Distribution of Silica-Coated Silver/Gold Nanostars in Soft- and Hardwood Applying SERS-Based Imaging.

Nanoparticles (NPs) in aqueous suspension have just begun to be exploited for the preservative treatment of wood. However, at present, there is very little information available on the distribution of NPs in wood after impregnation, due to associated analytical challenges. In this study, we present the detection of model NPs in softwood and hardwood by surface-enhanced Raman spectroscopy (SERS). SERS is a highly sensitive analytical method requiring no fluorescent labeling. The NP distribution after impregnation is evaluated with one representative species of the two wood types. To show the feasibility of the method, we prepared SERS-active Au/Ag nanostars coated with silica to act as a model NP system. We show herein that NPs can be imaged in very low quantities in both wood types without any matrix interactions. The presence of the NPs in the wood was confirmed by scanning electron microscopy (SEM) imaging and energy dispersive X-ray analysis (EDX). The fast detection of NPs in a complex matrix, without complicated sample preparation, marks a huge step forward in the development and application of nanotechnology for wood preservation and the quest to optimize the properties of one of the world's most important raw materials.

A critical review of the current knowledge regarding the biological impact of nanocellulose.

Several forms of nanocellulose, notably cellulose nanocrystals and nanofibrillated cellulose, exhibit attractive property matrices and are potentially useful for a large number of industrial applications. These include the paper and cardboard industry, use as reinforcing filler in polymer composites, basis for low-density foams, additive in adhesives and paints, as well as a wide variety of food, hygiene, cosmetic, and medical products. Although the commercial exploitation of nanocellulose has already commenced, little is known as to the potential biological impact of nanocellulose, particularly in its raw form. This review provides a comprehensive and critical review of the current state of knowledge of nanocellulose in this format. Overall, the data seems to suggest that when investigated under realistic doses and exposure scenarios, nanocellulose has a limited associated toxic potential, albeit certain forms of nanocellulose can be associated with more hazardous biological behavior due to their specific physical characteristics.

Effects and uptake of gold nanoparticles deposited at the air-liquid interface of a human epithelial airway model.

The impact of nanoparticles (NPs) in medicine and biology has increased rapidly in recent years. Gold NPs have advantageous properties such as chemical stability, high electron density and affinity to biomolecules, making them very promising candidates as drug carriers and diagnostic tools. However, diverse studies on the toxicity of gold NPs have reported contradictory results. To address this issue, a triple cell co-culture model simulating the alveolar lung epithelium was used and exposed at the air-liquid interface. The cell cultures were exposed to characterized aerosols with 15 nm gold particles (61 ng Au/cm2 and 561 ng Au/cm2 deposition) and incubated for 4 h and 24 h. Experiments were repeated six times. The mRNA induction of pro-inflammatory (TNFalpha, IL-8, iNOS) and oxidative stress markers (HO-1, SOD2) was measured, as well as protein induction of pro- and anti-inflammatory cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNFalpha, INFgamma). A pre-stimulation with lipopolysaccharide (LPS) was performed to further study the effects of particles under inflammatory conditions. Particle deposition and particle uptake by cells were analyzed by transmission electron microscopy and design-based stereology. A homogeneous deposition was revealed, and particles were found to enter all cell types. No mRNA induction due to particles was observed for all markers. The cell culture system was sensitive to LPS but gold particles did not cause any synergistic or suppressive effects. With this experimental setup, reflecting the physiological conditions more precisely, no adverse effects from gold NPs were observed. However, chronic studies under in vivo conditions are needed to entirely exclude adverse effects.

Leveraging proteomics to compare submerged versus air-liquid interface carbon nanotube exposure to a 3D lung cell model.

