Developmental toxicity of low generation PAMAM dendrimers in zebrafish

Biological molecules and intracellular structures operate at the nanoscale; therefore, development of nanomedicines shows great promise for the treatment of disease by using targeted drug delivery and gene therapies. PAMAM dendrimers, which are highly branched polymers with low polydispersity and high functionality, provide an ideal architecture for construction of effective drug carriers, gene transfer devices and imaging of biological systems. For example, dendrimers bioconjugated with selective ligands such as Arg-Gly-Asp (RGD) would theoretically target cells that contain integrin receptors and show potential for use as drug delivery devices. While RGD-conjugated dendrimers are generally considered not to be cytotoxic, there currently exists little information on the risks that such materials pose to human health. In an effort to compliment and extend the knowledge gleaned from cell culture assays, we have used the zebrafish embryo as a rapid, medium throughput, cost-effective whole-animal model to provide a more comprehensive and predictive developmental toxicity screen for nanomaterials such as PAMAM dendrimers. Using the zebrafish embryo, we have assessed the developmental toxicity of low generation (G3.5 and G4) PAMAM dendrimers, as well as RGD-conjugated forms for comparison. Our results demonstrate that G4 dendrimers, which have amino functional groups, are toxic and attenuate growth and development of zebrafish embryos at sublethal concentrations; however, G3.5 dendrimers, with carboxylic acid terminal functional groups, are not toxic to zebrafish embryos. Furthermore, RGD-conjugated G4 dendrimers are less potent in causing embryo toxicity than G4 dendrimers. RGD-conjugated G3.5 dendrimers do not elicit toxicity at the highest concentrations tested and warrant further study for use as a drug delivery device.     

Transport of Poly(Amidoamine) Dendrimers across Caco-2 Cell Monolayers: Influence of Size, Charge and Fluorescent Labeling

Purpose To investigate the transport of poly(amidoamine) (PAMAM) dendrimers with positive, neutral and negatively charged surface groups across Caco-2 cell monolayers. Methods Cationic PAMAM-NH₂ (G2 and G4), neutral PAMAM-OH (G2), and anionic PAMAM-COOH (G1.5–G3.5) dendrimers were conjugated to fluorescein isothiocyanate (FITC). The permeability of fluorescently labeled PAMAM dendrimers was measured in the apical-to-basolateral direction. ¹⁴C-Mannitol permeability was measured in the presence of unlabeled and FITC labeled PAMAM dendrimers. Caco-2 cells were incubated with the dendrimers followed by mouse anti-occludin or rhodamine phalloidin, and visualized using confocal laser scanning microscopy to examine tight junction integrity. Results The overall rank order of PAMAM permeability was G3.5COOH > G2NH₂ > G2.5COOH > G1.5COOH > G2OH. ¹⁴C-Mannitol permeability significantly increased in the presence of cationic and anionic PAMAM dendrimers with significantly greater permeability in the presence of labeled dendrimers compared to unlabeled. PAMAM dendrimers had a significant influence on tight junction proteins occludin and actin, which was microscopically evidenced by disruption in the occludin and rhodamine phalloidin staining patterns. Conclusions These studies demonstrate that enhanced PAMAM permeability is in part due to opening of tight junctions, and that by appropriate engineering of PAMAM surface chemistry it is possible to increase polymer transepithelial transport for oral drug delivery applications.     

Transport of Surface Engineered Polyamidoamine (PAMAM) Dendrimers Across IPEC-J2 Cell Monolayers

The aim of our study was to prepare arginine-and ornithine-conjugated Polyamidoamine (PAMAM) dendrimers and study their permeability across IPEC-J2 cell monolayers, a new intestinal cell line model for drug absorption studies. Arginine and ornithine were conjugated to the amine terminals of the PAMAM_G4 dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P_app) for the PAMAM dendrimers increased by conjugating the dendrimers with both of these polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. Correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of these surface-modified dendrimers was evaluated in IPEC-J2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were insignificantly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer-polyamine conjugates as the carriers for antigen/drug delivery through the oral mucosa.     

