MDCK Cell Monolayers Research Articles

Morphological Features and In Vitro Cytopathic Effect of Acanthamoeba griffini Trophozoites Isolated from a Clinical Case.

Light and transmission electron microscopy observations are reported on the structure and in vitro cytopathic effect of Acanthamoeba griffini trophozoites isolated from a clinical case. Live trophozoites were moderately active with a remarkable pleomorphism which changed from ovoid to quite elongated shapes. When moving, amoebae formed cytoplasmic projections such as wide lamellae and acanthopodia of diverse size and thickness which contain a significant amount of actin. Ultrastructurally, the cytoplasm showed the main organelles found in other free-living amoebae. Coincubation of trophozoites with MDCK cell monolayers resulted in a local damage to target cells after 24 h of interaction, suggesting that the cytopathic effect is contact-dependent. By transmission electron microscopy, amoebae appeared to engulf small portions of the MDCK cells; however, the cells that were not in contact with trophozoites had an unaltered morphology. When epithelial monolayers were incubated with conditioned medium for 24 h, small areas of cell injury were also observed. The phylogenetical analysis as well as the sequencing of the acquired amplified product for the DF3 region of the amoebae isolate confirmed that it belongs to genotype T3, which includes other pathogenic amoebae; besides the activity of two drugs currently used against Acanthamoeba was tested on A. griffini.

Enhancement effect of P-gp inhibitors on the intestinal absorption and antiproliferative activity of bestatin

Bestatin is an immunomodulator with antitumor activity. This study was performed to investigate the effect of P-gp on the intestinal absorption and antiproliferative activity of bestatin. Our results showed that P-gp inhibitors significantly increased rat intestinal absorption of bestatin in vivo and in vitro. The net efflux ratio of bestatin was 2.2 across mock-/MDR1–MDCK cell monolayers and was decreased by P-gp inhibitors, indicating bestatin was a substrate of P-gp. Furthermore, the IC50 values of bestatin on U937 and K562 cells were decreased dramatically and the intracellular concentrations of bestatin were increased by incubation of cells with verapamil or Cyclosporin A. K562/ADR cells exhibited a higher IC50 value and a lower intracellular level of bestatin. The bestatin level in K562/ADR cells was partially restored by incubation with doxorubicin. However, P-gp and APN mRNA levels were not changed by bestatin. These results suggested that the intestinal absorption and accumulation in cancer cells for bestatin were limited by P-gp-mediated efflux. Additional attention should be paid to the alternative exposure of bestatin when bestatin was coadministered with drugs as P-gp substrates in clinic.

The Cytokine Response of U937-Derived Macrophages Infected through Antibody-Dependent Enhancement of Dengue Virus Disrupts Cell Apical-Junction Complexes and Increases Vascular Permeability

Severe dengue (SD) is a life-threatening complication of dengue that includes vascular permeability syndrome (VPS) and respiratory distress. Secondary infections are considered a risk factor for developing SD, presumably through a mechanism called antibody-dependent enhancement (ADE). Despite extensive studies, the molecular bases of how ADE contributes to SD and VPS are largely unknown. This work compares the cytokine responses of differentiated U937 human monocytic cells infected directly with dengue virus (DENV) or in the presence of enhancing concentrations of a humanized monoclonal antibody recognizing protein E (ADE-DENV infection). Using a cytometric bead assay, ADE-DENV-infected cells were found to produce significantly higher levels of the proinflammatory cytokines interleukin 6 (IL-6), IL-12p70, and tumor necrosis factor alpha (TNF-α), as well as prostaglandin E2 (PGE2), than cells directly infected. The capacity of conditioned supernatants (conditioned medium [CM]) to disrupt tight junctions (TJs) in MDCK cell cultures was evaluated. Exposure of MDCK cell monolayers to CM collected from ADE-DENV-infected cells (ADE-CM) but not from cells infected directly led to a rapid loss of transepithelial electrical resistance (TER) and to delocalization and degradation of apical-junction complex proteins. Depletion of either TNF-α, IL-6, or IL-12p70 from CM from ADE-DENV-infected cells fully reverted the disrupting effect on TJs. Remarkably, mice injected intraperitoneally with ADE-CM showed increased vascular permeability in sera and lungs, as indicated by an Evans blue quantification assay. These results indicate that the cytokine response of U937-derived macrophages to ADE-DENV infection shows an increased capacity to disturb TJs, while results obtained with the mouse model suggest that such a response may be related to the vascular plasma leakage characteristic of SD.