With the emerging concern over the potential toxicity associated with carbon nanotube inhalation exposure, several in vitro methods have been developed to evaluate cellular responses. Since the major concern for adverse effects by carbon nanotubes is inhalation, various lung cell culture models have been established for toxicity testing, thus creating a wide variation of methodology. Limited studies have conducted side-by-side comparisons of common methods used for carbon nanotube hazard testing. The aim of this work was to use proteomics to evaluate global cellular response, including pro-inflammatory and pro-fibrotic mediators, of a 3D lung model composed of macrophages, epithelial cells, and fibroblasts which mimics the human alveolar epithelial tissue barrier. The cells were exposed to Mitsui 7 (M-7) multi-walled carbon nanotubes (MWCNT) under submerged and air-liquid interface (ALI) conditions and discovery proteomics identified 3500 proteins. The M-7 ALI exposure compared to control was found to increase expression in proteins related to oxidative stress that were not found to be enriched in submerged exposure. Comparison of MWCNT exposure methods, M-7 ALI exposure versus M-7 submerged exposure, yielded protein enrichment in pathways known to be associated with carbon nanotube exposure stress response, such as acute phase response signaling and NRF2-mediated oxidative stress response. This study demonstrates a comparison of commonly deployed carbon nanotube exposure methods. These data should be considered by the nanotoxicology community when interpreting or cross comparing in vitro exposure results.

A novel sample holder for 4D live cell imaging to study cellular dynamics in complex 3D tissue cultures.

Three dimensional (3D) co-cultures to mimic cellular dynamics have brought significant impacts in tissue engineering approaches for biomedical research. Herein, we present a novel sample holder combined with time-lapse fluorescence imaging technique, referred as 4D live cell imaging, allowing direct visualization of various cells up to 24 hours. We further extended our approach to monitor kinetics and dynamics of particle uptake by cells and translocation across tissue membranes.

A lock-in-based method to examine the thermal signatures of magnetic nanoparticles in the liquid, solid and aggregated states.

We propose a new methodology based on lock-in thermography to study and quantify the heating power of magnetic nanoparticles. Superparamagnetic iron oxide nanoparticles exposed to a modulated alternating magnetic field were used as model materials to demonstrate the potency of the system. Both quantitative and qualitative information on their respective heating power was extracted at high thermal resolutions under increasingly complex conditions, including nanoparticles in the liquid, solid and aggregated states. Compared to conventional techniques, this approach offers a fast, sensitive and non-intrusive alternative to investigate multiple and dilute specimens simultaneously, which is essential for optimizing and accelerating screening procedures and comparative studies.

Decoupling the shape parameter to assess gold nanorod uptake by mammalian cells.

The impact of nanoparticles (NPs) upon biological systems can be fundamentally associated with their physicochemical parameters. A further often-stated tenet is the importance of NP shape on rates of endocytosis. However, given the convoluted parameters concerning the NP-cell interaction, it is experimentally challenging to attribute any findings to shape alone. Herein we demonstrate that shape, below a certain limit, which is specific to nanomedicine, is not important for the endocytosis of spherocylinders by either epithelial or macrophage cells in vitro. Through a systematic approach, we reshaped a single batch of gold nanorods into different aspect ratios resulting in near-spheres and studied their cytotoxicity, (pro-)inflammatory status, and endocytosis/exocytosis. It was found that on a length scale of ∼10-90 nm and at aspect ratios less than 5, NP shape has little impact upon their entry into either macrophages or epithelial cells. Conversely, nanorods with an aspect ratio above 5 were preferentially endocytosed by epithelial cells, whereas there was a lack of shape dependent uptake following exposure to macrophages in vitro. These findings have implications both in the understanding of nanoparticle reshaping mechanisms, as well as in the future rational design of nanomaterials for biomedical applications.

Synthesis, characterization, antibacterial activity and cytotoxicity of hollow TiO2-coated CeO2 nanocontainers encapsulating silver nanoparticles for controlled silver release.