聚酰胺-胺PAMAM树状聚合物对灯盏花素的溶解性及其药代动力学的影响

测定聚酰胺-胺PAMAM树状聚合物对灯盏花素的增溶性能并探讨其增溶机制; 研究PAMAM树状聚合物对灯盏花素体内药代动力学的影响。测定和比较了G1、G1.5、G2、G2.5代PAMAM在不同浓度和不同pH时对灯盏花素的增溶量; 另将12只大鼠随机均分为2组, 每组6只, 分别以灯盏花素及灯盏花素-PAMAM灌胃, 采用反相高效液相色谱法检测血浆药物浓度。在pH小于7.0时, PAMAM树状聚合物对灯盏花素的增溶量随着PAMAM代数、浓度和溶液pH的增加而增大, 其增溶机制为灯盏花素的羧基与PAMAM的伯胺和叔胺发生静  电作用; 灯盏花素、灯盏花素-PAMAM口服给药的C_max分别为 (119.65 ± 9.36) 和 (518.17 ± 17.07) ng·mL^-1, AUC_{0-8 h}分别为 (370.09 ± 63.08) 和 (1 219.47 ± 201.87) ng·h·mL^-1, 两者具有极显著性差异 (P < 0.01)。说明PAMAM能显著提高灯盏花素在水中的溶解度; 大大改善灯盏花素口服给药的生物利用度。     

Polyamidomine dendrimers: an excellent drug carrier for improving the solubility and bioavailability of puerarin

The potential of polyamidoamine (PAMAM) dendrimers as solubility enhancers and oral drug delivery system was well known. Herein, we investigated the possibility of PAMAM dendrimers for promoting the solubility and oral bioavailability of puerarin. In the present study, the effect of PAMAM dendrimers with different generations (G1.5, G2, G2.5, and G3) on the solubility of puerarin was evaluated at different concentrations and pH conditions. Further more, the puerarin–G2 dendrimer complex was conducted for the in vitro hemolytic toxicity studies and pharmacokinetics studies in rats. The solubility of puerarin was significantly higher in the presence of the full generation dendrimers (e.g. G2 and G3). No significant hemolysis was observed on erythrocytes (G2, 0–2.5?mg/mL) in the hemolytic toxicity studies. The pharmacokinetics parameters T_max, C_max, and AUC_{0–8 h} of puerarin suspension solution and puerarin–G2 dendrimer complex solution were 0.76?h, 1.50 µg/mL, 7.33 µg·h/mL and 0.33?h, 6.49 µg/mL, 14.02 µg·h/mL, respectively. These studies demonstrate that PAMAM dendrimers may be a promising strategy for peroral delivery of puerarin.     

甲氨蝶呤-G4.0聚酰胺胺树状大分子体外释放初探

目的探讨新型纳米级生物材料G4 0聚酰胺胺(polyamidoamine ,PAMAM )树状大分子对抗癌药物甲氨蝶呤(methotrexate ,MTX)体外释放的影响及释药机理。方法用紫外分光光度法测定G4 0聚酰胺胺树状大分子与甲氨蝶呤复合物在不同浓度和离子强度缓冲溶液( 37℃、pH =7 4 )中甲氨蝶呤的释放行为。结果确定了一个G4 0聚酰胺胺树状大分子可复合14个甲氨蝶呤,在37℃、pH为7 4的10mmol·L-1Tris HCl缓冲溶液中释放甲氨蝶呤80 %需2 0 0h ,但这种缓释作用随离子强度的增加而减弱。结论G4 0聚酰胺胺树状大分子对甲氨蝶呤具有明显的缓释作用,二者的复合作用是聚酰胺胺树状大分子的氨基阳离子与甲氨蝶呤的羧基阴离子之间形成的静电作用导致的。     

Intracellular Ca2+ Release Mediates Cationic but Not Anionic Poly(amidoamine) (PAMAM) Dendrimer-Induced Tight Junction Modulation

Purpose

Poly(amidoamine) (PAMAM) dendrimers show great promise for utilization as oral drug delivery vehicles. These polymers are capable of traversing epithelial barriers, and have been shown to translocate by both transcellular and paracellular routes. While many proof-of-concept studies have shown that PAMAM dendrimers improve intestinal transport, little information exists on the mechanisms of paracellular transport, specifically dendrimer-induced tight junction modulation.

Methods

Using anionic G3.5 and cationic G4 PAMAM dendrimers with known absorption enhancers, we investigated tight junction modulation in Caco-2 monolayers by visualization and mannitol permeability and compared dendrimer-mediated tight junction modulation to that of established permeation enhancers. [¹⁴C]-Mannitol permeability in the presence and absence of phospholipase C-dependent signaling pathway inhibitors was also examined and indicated that this pathway may mediate dendrimer-induced changes in permeability.