Reevaluating the role of Acanthamoeba proteases in tissue invasion: observation of cytopathogenic mechanisms on MDCK cell monolayers and hamster corneal cells.

The morphological analysis of the cytopathic effect on MDCK cell monolayers and hamster cornea and qualitative and quantitative analyses of conditioned medium and proteases were evaluated and compared between two strains of Acanthamoeba genotype T4. Further than highlighting the biological differences found between both strains, the most important observation in this study was the fact that proteases both in total extracts and in conditioned medium are apparently not determinant in tissue destruction. An interestingly finding was that no lysis of corneal tissue was observed as it was previously suggested. These results, together with previous studies, allow us to conclude that the invasion and disruption of corneal tissue is performed by the penetration of the amoebae through cell junctions, either by the action of proteases promoting cellular separation but not by their destruction and/or a mechanical effect exerted by amoebae. Therefore, contact-dependent mechanisms in Acanthamoeba pathogenesis are more relevant than it has been previously considered. This is supported because the phagocytosis of recently detached cells as well as those attached to the corneal epithelium leads to the modification of the cellular architecture facilitating the migration and destruction of deeper layers of the corneal epithelium.

The Serological and Virological Investigation of Canine Adenovirus Infection on the Dogs

Two types of Canine Adenovirus (CAVs), Canine Adenovirus type 1 (CAV-1), the virus which causes infectious canine hepatitis, and Canine Adenovirus type 2 (CAV-2), which causes canine infectious laryngotracheitis, have been found in dogs. In this study, blood samples taken from 111 dogs, which were admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, with clinical symptoms. Seventy-seven dogs were sampled from Isparta and Burdur dog shelters by random sampling, regardless of the clinical findings. Dogs showed a systemic disease, characterized by fever, diarrhea, vomiting, oculonasal discharge, conjunctivitis, severe moist cough, signs of pulmonary disease and dehydration. Two dogs had corneal opacity and photophobia. In serological studies, 188 serum samples were investigated on the presence of CAV antibodies by ELISA. Total 103 (103/188–54.7%) blood samples were detected to be positive for CAV antibodies by ELISA. However, 85 (85/188–45.2%) blood samples were negative. Blood leukocyte samples from dogs were processed and inoculated onto confluent monolayers of MDCK cells using standard virological techniques. After third passage, cells were examined by direct immunoflourescence test for virus isolation. But positive result was not detected. In conclusion, this study clearly demonstrates the high prevalence of CAV infection in dogs.

Combination of vitamin C and zinc gluconate prevented vanadium-induced tight junction leakage of MDCK cell monolayer

Combination of vitamin C and zinc gluconate prevented vanadium-induced tight junction leakage of MDCK cell monolayer

Acute Activation of the Renal Betaine/GABA Transporter in Response to a Decrease in Extracellular Calcium

The betaine/GABA transporter (BGT1) is important for osmoprotection in kidney medullary cells. We previously reported an acute (30 min) increase in extracellular Ca2+caused dose dependent inhibition of BGT-1 in renal MDCK cells. To determine if extracellular Ca2+might be a local regulator of BGT-1, we have tested the response to low Ca2+serum-free growth medium (LCM, 0.05 mM Ca2+). Chronic treatment (8–24 h) of MDCK cell monolayers completely blocked hypertonic adaptation of BGT1 and disrupted tight junctions. In contrast, acute treatment activated BGT1 transport within 30 min in MDCK cells previously adapted to hypertonic growth medium containing normal Ca2+(1.6 mM). Activation was significant after 60–90 min and was independent of medium osmolarity. Peak transport was increased 50% in isotonic LCM and 100% in hypertonic (500 mOsm) LCM over controls. The activation was reversed by restoration of normal Ca2+. Perfusion of Fura-2-loaded MDCK cells with LCM decreased intracellular Ca2+by 31% within 6-7 min. Inclusion of staurosporine (0.6 μM), a protein kinase C inhibitor, potentiated the action of LCM. We suggest that activation of BGT1 by LCM may be due in part to inhibition of protein kinase C.