Biomaterials as implants are being applied more extensively in medicine due to their on-going development and associated improvements, and the increase in human life expectancy. Nonetheless, biomaterial-related infections, as well as propagating bacterial resistance, remain significant issues. Therefore, there is a growing interest for silver-based drugs because of their efficient and broad-range antimicrobial activity and low toxicity to humans. Most newly-developed silver-based drugs have an extremely fast silver-ion release, increasing adverse biological impact to the surrounding tissue and achieving only short-term antimicrobial activity. Nanoencapsulation of these drugs is hypothesized as beneficial for controlling silver release, and thus is the aim of the present study. Initially, an amorphous or crystalline (anatase) titania (TiO2) coating was synthesized around silver nanoparticle-containing (AgNP) ceria (CeO2) nanocontainers using a sonication method forming AgNP/CeO2/TiO2 nanocontainers. These nanocontainers were characterized by high-resolution transmission electron microscopy, scanning electron microscopy, powder X-ray diffraction, gas sorption experiments and energy-dispersive X-ray spectroscopy. Silver release, monitored by using inductively coupled plasma optical emission spectroscopy, showed that these containers prevented silver release in water at neutral pH, and released the silver in concentrated nitric acid solution (pH = 1.1). The AgNP/CeO2/TiO2 nanocontainers showed an antibacterial activity against E. coli, however a concentration-dependent cytotoxicity towards a model epithelial barrier cell type (A549 cells) was observed. These nanocontainers offer the concept of potentially controlling silver delivery for the prevention of implant-associated infections.

Characterizing nanoparticles in complex biological media and physiological fluids with depolarized dynamic light scattering.

Light scattering is one of the few techniques available to adequately characterize suspended nanoparticles (NPs) in real time and in situ. However, when it comes to NPs in multicomponent and optically complex aqueous matrices - such as biological media and physiological fluids - light scattering suffers from lack of selectivity, as distinguishing the relevant optical signals from the irrelevant ones is very challenging. We meet this challenge by building on depolarized scattering: Unwanted signals from the matrix are completely suppressed. This approach yields information with an unprecedented signal-to-noise ratio in favour of the NPs and NP-biomolecule corona complexes, which in turn opens the frontier to scattering-based studies addressing the behaviour of NPs in complex physiological/biological fluids.

Integrating silver compounds and nanoparticles into ceria nanocontainers for antimicrobial applications.

Silver compounds and nanoparticles (NPs) are gaining increasing interest in medical applications, specifically in the treatment and prevention of biomaterial-related infections. However, the silver release from these materials, resulting in a limited antimicrobial activity, is often difficult to control. In this paper, ceria nanocontainers were synthesized by a template-assisted method and were then used to encapsulate silver nitrate (AgNO3/CeO2 nanocontainers). Over the first 30 days, a significant level of silver was released, as determined using inductively coupled plasma optical emission spectroscopy (ICP-OES). A novel type of ceria container containing silver NPs (AgNP/CeO2 containers) was also developed using two different template removal methods. The presence of AgNPs was confirmed both on the surface and in the interior of the ceria containers by X-ray diffraction (XRD), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Upon removal of the template by calcination, the silver was released over a period exceeding three months (>90 days). However, when the template was removed by dissolution, the silver release was shortened to ≤14 days. The antimicrobial activity of the silver-containing CeO2 containers was observed and the minimum bactericidal concentration (MBC) was determined using the broth dilution method. Investigation on human cells, using a model epithelial barrier cell type (A549 cells), highlighted that all three samples induced a heightened cytotoxicity leading to cell death when exposed to all containers in their raw form. This was attributed to the surface roughness of the CeO2 nanocontainers and the kinetics of the silver release from the AgNO3/CeO2 and AgNP/CeO2 nanocontainers. In conclusion, despite the need for further emphasis on their biocompatibility, the concept of the AgNP/CeO2 nanocontainers offers a potentially alternative long-term antibactericidal strategy for implant materials.

Novel peptide conjugates for tumor-specific chemotherapy.

