polyethylene glycols (PEGS) 900 and 4000, were assessed. Simple micelle systems of the unconjugated bile salt, NaC, caused greater enhancement of the hydrophilic markers than the conjugated bile salt, NaTC. In the case of NaC systems the enhancement was coincident with excess membrane disruption and toxicity as indicated by altered TEERs, TEMs, MTT values, and, the lack of recovery following removal of the enhancer. In contrast, the NaTC systems were less toxic, and, in the simple micelle form the likely mechanism of enhancement of the hydrophilic markers is via a transient effect on the tight junctions. Formation of mixed micellar systems with linoleic acid (LA) accentuated the toxic effects of NaC. In comparison, NaTC:LA mixed micelles showed superior permeability enhancement versus simple micelles without increasing membrane toxicity. The mechanism of enhancement of NaTC:LA appears more complex and involves a possible combination effect on both the paracellular and transcellular routes.

Click-modified cyclodextrins as nonviral vectors for neuronal siRNA delivery.

RNA interference (RNAi) holds great promise as a strategy to further our understanding of gene function in the central nervous system (CNS) and as a therapeutic approach for neurological and neurodegenerative diseases. However, the potential for its use is hampered by the lack of siRNA delivery vectors which are both safe and highly efficient. Cyclodextrins have been shown to be efficient and low toxicity gene delivery vectors in various cell types in vitro. However, to date, they have not been exploited for delivery of oligonucleotides to neurons. To this end, a modified β-cyclodextrin (CD) vector was synthesized, which complexed siRNA to form cationic nanoparticles of less than 200 nm in size. Furthermore, it conferred stability in serum to the siRNA cargo. The in vitro performance of the CD in both immortalized hypothalamic neurons and primary hippocampal neurons was evaluated. The CD facilitated high levels of intracellular delivery of labeled siRNA, while maintaining at least 80% cell viability. Significant gene knockdown was achieved, with a reduction in luciferase expression of up to 68% and a reduction in endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) expression of up to 40%. To our knowledge, this is the first time that a modified CD has been used as a safe and efficacious vector for siRNA delivery into neuronal cells.

Cationic cyclodextrin amphiphiles as gene delivery vectors

Amphiphilic polycationic cyclodextrins, heptakis[2-(m-amino-oligoethyleneglycol)-6-deoxy-6-hexylthio]-p-cyclodextrin and heptakis[2-(m- amino-oligoethyleneglycol)-6-deoxy-6-hexadecylthio]-p-cyclodextrin, were assessed for condensation of plasmid DNA and cell transfection. These cyclodextrins can self-assemble into cationic vesicles or nanoparticles and, unlike their amphiphilic non-aminated analogues, form DNA lipoplexes which efficiently transfected COS-7 cells. Levels of luciferase expression with the hexadecylthiopolyamino-CD vector were 2 x 10 4 times greater than for DNA alone. These levels are a significant improvement on non-amphiphilic polyamino CDs as vectors and are of the same order as the widely used transfection agent Lipofectin.

A comparison of intestinal lymphatic transport and systemic bioavailability of saquinavir from three lipid-based formulations in the anaesthetised rat model

Saquinavir is a lipophilic, poorly water‐soluble HIV protease inhibitor that undergoes extensive first‐pass metabolism and exhibits poor oral bioavailability. Redirection of the absorption pathway of anti‐HIV compounds from the portal blood to the HIV‐rich intestinal lymphatics may enhance therapeutic efficacy and reduce the extent of the first‐pass effect. This study investigates the potential of targeted intestinal lymphatic transport of saquinavir via a lipid formulation approach. Three formulations containing oleic acid were examined: cremophor‐oleic acid mixed micelles, d‐alpha tocopheryl polyethylene glycol 1000 succinate (TPGS)‐oleic acid mixed micelles and an oleic acid microemulsion. The mesenteric lymph duct cannulated anaesthetised rat model was employed. Plasma and lymph samples were analysed by HPLC. Lymph triglyceride was measured using an enzymatic colorimetric technique. The extent of lymphatic transport from the lipid vehicles was 0.025‐0.05% of the dose administered. The microemulsion produced higher and more prolonged mesenteric lymph concentrations than the micellar formulations. A strong correlation existed between the concentration of saquinavir in intestinal lymph and lymph triglyceride levels. The systemic bioavailability was estimated to be 8.5% and 4.8% for the cremophor mixed micelle and the microemulsion, respectively. The cremophor mixed micelles produced higher bioavailability than TPGS mixed micelles, implying that the nature of the surfactant can influence the distribution of drug between lymph and plasma.

