Jeffrey Penny

The influence of surface modification on the cytotoxicity of PAMAM dendrimers

The influence of surface modification on the cytotoxicity of PAMAM dendrimers was examined using Caco-2 cells. Dendrimers were modified by conjugating either lauroyl chains or polyethylene glycol (PEG) 2000 onto the surface of cationic PAMAM dendrimers (G2, G3, G4). The cytotoxicity of unmodified dendrimers towards Caco-2 cells was appreciably higher for cationic (whole generation) compared with anionic (half generation) dendrimers and for both types increased with increasing size (generation) and concentration. A marked decrease in the cytotoxicity of cationic PAMAM dendrimers was noted when the surface was modified, with the addition of six lauroyl or four PEG chains being particularly effective in decreasing cytotoxicity. This decrease in cytotoxicity is thought to be due to a reduction/shielding of the positive charge on the dendrimer surface by the attached chains. The cytotoxicity of dendrimer-based delivery systems is likely to be very different from the parent dendrimer.

Engineering of Dendrimer Surfaces to Enhance Transepithelial Transport and Reduce Cytotoxicity

AbstractPurpose. To evaluate the cytotoxicity, permeation, and transport mechanisms of PAMAM dendrimers and surface-modified cationic PAMAM dendrimers using monolayers of the human colon adenocarcinoma cell line, Caco-2. Methods. Cytotoxicity was determined using the MTT assay. The effect of dendrimers on monolayer integrity was determined from measurements of transepithelial electrical resistance (TEER) and [14C]mannitol apparent permeability coefficient (Papp). The Papp of dendrimers through monolayers was measured in both the apical (A)-to-basolateral (B) and B→A directions at 4°C and 37°C and also in the presence and absence of ethylenediamine tetraacetic acid (EDTA) and colchicine. Results. The cytotoxicity and permeation of dendrimers increased with both concentration and generation. The cytotoxicity of cationic dendrimers (G2, G3, G4) was greater than that of anionic dendrimers (G2.5, G3.5) but was reduced by conjugation with lauroyl chloride; the least cytotoxic conjugates were those with six attached lauroyl chains. At 37°C the Papp of cationic dendrimers was higher than that of anionic dendrimers and, in general, increased with the number of attached lipid chains. Cationic dendrimers decreased TEER and significantly increased the Papp of mannitol. Modified dendrimers also reduced TEER and caused a more marked increase in the Papp of mannitol. The Papp values of dendrimers and modified dendrimers were higher in the presence of EDTA, lower in the presence of colchicine, and lower at 4°C than at 37°C. Conclusions. The properties of dendrimers may be significantly modified by surface engineering. Conjugation of cationic PAMAM dendrimers with lauroyl chloride decreased their cytotoxicity and increased their permeation through Caco-2 cell monolayers. Both PAMAM dendrimers and lauroyl-PAMAM dendrimer conjugates can cross epithelial monolayers by paracellular and transcellular pathways.

Intraerythrocytic Plasmodium falciparum Expresses a High Affinity Facilitative Hexose Transporter

Asexual stages of Plasmodium falciparum cause severe malaria and are dependent upon host glucose for energy. We have identified a glucose transporter ofP. falciparum (PfHT1) and studied its function and expression during parasite development in vitro. PfHT1 is a saturable, sodium-independent, and stereospecific transporter, which is inhibited by cytochalasin B, and has a relatively high affinity for glucose (K m = 0.48 mm) when expressed in Xenopus laevis oocytes. Competition experiments with glucose analogues show that hydroxyl groups at positions C-3 and C-4 are important for ligand binding. mRNA levels for PfHT1, assessed by the quantitative technique of tandem competitive polymerase chain reaction, are highest during the small ring stages of infection and lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host structures. These findings have implications for development of new drug targets in malaria as well as for understanding of the pathophysiology of severe infection. When hypoglycemia complicates malaria, modeling studies suggest that the high affinity of PfHT1 is likely to increase the relative proportion of glucose taken up by parasites and thereby worsen the clinical condition.

Surface modification of PAMAM dendrimers modulates the mechanism of cellular internalization.