One of the major problems in cancer chemotherapy are the severe side effects that limit the dose of the anticancer drugs because of their unselectivity for tumor versus normal cells. In the present work, we show that coupling of anthracyclines to peptides is a promising approach to obtain selectivity. The peptide-drug conjugate was designed to bind to specific receptors expressed on the tumor cells with subsequent internalization of the ligand-receptor complex. Neuropeptide Y (NPY), a 36-amino acid peptide of the pancreatic polypeptide family, was chosen as model peptide because NPY receptors are overexpressed in a number of neuroblastoma tumors and the thereof derived cell lines. Daunorubicin and doxorubicin, two widely used antineoplastic agents in tumor therapy, were covalently linked to NPY via two spacers that differ in stability: an acid-sensitive hydrazone bond at the 13-keto position of daunorubicin and a stable amide bond at the 3'-amino position of daunorubicin and doxorubicin. Receptor binding of these three conjugates ([C(15)]-NPY-Dauno-HYD, [C(15)]-NPY-Dauno-MBS, and [C(15)]-NPY-Doxo-MBS) was determined at the human neuroblastoma cell line SK-N-MC, which selectively expresses the NPY Y(1) receptor subtype, and cytotoxic activity was evaluated using a XTT-based colorimetric cellular cytotoxicity assay. The different conjugates were able to bind to the receptor with affinities ranging from 25 to 51 nM, but only the compound containing the acid-sensitive bond ([C(15)]-NPY-Dauno-HYD) showed cytotoxic activity comparable to the free daunorubicin. This cytotoxicity is Y(1) receptor-mediated as shown in blocking studies with BIBP 3226, because tumor cells that do not express NPY receptors were sensitive to free daunorubicin, but not to the peptide-drug conjugate. The intracellular distribution was investigated by confocal laser scanning microscopy. We found evidence that the active conjugate [C(15)]-NPY-Dauno-HYD releases daunorubicin, which is localized close to the nucleus, whereas the inactive conjugate [C(15)]-NPY-Dauno-MBS is distributed distantly from the nucleus and does not seem to release the drug within the cell.

Cell-cycle arrest and p53-independent induction of apoptosis in vitro by the new anticancer drugs 5-FdUrd-P-FdCydOct and dCydPam-P-FdUrd in DU-145 human prostate cancer cells.

PURPOSE: Current therapies have limited impact on the progression of metastatic hormone-refractory prostate cancer. Therefore, we investigated the utility of new heterodinucleoside phosphate dimers of 5-fluorodeoxyuridine (5-FdUrd) in p53-mutated and androgen-independent DU-145 human prostate tumour cells. METHODS: The effects of the dimers were assessed in vitro by a cell proliferation assay for cytotoxicity, flow cytometry for cell cycle distribution, confocal laser scanning microscopy for the detection of apoptotic bodies, poly(ADP-ribose) polymerase cleavage for caspase 3 activity and by a thymidylate synthetase assay. RESULTS: The new dimers N4-palmitoyl-2'-deoxycytidylyl-(3'-->5')-5-fluoro-2'-deoxyuridine (dCydPam-P-FdUrd) and 2'-deoxy-5-fluorouridylyl-(3'-->5')-2'-deoxy-5-fluoro-N4-octade cylcytidine (5-FdUrd-P-FdCydOct) caused marked cytotoxicity with IC50 values of 3-4 microM. 5-FdUrd-P-FdCydOct at 200 microM was capable of eradicating 100% of tumour cells whereas 10% of the cells were resistant to 5-FdUrd. Cytotoxicity was caused by a dramatic S-phase arrest, resulting in an increase of this cell population from 34% to 85% with 5-FdUrd-P-FdCydOct and to 81% with dCydPam-P-FdUrd. S-phase arrest was followed by apoptosis, as shown by 85% of the cells staining positive for Apo 2.7 antibody, a six- to eight-fold increased caspase 3 activity and DNA fragmentation. Thymidylate synthase activity was inhibited by 50% at 0.6-0.7 microM dimer concentration. The dimers were hydrolysed in vitro by phosphodiesterase I and human serum to the corresponding nucleosides and nucleoside monophosphates. CONCLUSIONS: The new dimers dCydPam-P-FdUrd and 5-FdUrd-P-FdCydOct are effective prodrugs of 5-FdUrd and have potential value for the treatment of p53-mutated and hormone-independent human prostate carcinomas.