Cilia develop long-lasting contacts, with other cilia

Cilia develop long-lasting contacts, with other cilia

291 Development and Testing of a Novel Fluorinated Bile Acid for Detection of Bile Acid Malabsorption by Magnetic Resonance Imaging (MRI)

Bile Acid Malabsorption (BAM) resulting from bile acid overproduction or reduced transport can increase fecal bile acids and cause chronic diarrhea. BAM is thought to account for up to 30% of patients diagnosed with irritable bowel syndrome (IBS-D). Current methods of diagnosing BAM are limited; it is likely under-diagnosed. Our objective was to develop a non-invasive method of detecting BAM by synthesizing and testing a multi-fluorinated bile acid (MFBA) that can be used for fluorine magnetic resonance imaging (MRI) of bile acid enterohepatic circulation. A prototype MFBA was synthesized by conjugating trifluoroacetyl lysine to cholic acid (19F-CA-lys). This MFBA was tested for transport and inhibition of the apical sodium dependent bile acid transporter (ASBT) using stably transfected MDCK cell monolayers and of Na+/taurocholate cotransporting polypeptide (NTCP) using stably transfected HEK cell monolayers. In Vitro, 19F-CA-lys was a potent inhibitor and substrate of both ASBT (Ki = 20.0±3.9 μM, Kt = 39.4±23.8 μM, normalized Vmax =0.853 ± 0.197) and NTCP (Ki = 2.93±0.60 μM, Kt = 8.99 ± 2.79 μM, normalized Vmax = 0.281±0.052). Stability of 19F-CA-lys (5 μM) was measured after treatment with Caco-2 cell homogenates, rat plasma, rat liver microsomal S9 pools, simulated intestinal fluid with pancreatic enzymes, 1 M HCl, buffers at pH 7.4 and 6.8, and choloylglycine hydrolase (CGH) (N=3 for each condition). Except for CGH, 19F-CA-lys was stable in each condition. After 1 h in CGH, 60.4±4.0% 19F-CA-lys remained compared to 2.4±0.5% naturally-occurring taurocholic acid (P<0.005). To test In Vivo stability and utility of 19F-CA-lys for MRI, we examined its distribution in 8 male C57BL/6 mice (average weight, 25.7 g). After overnight fasting, mice were gavaged with 1:1 polyethylene glycol (PEG):water vehicle control (2 mice), 150 mg/ kg 19F-CA-lys (3 mice) and 300 mg/kg 19F-CA-lys (3 mice). Four hours after dosing, 19FCA-lys in gallbladder, liver and plasma was measured by LC/MS. 19F-CA-lys was not detected in control mice receiving vehicle alone. With 150 and 300 mg/kg 19F-CA-lys, 4 h gallbladder 19F-CA-lys concentrations were 27.0±2.5 and 36.8±8.8 μmol/g, liver concentrations were 0.079±0.035 and 0.057±0.010 μmol/g, and plasma concentrations were 0.444±0.170 and 0.418 ± 0.015 μM, respectively. We calculated that at both doses, 10.8±1.9% (95% CI, 5.915.8%) of administered 19F-CA-lys was present in the gallbladder. We conclude that 19FCA-lys, a novel MFBA, is stable In Vitro and In Vivo, and accumulates in the gallbladder at concentrations that will provide a 19F signal in excess of that we previously determined to be the minimum detectable by 19F MRI (Drug Metab. Disp. 39:736, 2011). These findings suggest that non-radioactive MFBAs have excellent potential to measure In Vivo bile acid transport and serve as clinical imaging tools to diagnosis BAM.