Results

Differences between G3.5 and G4 in tight junction protein staining and permeability with inhibitors were evident, suggesting divergent mechanisms were responsible for tight junction modulation. These dissimilarities are further intimated by the intracellular calcium release caused by G4 but not G3.5. Based on our results, it is apparent that the underlying mechanisms of dendrimer permeability are complex, and the complexities are likely a result of the density and sign of the surface charges of PAMAM dendrimers.

Conclusions

The results of this study will have implications on the future use of PAMAM dendrimers for oral drug delivery.     

Metabolism of the masked mycotoxin deoxynivalenol-3-glucoside in pigs

Plants can metabolize the Fusarium mycotoxin deoxynivalenol (DON) by forming the masked mycotoxin deoxynivalenol-3-β-d-glucoside (D3G). D3G might be cleaved during digestion, thus increasing the total DON burden of an individual. Due to a lack of invivo data, D3G has not been included in the various regulatory limits established for DON so far. The aim of our study was to contribute to the risk assessment of D3G by determination of its metabolism in pigs. Four piglets received water, D3G (116 μg/kg b.w.) and the equimolar amount of DON (75 μg/kg b.w.) by gavage on day 1, 5 and 9 of the experiment, respectively. Additionally, 15.5 μg D3G/kg b.w. were administered intravenously on day 13. Urine and feces were collected for 24 h and analyzed for DON, D3G, deoxynivalenol-3-glucuronide (DON-3-GlcA), deoxynivalenol-15-GlcA (DON-15-GlcA) and deepoxy-deoxynivalenol (DOM-1) by UHPLC–MS/MS. After oral application of DON and D3G, in total 84.8 ± 9.7% and 40.3 ± 8.5% of the given dose were detected in urine, respectively. The majority of orally administered D3G was excreted in form of DON, DON-15-GlcA, DOM-1 and DON-3-GlcA, while urinary D3G accounted for only 2.6 ± 1.4%. In feces, just trace amounts of metabolites were found. Intravenously administered D3G was almost exclusively excreted in unmetabolized form via urine. Data indicate that D3G is nearly completely hydrolyzed in the intestinal tract of pigs, while the toxin seems to be rather stable after systemic absorption. Compared to DON, the oral bioavailability of D3G and its metabolites seems to be reduced by a factor of up to 2, approximately.     

Evaluation of polyamidoamine dendrimers as potential carriers for quercetin,a versatile flavonoid

The aim of the present research work was to investigate the potential of polyamidoamine (PAMAM) dendrimers as oral drug delivery carriers for quercetin, a Biopharmaceutical Classification System (BCS) class II molecule. The aqueous solubility of quercetin was investigated in different generations of dendrimers, i.e. G0, G1, G2 and G3, with varying concentrations (0.1, 0.5, 1, 2 and 4?µM). Then, it was successfully incorporated in PAMAM dendrimers and they were characterized for incorporation efficacy, nature of nanoformulations, size, size distribution, surface morphology and stability. In vitro release characteristics of quercetin from all quercetin-PAMAM complexes were studied at 37?°C in phosphate buffer saline (PBS; pH 7.4). Furthermore, the efficacy of quercetin-loaded PAMAM dendrimer was assessed by pharmacodynamic experiment, namely, a carrageenan-induced paw edema model to evaluate the acute activity of this nanocarrier in response to inflammation. It was observed that both generation and the respective concentrations of PAMAM dendrimers showed potential positive effects on solubility enhancement of quercetin. All the quercetin-PAMAM complexes were found to be in nanometeric range (<100?nm) with narrow polydispersity index. In vitro study revealed a biphasic release pattern of quercetin which was characterized by an initial faster release followed by sustained release phase and pharmacodynamic study provided the preliminary proof of concept about the potential of quercetin-PAMAM complexes. The study concludes that the dendrimer-based drug delivery system for quercetin has enormous potential to resolve the drug delivery issues associated with it.     

Volume to dissolve applied dose (VDAD) and apparent dissolution rate (ADR): Tools to predict in vivo bioavailability from orally applied drug suspensions

Low solubility of drug candidates generated in research contributes to their elimination during subsequent development due to insufficient oral bioavailability (BA) of crystalline compound. Therefore, the purpose of the study was to identify critical in vitro solubility and dissolution parameter that would predict critical in vivo dissolution by means of in vitro-in vivo correlation. Thermodynamic solubility and apparent dissolution rate (ADR) were determined using the shake-flask method and mini-flow-through-cell, respectively. Oral BA studies in rats and humans were conducted from drug solution and suspension/tablets. Relative BA was calculated using F_rel [%] = AUC_suspension/AUC_solution * 100, representing a measure of in vivo dissolution. Roughly, F_rel rat >50% translates into F_rel human of >90%. Both, ADR and log volume to dissolve applied dose (VDAD), when plotted against F_rel rat, revealed certain threshold levels, (ADR, ∼150-200 μg of compound dissolved under respective assay conditions; VDAD, ∼100-500 ml/kg) which translate into F_rel in rats of >50%.Thus, assuming that F_rel > 50% in rats is indicative of sufficient in vivo dissolution in humans after oral application, drugs should exhibit a VDAD of ∼100-500 ml/kg or less in aqueous media to avoid insufficient or varying drug absorption.     