Permeation and pathways of human calcitonin (hCT) across excised bovine nasal mucosa.

In vitro permeation of human calcitonin (hCT), salmon calcitonin (sCT), and the somatostatin analog octreotide (SMS) through excised bovine nasal mucosa was studied applying donor/receiver experiments and confocal laser scanning microscopy. Permeabilities of gonadorelin, buserelin, Hoe013, and of thymopoietin fragments TP5 and TP4 were also included. Apparent permeability coefficients (Peff) ranged between 4 x 10(-5) (SMS) and 1.7 x 10(-5) cm s(-1) (TP4). Such Peff are typical for leaky-type airway epithelia. The order of permeabilities was: SMS >> hCT, sCT > buserelin, Hoe013 >> TP5 > TP4, LHRH. The relatively high permeability of hCT and sCT contrasted to their high molecular weight. At 37 degrees C, the permeability of hCT from mucosal to serosal (m-to-s) was found two-fold higher (p < 0.05) than from serosal to mucosal (s-to-m). Controls using 3H-mannitol showed equal permeabilities in both directions. At 4 degrees C, permeation of hCT was reduced but equal in both directions (m-to-s and s-to-m). As evaluated by confocal laser scanning microscopy, uptake studies with FITC-18-hCT revealed intracellular fluorescence in the epithelial cells, at 10 min/10 microM exposure in the form of fluorescent vesicles. By combination of these findings, an endocytotic pathway is suggested to contribute to the transport of hCT through nasal epithelium.

Evaluation of particle uptake in human blood monocyte-derived cells in vitro. Does phagocytosis activity of dendritic cells measure up with macrophages?

This work focuses on microparticles as potential antigen delivery systems to target professional antigen-presenting cells. Surface modified polystyrene microparticles were administered to human-derived macrophages (MPhis) and dendritic cells (DCs) in vitro to evaluate the phagocytosis activity of each cell type. To discriminate between internalised particles and those closely attached to the outside of the cells, particle internalisation was verified by confocal laser scanning microscopy. Especially positively charged particles tend to stick to the outer cell membrane and may lead to false positive results when measured by conventional microscopy. In contrast, fluorescence microscopy in combination with an extracellular fluorescence quenching agent (trypan blue) allows the unequivocal assessment of particle uptake for screening purposes. For this assay, the fluorescent label needs to be in direct contact to the quenching agent and cannot be localised inside the particle core. Different types of microparticles varying in size, surface-material and zeta potential resulted in vast differences regarding their uptake by MPhis and DCs as well as the maturation of DCs. Negatively-charged carboxylated and bovine serum albumin-coated particles were phagocytosed by MPhis to a relatively small extent. Interestingly, phagocytosis of these particles was still significantly lower in DCs while positively charged poly-L-lysine (PLL) coated particles induced high phagocytosis activity in both cell types. By comparing our results with literature data, we conclude that phagocytosis activity of DCs and MPhis largely depends on particle size and surface charge and is also influenced by the character of bulk and coating material. PLL can be directed to DCs and MPhis with comparable efficiency and, in addition, induce maturation of DCs.

Structure-permeation relations of met-enkephalin peptide analogues on absorption and secretion mechanisms in Caco-2 monolayers.