The &lt;I&gt;In Vitro&lt;/I&gt; and &lt;I&gt;In Vivo&lt;/I&gt; Study on Self-Nanoemulsifying Drug Delivery System (SNEDDS) Based on Insulin-Phospholipid Complex

Self-nanoemulsifying drug delivery system (SNEDDS) was developed to enhance the absorption of insulin after oral administration, where insulin was complexed with phospholipid to achieve a better liposolubility in the form of insulin-phospholipid complex (IPC). IPC was formulated into the oil phase of SNEDDS by solvent-evaporation method. The formula of IPC-SNEDDS was optimized and characterized. Data showed that SNEDDS as a drug vehicle did not exhibit an obvious inhibition over MDCK cells, and it can facilitate the transport of IPC across MDCK cell monolayer. IPC-SNEDDS could enhance the absorption of insulin after oral administration and yielded a pronounced hypoglycemic effect on diabetic Wistar rats. All these suggested that IPC-SNEDDS has a great potential for oral delivery of insulin.

Antibodies to Plasmodium circumsporozoite protein (CSP) inhibit sporozoite's cell traversal activity

Plasmodium sporozoites are deposited in the skin of the mammalian host by Anopheles mosquitoes. To continue the life cycle, the sporozoites have to invade the host's hepatocytes, where they transform into exoerythrocytic forms (EEFs) inside a parasitophorous vacuole. During their route from the skin to the liver, the parasites traverse the capillary epithelium in the dermis to enter the blood circulation, and cross the endothelium of liver sinusoids to enter the parenchyma. Cell traversal by sporozoites is usually measured by quantifying dyes that enter or are released from cells during incubation with salivary gland sporozoites. These methods do not distinguish cell traversal from cell wounding. Here we validate an assay that quantifies cell traversal of sporozoites through monolayers of MDCK cells that form tight junctions. We compared cell traversal of wt sporozoites and of parasites lacking the Type I membrane protein TLP (TRAP-like protein) previously implicated in cell traversal. We provide direct evidence that TLP ko sporozoites are defective in cell traversal and that they are retained inside the MDCK cytoplasm. We then used the MDCK assay to study the effect of a monoclonal antibody (3D11) to the circumsporozoite protein (CSP) on the parasite's cell traversal. We show that 3D11 inhibits cell traversal at nanomolar concentrations. We conclude that antibodies elicited by CSP-based vaccines are likely to inhibit the migration of sporozoites from the skin to the liver.

Tubulin detyrosination promotes monolayer formation and apical trafficking in epithelial cells

The role of post-translational tubulin modifications in the development and maintenance of a polarized epithelium is not well understood. We studied the balance between detyrosinated (detyr-) and tyrosinated (tyr-) tubulin in the formation of MDCK cell monolayers. Increased quantities of detyrosinated microtubules were detected during assembly into confluent cell sheets. These tubules were composed of alternating stretches of detyr- and tyr-tubulin. Constant induction of tubulin tyrosination, which decreased the levels of detyr-tubulin by overexpression of tubulin tyrosine ligase (TTL), disrupted monolayer establishment. Detyr-tubulin-depleted cells assembled into isolated islands and developed a prematurely polarized architecture. Thus, tubulin detyrosination is required for the morphological differentiation from non-polarized cells into an epithelial monolayer. Moreover, membrane trafficking, in particular to the apical domain, was slowed down in TTL-overexpressing cells. This effect could be reversed by TTL knockdown, which suggests that detyr-tubulin-enriched microtubules serve as cytoskeletal tracks to guide membrane cargo in polarized MDCK cells.

Dose-independent pharmacokinetics of a new peroxisome proliferator-activated receptor-γ agonist, KR-62980, in Sprague-Dawley rats and ICR mice

The pharmacokinetics of a novel peroxisome proliferator-activated receptor-γ agonist, KR-62980, were characterized in vitro with respect to liver metabolic stability, cell permeability, and plasma protein binding and in vivo using Sprague-Dawley rats and ICR mice. The metabolic half-life of 0.1-10 μM KR-62980 was 11.5-15.2 min in rat liver microsomes and 25.8-28.8 min in human liver microsomes. KR-62980 showed high permeability across MDCK cell monolayers, with apparent permeability coefficients of 20.4 × 10(-6) to 30.8 × 10(-6) cm/sec. The plasma protein binding rate of KR-62980 was 89.4%, and most was bound to serum albumin. After intravenous administration of KR-62980 (2 mg/kg), the systemic clearance was 2.50 L/h/kg, and the volume of distribution at steady-state was 9.16 L/kg. The bioavailability after oral administration was approximately 60.9%. The dose-normalized AUC values were 0.50 ± 0.09, 0.41 ± 0.20, and 0.62 ± 0.08 h · μg/mL after oral administration of 2, 5, and 10 mg/kg KR-62980, respectively, showing no dose-dependency. The in vivo pharmacokinetic parameters in ICR mice were also dose independent. These data suggest that KR-62980 is not significantly dose dependent in rats or mice, although it may disappear rapidly from the systemic circulation via metabolism in the liver.