Anionic PAMAM Dendrimers Rapidly Cross Adult Rat Intestine In Vitro: A Potential Oral Delivery System?

Purpose. To investigate systematically the effect ofpolyamidoamine (PAMAM) dendrimer size, charge, and concentration on uptakeand transport across the adult rat intestine in vitro using theeverted rat intestinal sac system. Methods. Cationic PAMAM dendrimers (generations 3 and 4)and anionic PAMAM dendrimers (generations 2.5, 3.5, and 5.5) that weremodified to include on average a single pendant amino group wereradioiodinated using the Bolton and Hunter Reagent. ¹²⁵I-Labelleddendrimers were incubated with everted sacs in vitro and thetransfer of radioactivity into the tissue and serosal fluid was followedwith time. Results. The serosal transfer rates seen for all anionicgenerations were extremely high with Endocytic Indices (EI) in the range3.4–4.4 mL/mg protein/h. The concentration-dependence of serosaltransfer was linear over the dendrimer concentration range 10–100mg/mL. For ¹²⁵I-labelled generation 5.5 the rate of tissueuptake was higher (EI = 2.48 ± 0.51 mL/mg protein/h) thanseen for ¹²⁵I-labelled generations 2.5 and 3.5 (0.6–0.7mL/mg protein/h) (p < 0.05). The ¹²⁵I-labelled cationicPAMAM dendrimers (generations 3 and 4) displayed a tissue uptake (EI= 3.3–4.8 mL/mg protein/h) which was higher (p < 0.05)than the rate of serosal transfer (EI = 2.3–2.7 mL/mgprotein/h), probably due to nonspecific adsorption of cationic dendrimer tothe mucosal surface. Conclusions. As the anionic PAMAM dendrimers displayedserosal transfer rates that were faster than observed for other syntheticand natural macromolecules (including tomato lectin) studied in the evertedsac system, these interesting nanoscale structures may have potential forfurther development as oral drug delivery systems.     

Engineering of Dendrimer Surfaces to Enhance Transepithelial Transport and Reduce Cytotoxicity

Purpose. 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 (P_app). The P_app of dendrimers through monolayers was measured in both the apical (A)-to-basolateral (B) and BA 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 P_app 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 P_app of mannitol. Modified dendrimers also reduced TEER and caused a more marked increase in the P_app of mannitol. The P_app 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.     

Endocytosis and Interaction of Poly (Amidoamine) Dendrimers with Caco-2 Cells

Purpose To investigate the internalization and subcellular trafficking of fluorescently labeled poly (amidoamine) (PAMAM) dendrimers in intestinal cell monolayers. Materials and methods PAMAM dendrimers with positive or negative surface charge were conjugated to fluorescein isothiocyanate (FITC) and visualized for colocalization with endocytosis markers using confocal microscopy. Effect of concentration, generation and charge on the morphology of microvilli was observed using transmission electron microscopy. Results Both cationic and anionic PAMAM dendrimers internalized within 20 min, and differentially colocalized with endocytosis markers clathrin, EEA-1, and LAMP-1. Transmission electron microscopy analysis showed a concentration-, generation- and surface charge-dependent effect on microvilli morphology. Conclusion These studies provide visual evidence that endocytic mechanism(s) contribute to the internalization and subcellular trafficking of PAMAM dendrimers across the intestinal cells, and that appropriate selection of PAMAM dendrimers based on surface charge, concentration and generation number allows the application of these polymers for oral drug delivery.     