Due to the low effective permeabilities of peptides at many absorption sites, their structure-permeation relations are of high interest. In this work structure-permeation relations of Met-enkephalin analogues are presented using confluent Caco-2 cells as an in vitro permeation model. Four model peptides (Met-enkephalin, [D-Ala2]Met-enkephalin, [D-Ala2]Met-enkephalinamide, and metkephamid) were tested in terms of permeability, lipophilicity, charge, and molecular size. Permeability coefficients (P(eff)) across Caco-2 cells were low, 3.3 x 10(-8) to 9.5 x 10(-8) cm s-1, and were similar to typical paracellular markers. No correlation of permeability and the log(apparent octanol/buffer partition coefficient) was observed. A 40-fold increase of the permeability of metkephamid in the presence of 10 mM EDTA suggested a significant contribution of paracellular transport. Independent support for this conclusion was obtained by visualizing the pathway of the fluorescein isocyanate isomer I 1-metkephamid by confocal laser scanning microscopy (CLSM). The fluorophore-labeled peptide was observed in the intercallular space only. Metkephamid permeabilities were found to be direction-specific. Permeabilities from basolateral to apical (b-to-a) were significantly higher (ca. 4-fold) than in the opposite (a-to-b) direction. The addition of verapamil equalized the permeabilities in the a-to-b and b-to-a directions, suggesting the involvement of a P-glycoprotein-mediated secretion mechanism. Similar observations were obtained with [D-Ala2]Met-enkephalinamide, but not with Met-enkephalin and [D-Ala2]Met-enkephalin. In contrast to the other analogues, metkephamid and [D-Ala2]Met-enkephalinamide are positively charged at neutral pH, as demonstrated by their isoelectric points (pl = 8.6 for [D-Ala2]Met-enkephalinamide and metkephamid and 5.3 for [D-Ala2]Met-enkephalin and Met-enkephalin). The data is in agreement with the literature showing that most compounds secreted by the P-glycoprotein transporter carry a positive charge.

Phenotypic characterization of human umbilical vein endothelial (ECV304) and urinary carcinoma (T24) cells: endothelial versus epithelial features.

ECV304 cells reported as originating from human umbilical vein endothelial cells by spontaneous transformation have been used as a model cell line for endothelia over the last decade. Recently, deoxyribonucleic acid fingerprinting revealed an identical genotype for ECV304 and T24 cells (urinary bladder carcinoma cell line). In order to resolve the apparent discrepancy between the identical genotype and the fact that ECV304 cells phenotypically show important endothelial characteristics, a comparative study was performed. Immortalized porcine brain microvascular endothelial cells/C1-2, and Madin Darby canine kidney cells were included as typical endothelial and epithelial cells, respectively. Various methods, such as confocal laser scanning microscopy. Western blot, and protein activity tests, were used to study the cell lines. ECV304 and T24 cells differ in criteria, such as growth behavior, cytoarchitecture, tight junction arrangement. transmembrane electrical resistance, and activity of gamma-glutamyltransferase. Several endothelial markers (von Willebrand factor, uptake of low-density lipoprotein, vimentin) could clearly be identified in ECV304, but not in T24 cells. Desmoglein and cytokeratin, both known as epithelial markers, were found in ECV304 as well as in T24 tells. However, differences were found for the two cell lines with respect to the type of cytokeratin: in ECV304 cells mainly cytokeratin 18 (45 kDa) is found, whereas in T24 cells cytokeratin 8 (52 kDa) is predominant. As we could demonstrate, the ECV304 cell line exposes many endothelial features which, in view of the scarcity of suitable endothelial cell lines, still make it an attractive in vitro model for endothelia.

P-Glycoprotein in cell cultures: a combined approach to study expression, localisation, and functionality in the confocal microscope.

Madin Darby canine kidney (MDCK) cells transfected with the multidrug resistance mdr1 gene, MDR1-MDCK (Pastan et al., 1988, Proc. Natl. Acad. Sci. USA 85 4486-4470), were used in a combined approach to study expression, localisation and functionality of the P-glycoprotein (P-gp) membrane transporter in the same cell culture preparations. Cells were characterised with regard to their growth curve, transepithelial electrical resistance (TEER), and cytoarchitecture. Efflux of the P-gp substrate rhodamine123 (rho123) was monitored with confocal laser scanning microscopy (CLSM). The transfected cells grew in multilayers. After reaching confluence they exhibited a complete tight junction (TJ) network. P-gp was strongly expressed at the uppermost apical surface of the multilayer already after 4 days in culture. The lower cell layers were not clearly polarised. P-gp-mediated transport could be followed by efflux of the fluorescent rho123 from the cells into the apical extracellular space. Verapamil, a P-gp inhibitor, significantly decreased efflux. For MDCK parent cells the rho123 assay was negative up to about day 20, and only at later times (day 25) low P-gp activity was detected. These results clearly show that despite the fact that the transfected cells form irregular layers, they provide a good model for screening of P-gp substrates and inhibitors.