Myosin II activity dependent and independent vinculin recruitment to the sites of E-cadherin-mediated cell-cell adhesion

BackgroundMaintaining proper adhesion between neighboring cells depends on the ability of cells to mechanically respond to tension at cell-cell junctions through the actin cytoskeleton. Thus, identifying the molecules involved in responding to cell tension would provide insight into the maintenance, regulation, and breakdown of cell-cell junctions during various biological processes. Vinculin, an actin-binding protein that associates with the cadherin complex, is recruited to cell-cell contacts under increased tension in a myosin II-dependent manner. However, the precise role of vinculin at force-bearing cell-cell junctions and how myosin II activity alters the recruitment of vinculin at quiescent cell-cell contacts have not been demonstrated.ResultsWe generated vinculin knockdown cells using shRNA specific to vinculin and MDCK epithelial cells. These vinculin-deficient MDCK cells form smaller cell clusters in a suspension than wild-type cells. In wound healing assays, GFP-vinculin accumulated at cell-cell junctions along the wound edge while vinculin-deficient cells displayed a slower wound closure rate compared to vinculin-expressing cells. In the presence of blebbistatin (myosin II inhibitor), vinculin localization at quiescent cell-cell contacts was unaffected while in the presence of jasplakinolide (F-actin stabilizer), vinculin recruitment increased in mature MDCK cell monolayers.ConclusionThese results demonstrate that vinculin plays an active role at adherens junctions under increased tension at cell-cell contacts where vinculin recruitment occurs in a myosin II activity-dependent manner, whereas vinculin recruitment to the quiescent cell-cell junctions depends on F-actin stabilization.

Structure–Activity Relationships of 4-Position Diamine Quinoline Methanols as Intermittent Preventative Treatment (IPT) against Plasmodium falciparum

A library of diamine quinoline methanols were designed based on the mefloquine scaffold. The systematic variation of the 4-position amino alcohol side chain led to analogues that maintained potency while reducing accumulation in the central nervous system (CNS). Although the mechanism of action remains elusive, these data indicate that the 4-position side chain is critical for activity and that potency (as measured by IC(90)) does not correlate with accumulation in the CNS. A new lead compound, (S)-1-(2,8-bis(trifluoromethyl)quinolin-4-yl)-2-(2-(cyclopropylamino)ethylamino)ethanol (WR621308), was identified with single dose efficacy and substantially lower permeability across MDCK cell monolayers than mefloquine. This compound could be appropriate for intermittent preventative treatment (IPTx) indications or other malaria treatments currently approved for mefloquine.

Characterization of in vivo metabolites of WR319691, a novel compound with activity against Plasmodium falciparum

WR319691 has been shown to exhibit reasonable Plasmodium falciparum potency in vitro and exhibits reduced permeability across MDCK cell monolayers, which as part of our screening cascade led to further in vivo analysis. Single-dose pharmacokinetics was evaluated after an IV dose of 5 mg/kg in mice. Maximum bound and unbound brain levels of WR319691 were 97 and 0.05 ng/g versus approximately 1,600 and 3.2 ng/g for mefloquine. The half-life of WR319691 in plasma was approximately 13 h versus 23 h for mefloquine. The pharmacokinetics of several N-dealkylated metabolites was also evaluated. Five of six of these metabolites were detected and maximum total and free brain levels were all lower after an IV dose of 5 mg/kg WR319691 compared to mefloquine at the same dose. These data provide proof of concept that it is feasible to substantially lower the brain levels of a 4-position modified quinoline methanol in vivo without substantially decreasing potency against P. falciparum in vitro.