The aim of this study was to investigate the influence of dendrimer surface properties on cellular internalization and intracellular trafficking in the human colon adenocarcinoma HT-29 cell line. Third-generation (G3) polyamidoamine (PAMAM) dendrimers were modified to contain either two lauroyl chains (G3L2), two propranolol molecules (G3P2), or two lauroyl and two propranolol molecules (G3L2P2) at the dendrimer surface. Surface-modified and unmodified dendrimers were labeled with fluorescein isothiocyanate (FITC) at an average molar ratio of 1:1. The mechanisms of cellular internalization and intracellular trafficking of dendrimers were analyzed by confocal laser scanning microscopy and flow cytometry. The internalization of G3 and G3P2 dendrimers involved both caveolae-dependent endocytosis and macropinocytosis pathways; internalization of G3L2P2 dendrimer appeared to involve caveolae-dependent, and possibly clathrin-dependent, endocytosis pathways; and internalization of G3L2 dendrimer occurred via caveolae-dependent, clathrin-dependent, and macropinocytosis pathways. Subcellular colocalization data indicated that unmodified and all surface-modified G3 PAMAM dendrimers were internalized and trafficked to endosomes and lysosomes. It is therefore apparent that the initial mode of dendrimer internalization into HT-29 cells is influenced by the surface properties of G3 PAMAM dendrimer.

Transport of interleukin‐1 across cerebromicrovascular endothelial cells

Background and purpose:  The inflammatory cytokine interleukin‐1 (IL‐1) has profound actions in the brain, causing neuronal cell death and exacerbating brain damage. While circulating levels are normally low, IL‐1 can be produced on the vascular side of the brain endothelium, and within the brain. The naturally occurring IL‐1 receptor antagonist has been administered peripherally in a Phase II trial in acute stroke patients; understanding how IL‐1 and IL‐1 receptor antagonist penetrate the brain is, therefore, of considerable importance.

Methodology for development of a physiological model incorporating CYP3A and P-glycoprotein for the prediction of intestinal drug absorption

The small intestine poses a major barrier to the efficient absorption of orally administered therapeutics. Intestinal epithelial cells are an extremely important site for extrahepatic clearance, primarily due to prominent P-glycoprotein-mediated active efflux and the presence of cytochrome P450s. We describe a physiologically based pharmacokinetic model which incorporates geometric variations, pH alterations and descriptions of the abundance and distribution of cytochrome 3A and P-glycoprotein along the length of the small intestine. Simulations using preclinical in vitro data for model drugs were performed to establish the influence of P-glycoprotein efflux, cytochrome 3A metabolism and passive permeability on drug available for absorption within the enterocytes. The fraction of drug escaping the enterocyte (F(G)) for 10 cytochrome 3A substrates with a range of intrinsic metabolic clearances were simulated. Following incorporation of P-glycoprotein in vitro efflux ratios all predicted F(G) values were within 20% of observed in vivo F(G). The presence of P-glycoprotein increased the level of cytochrome 3A drug metabolism by up to 12-fold in the distal intestine. F(G) was highly sensitive to changes in intrinsic metabolic clearance but less sensitive to changes in intestinal drug permeability. The model will be valuable for quantifying aspects of intestinal drug absorption and distribution.

An immortalised astrocyte cell line maintains the in vivo phenotype of a primary porcine in vitro blood–brain barrier model

Whilst it is well documented that all components of the neurovascular unit contribute to the restrictive nature of the blood-brain barrier (BBB), astrocytes have been identified as the cellular component most likely to play an essential role in maintaining the barrier properties. The aim of this study was to examine the impact of the rat astrocyte cell line, CTX-TNA2, on the structural and functional characteristics of an in vitro BBB and determine the capacity of this astrocyte cell line to maintain the BBB phenotype. Co-culture of the CTX-TNA2 cells with primary porcine brain endothelial cells produced an in vitro BBB model which retains key features of the in vivo BBB. High transendothelial electrical resistances, comparable to those reported in vivo, were obtained. Ultrastructural analysis revealed distinct intercellular tight junction protein complexes and immunocytochemistry confirmed expression of the tight junction proteins ZO-1 and occludin. Western blotting and fluorescent tracer assays confirmed expression and functional activity of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) efflux transporters. Studies employing Alexa-fluor 555-conjugated human transferrin revealed temperature-sensitive internalisation indicating the BBB model retains functional receptor-mediated transferrin uptake. The findings of this study indicate that a robust BBB model has been produced and this is the first report of the inductive capacity of the CTX-TNA2 cell line. Since this in vitro BBB model possesses many key characteristics of the BBB in vivo it has the potential to be a valuable tool for the study of biochemical and physiological processes associated with the BBB.