Gene silencing of TNF-alpha in a murine model of acute colitis using a modified cyclodextrin delivery system

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the gastrointestinal tract. The cytokine TNF-alpha (TNF-α) plays a pivotal role in mediating this inflammatory response. RNA interference (RNAi) holds great promise for the specific and selective silencing of aberrantly expressed genes, such as TNF-α in IBD. The aim of this study was to investigate the efficacy of an amphiphilic cationic cyclodextrin (CD) vector for effective TNF-α siRNA delivery to macrophage cells and to mice with induced acute-colitis. The stability of CD.siRNA was examined by gel electrophoresis in biorelevant media reflecting colonic fluids. RAW264.7 cells were transfected with CD.TNF-α siRNA, stimulated with lipopolysaccharide (LPS) and TNF-α and IL-6 responses were measured by PCR and ELISA. Female C57BL/6 mice were exposed to dextran sodium sulphate (DSS) and treated by intrarectal administration with either CD.siRNA TNF-α or a control solution. In vitro, siRNA in CD nanocomplexes remained intact and stable in both fed and fasted simulated colonic fluids. RAW264.7 cells transfected with CD.TNF-α siRNA and stimulated with LPS displayed a significant reduction in both gene and protein levels of TNF-α and IL-6. CD.TNF-α siRNA-treated mice revealed a mild amelioration in clinical signs of colitis, but significant reductions in total colon weight and colonic mRNA expression of TNF-α and IL-6 compared to DSS-control mice were detected. This data indicates the clinical potential of a local CD-based TNF-α siRNA delivery system for the treatment of IBD.

The effect of mixed micellar systems, bile salt/fatty acids, on the solubility and intestinal absorption of clofazimine (B663) in the anaesthetised rat

Abstract Clofazimine (B663) is a highly lipophilic drug used in the treatment of leprosy. The solubility and gastrointestinal membrane permeability (Papp) of B663 in mixed micellar systems were examined. Membrane permeability was determined using a rat gut perfusion model and, in addition, these studies incorporated the hydrophilic marker PEG 4000. The mixed micellar systems studied contained the bile salt, sodium cholate (NaC), in association with different fatty acids including caprylic acid, oleic acid and linoleic acid. At a set concentration of NaC (40 mM) the solubility of B663 increased with increasing concentration of each fatty acid. Relative to NaC, the maximum enhancement in solubility (16-fold) was obtained with the NaC/linoleic acid (40:40 mM) system. An optimum bile salt/fatty acid ratio of 1:1 existed for maximum solubility enhancement. All mixed micellar systems enhanced the absorption of B663 relative to the simple micelle. The Papp tended to increase with increasing fatty acid concentration, maximum enhancement being obtained with the NaC/linoleic acid 40:40 mM system. With each mixed micellar system a higher Papp was obtained with lower drug loading. The effects of the mixed micellar systems on the absorption of PEG 4000 varied with fatty acid loading. These results have shown that mixed micelles can enhance the absorption of B663 to a greater extent relative to non-micellar and simple micellar systems. Maximum enhancement (> 800-fold) in the rate of B663 absorption was obtained with the NaC/linoleic acid 40:40 mM system. These results offer a possible explanation for the reported enhancement in gastrointestinal absorption of B663 when co-administered with fatty materials.