Cell cultures as tools in biopharmacy.

A survey is given on a few selected cell culture models that are used for transport studies. They are characterised for growth, transcellular electrical resistance and cytoarchitecture. The importance of standardisation in view of their use as transport models is documented. Their potential for studies on passive permeation and P-glycoprotein-mediated transport is explored and related to published data. Transport studies are presented that were performed in a two-chamber set-up, the Costar "vertical diffusion system". A series of non-homologous compounds showed similar permeability data (P(app)) in the different cell cultures. The origin of the cell type had no remarkable influence on passive transcellular permeation. MDCK cells, an epithelial cell line of canine kidney origin, are perfectly suited to screen for passive permeation. They have low expression of transporter proteins and low metabolic activity. In general, they probably represent the best-known epithelial cell line with respect to genetics as well as lipid and protein composition. MDCK cells are easy to handle. Transport experiments can be done between 7 and 14 days after seeding, when the stationary growth phase is reached. To screen for P-glycoprotein substrates, efflux and uptake studies were performed with mdr1-transfected MDCK cells (MDR1-MDCK) in a one-chamber system in the presence or absence of verapamil or cyclosporin A as inhibitor. Evidence is presented why the transfected cells, which express large amounts of P-glycoprotein, are not suitable for two-chamber transport studies.

Function and immunolocalization of overexpressed human intestinal H+/peptide cotransporter in adenovirus-transduced Caco-2 cells.

PURPOSE: To determine the localization of the human intestinal H+/peptide cotransporter (hPepT1) and its function in intestinal epithelial cells after adenoviral transduction. METHODS: Caco-2 cells grown on Transwell membrane filters were transduced with a recombinant replication-deficient adenovirus carrying the hPepT1 gene. The transport of Gly-Sar across both apical and basolateral membranes was measured after adenoviral transduction as a function of pH, temperature, inhibitors, and substrate concentration. The localization of hPepT1 was examined by immunocytochemistry using confocal laser scanning microscopy. RESULTS: The apical-to-basolateral and basolateral-to-apical transport of Gly-Sar in Caco-2 cells after viral transduction was increased 3.3 and 3.5-fold, respectively. The similar magnitude of Gly-Sar permeability from either direction indicates involvement of identical transport pathways in both membranes. This was further confirmed by immunocytochemistry showing that hPepT1 was localized in the apical and basolateral membrane of Caco-2 cells after adenoviral transduction. In both directions, Gly-Sar transport was enhanced in the presence of a pH gradient. In addition, the basolateral-to-apical Gly-Sar transport was dependent on temperature, multiplicity of infection (MOI), and Gly-Sar concentration. It was inhibited in the presence of excess Gly-Pro and cephalexin. CONCLUSIONS: Caco-2 cell monolayers represent an appropriate model to study gene expression in intestinal epithelial cells. Transport characteristics of Gly-Sar from the basolateral to the apical side in adenovirus-transduced Caco-2 cells are in agreement with those from the apical to the basolateral side, indicating that hPepT1 is also expressed in the basolateral membrane and displays a similar level of transport enhancement after adenovirus mediated hPepT1 gene expression.

In vitro dosimetry of agglomerates.

Agglomeration of nanoparticles in biological fluids is a pervasive phenomenon that leads to difficulty in the interpretation of results from in vitro exposure, primarily due to differing particokinetics of agglomerates to nanoparticles. Therefore, well-defined small agglomerates were designed that possessed different particokinetic profiles, and their cellular uptake was compared to a computational model of dosimetry. The approach used here paves the way for a better understanding of the impact of agglomeration on the nanoparticle-cell interaction.