Expression of substrate-specific transporters encoded by Plasmodium falciparum in Xenopus laevis oocytes

When the malarial parasite Plasmodium falciparum multiplies in erythrocytes it dramatically increases uptake of essential metabolic precursors (nucleosides, nucleobases and glucose) and export of lactic acid by undefined mechanisms. The first evidence is provided here, by a detailed study in Xenopus laevis oocytes, that several specific nutrient transporters are the product of P. falciparum genes. We report the expression of nucleoside, nucleobase, hexose and monocarboxylate transport systems in Xenopus oocytes when injected with mRNA isolated from asexual stages of developing P. falciparum parasites. Their properties are distinct from transport events occurring at the infected erythrocyte membrane or the electrophysiologically identified channel localised to the parasitophorous vacuolar membrane. These novel transporters are substrate-specific and stereoselective, and represent a key regulatory step in the acquisition and export of metabolites by intraerythrocytic P. falciparum.

The extracellular matrix protein laminin-10 promotes blood–brain barrier repair after hypoxia and inflammation in vitro

BackgroundThe blood–brain barrier (BBB) of the central nervous system (CNS) is essential for normal brain function. However, the loss of BBB integrity that occurs after ischaemic injury is associated with extracellular matrix (ECM) remodelling and inflammation, and contributes to poor outcome. ECM remodelling also contributes to BBB repair after injury, but the precise mechanisms and contribution of specific ECM molecules involved are unknown. Here, we investigated the mechanisms by which hypoxia and inflammation trigger loss of BBB integrity and tested the hypothesis ECM changes could contribute to BBB repair in vitro.MethodsWe used an in vitro model of the BBB, composed of primary rat brain endothelial cells grown on collagen (Col) I-, Col IV-, fibronectin (FN)-, laminin (LM) 8-, or LM10-coated tissue culture plates, either as a single monolayer culture or on Transwell® inserts above mixed glial cell cultures. Cultures were exposed to oxygen-glucose deprivation (OGD) and/or reoxygenation, in the absence or the presence of recombinant interleukin-1β (IL-1β). Cell adhesion to ECM molecules was assessed by cell attachment and cell spreading assays. BBB dysfunction was assessed by immunocytochemistry for tight junction proteins occludin and zona occludens-1 (ZO-1) and measurement of trans-endothelial electrical resistance (TEER). Change in endothelial expression of ECM molecules was assessed by semi-quantitative RT-PCR.ResultsOGD and/or IL-1 induce dramatic changes associated with loss of BBB integrity, including cytoplasmic relocalisation of membrane-associated tight junction proteins occludin and ZO-1, cell swelling, and decreased TEER. OGD and IL-1 also induced gene expression of key ECM molecules associated with the BBB, including FN, Col IV, LM 8, and LM10. Importantly, we found that LM10, but not FN, Col IV, nor LM8, plays a key role in maintenance of BBB integrity and reversed most of the key hallmarks of BBB dysfunction induced by IL-1.ConclusionsOur data unravel new mechanisms of BBB dysfunction induced by hypoxia and inflammation and identify LM10 as a key ECM molecule involved in BBB repair after hypoxic injury and inflammation.

Identification of putative steroid-binding sites in human ABCB1 and ABCG2

Homology modelling was used to generate three-dimensional structures of the nucleotide-binding domains (NBDs) of human ABCB1 and ABCG2. Interactions between a series of steroidal ligands and transporter NBDs were investigated using an in silico docking approach. C-terminal ABCB1 NBD (ABCB1 NBD2) was predicted to bind steroids within a cavity formed partly by the P-Loop, Tyr1044 and Ile1050. The P-Loop within ABCG2 NBD was also predicted to be involved in steroid binding. No overlap between ATP- and RU-486-binding sites was predicted in either NBD, though overlaps between ATP- and steroid-binding sites were predicted in the vicinity of the P-Loop in both nucleotide-binding domains.