Hetero-oligomerization of Neuronal Glutamate Transporters

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo- and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.

Shear Stress Reverses Dome Formation in Confluent Renal Tubular Cells

Background/Aims. It has been shown that MDCK cells, a cell line derived from canine renal tubules, develop cell domes due to fluid pumped under cell monolayer and focal detachment from the adhesion surface. In vitro studies have shown that primary cilia of kidney tubular epithelial cells act as mechanosensors, increasing intracellular calcium within seconds upon changes in fluid shear stress (SS) on cell membrane. We then studied the effect of prolonged SS exposure on cell dome formation in confluent MDCK cell monolayers. Methods. A parallel plate flow chamber was used to apply laminar SS at 2 dynes/cm² to confluent cell monolayers for 6 hours. Control MDCK cell monolayers were maintained in static condition. The effects of Ca²⁺ blockade and cell deciliation on SS exposure were also investigated. Results. Seven days after reaching confluence, static cultures developed liquid filled domes, elevating from culture plate. Exposure to SS induced almost complete disappearance of cell domes (0.4±0.8 vs. 11.4±2.8 domes/mm², p < 0.01, n=14). SS induced dome disappearance took place within minutes to hours, as shown by time-lapse videomicroscopy. Exposure to SS importantly affected cell cytoskeleton altering actin stress fibers expression and organization, and the distribution of tight junction protein ZO-1. Dome disappearance induced by flow was completely prevented in the presence of EGTA or after cell deciliation. Conclusions. These data indicate that kidney tubular cells are sensitive to apical flow and that these effects are mediated by primary cilia by regulation of Ca²⁺ entry in to the cell. SS induced Ca²⁺ entry provokes contraction of cortical actin ring that tenses cell-cell borders and decreases basal stress fibers. These processes may increase paracellular permeability and decrease basal adhesion making dome disappear. Elucidation of the effects of apical fluid flow on tubular cell function may open new insights on the pathophysiology of kidney diseases associated with cilia dysfunction.

Hemolytic anemia and distal renal tubular acidosis in two Indian patients homozygous for SLC4A1/AE1 mutation A858D

Familial distal renal tubular acidosis (dRTA) can be caused by mutations in the Cl2/HCO32 exchanger of the renal Type A intercalated cell, kidney AE1/SLC4A1. dRTA-associated AE1 mutations have been reported in families from North America, Europe, Thailand, Malaysia, Papua-New Guinea, Taiwan, and the Philippines, but not India. The dRTA mutation AE1 A858D has been detected only in the context of compound heterozygosity. We report here two unrelated Indian patients with combined hemolytic anemia and dRTA who share homozygous A858D mutations of the AE1/SLC4A1 gene. The mutation creates a novel restriction site that is validated for diagnostic screening.

Nanoparticulate Formulations for Paclitaxel Delivery Across MDCK Cell Monolayer

Paclitaxel appears to be a potential substrate of the multidrug resistance protein p-glycoprotein, thus preventing itself from entry into the brain and penetrating blood-brain barrier poorly. In this study, the main objective was to design paclitaxel formulation using PLGA-based nanoparticles with different additives and surface coatings to facilitate the paclitaxel transport through MDCK cell monolayer. PLGA-based nanoparticles of around 200 nm without and with additives and surface coatings were developed by direct dialysis. The transendothelial electrical resistance (TEER) variation of MDCK cell monolayer on the cell inserts imposed by paclitaxel-loaded nanoparticles with and without additives was investigated. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) (MTT) assay was used to quantify the cell viability of C6 glioma cells after administration of formulations on the topical side. Investigations showed that particles with additives were able to enhance cellular uptake more than surface-coated particles. TEER values dropped upon the introduction of paclitaxel-loaded PLGA nanoparticles to the cell inserts. After incubation for 24 h, the cell viability of C6 glioma cells in the wells treated with nanoparticles was lower than that of the control wells without particles. Taken together, PLGA nanoparticles with vitamin E TPGS and polysorbate 80 as additives were successfully obtained as paclitaxel formulations, demonstrating great potential for the delivery of paclitaxel through MDCK cell monolayer in vitro.