Effects of PAMAM dendrimers in the mouse brain after a single intranasal instillation

Dendrimers are highly branched spherical nanomaterials produced for use in diagnostic and therapeutic applications such as a drug delivery system. The toxicological profiles of dendrimers are largely unknown. We investigated the in vivo effects of nasal exposure to polyamidoamine (PAMAM) dendrimers on their effects on neurological biomarkers in the mouse brain. A single dose of PAMAM dendrimers (3 or 15 μg/mouse) was intranasally administered to 8-week old male BALB/c mice. Twenty-four hours after administration, the olfactory bulb, hippocampus, and cerebral cortex were collected and potential biomarkers in the blood and brain were examined using blood marker, microarray and real-time RT-PCR analyses. No remarkable changes in standard serum biochemical markers were observed in the blood. A microarray analysis showed the alterations of the genes expression level related to pluripotent network, serotonin-anxiety pathway, TGF-beta receptor signaling, prostaglandin synthesis-regulation, complement-coagulation cascades, and chemokine-signaling pathway and non-odorant GPCR signaling pathways in brain tissues. Brain derived-neurotrophic factor mRNA was up-regulated in the hippocampus and cerebral cortex in mice treated with a high dose of dendrimers. These findings suggest that PAMAM dendrimers may reach the brain via the systemic circulation or an olfactory nerve route after intranasal instillation, and indicate that a single intranasal administration of PAMAM dendrimers may potentially lead to neuronal effects by modulating the gene expression of brain-derived neurotrophic factor signaling pathway.     

Alginate coated chitosan core shell nanoparticles for oral delivery of enoxaparin: In vitro and in vivo assessment

The objective of present research work was to develop alginate coated chitosan core shell nanoparticles (Alg-CS-NPs) for oral delivery of low molecular weight heparin, enoxaparin. Chitosan nanoparticles (CS-NPs) were synthesized by ionic gelation of chitosan using sodium tripolyphosphate. Core shell nanoparticles were prepared by coating CS-NPs with alginate solution under mild agitation. The Alg-CS-NPs were characterized for surface morphology, surface coating, particle size, polydispersity index, zeta potential, drug loading and entrapment efficiency using SEM, Zeta-sizer, FTIR and DSC techniques. Alginate coating increased the size of optimized chitosan nanoparticles from around 213 nm to about 335 nm as measured by dynamic light scattering in zeta sizer and further confirmed by SEM analysis. The performance of optimized enoxaparin loaded Alg-CS-NPs was evaluated by in vitro drug release studies, in vitro permeation study across intestinal epithelium, in vivo venous thrombosis model, particulate uptake by intestinal epithelium using fluorescence microscopy and pharmacokinetic studies in rats. Coating of alginate over the CS-NPs improved the release profile of enoxaparin from the nanoparticles for successful oral delivery. In vitro permeation studies elucidated that more than 75% enoxaparin permeated across the intestinal epithelium with Alg-CS-NPs. The Alg-CS-NPs significantly increased (p < 0.05) the oral bioavailability of enoxaparin in comparison to plain enoxaparin solution as revealed by threefold increase in AUC of plasma drug concentration time curve and around 60% reduction in thrombus formation in rat venous thrombosis model. The core shell Alg-CS-NPs showed promising potential for oral delivery and significantly enhanced the in vivo oral absorption of enoxaparin.     

Potential Oral Delivery of 7-Ethyl-10-Hydroxy-Camptothecin (SN-38) using Poly(amidoamine) Dendrimers

Purpose  To investigate potential application of poly(amidoamine) (PAMAM) dendrimers for improving the delivery of SN-38. Methods  Complexes of SN-38 with generation 4 amine terminated PAMAM dendrimers were synthesized with varying amounts of drug. Stability of the complexes as well as influence of complexation on permeability across and cellular uptake by Caco-2 cells was evaluated. Results  The complexes were stable at pH 7.4 and drug was released at pH 5. A tenfold increase in permeability and more than hundredfold increase in cellular uptake of the complexes with respect to free SN-38 was observed. Conclusions  Studies suggest that complexation with PAMAM dendrimers has the potential to improve the oral bioavailability of SN-38.     

Permeability of surface-modified polyamidoamine (PAMAM) dendrimers across Caco-2 cell monolayers

Aim of this study was to prepare polyamine-conjugated PAMAM dendrimers and study their permeability across Caco-2 cell monolayers. Polyamines, namely, arginine and ornithine were conjugated to the amine terminals of the G4 PAMAM dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P_app) for the PAMAM dendrimers increased by conjugating the dendrimers with both of the polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. The correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with both the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of the polyamine-conjugated dendrimers was evaluated in Caco-2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were slightly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer–polyamine conjugates as drug carriers to increase the oral absorption of drugs.     

Transport of surface engineered polyamidoamine (PAMAM) dendrimers across IPEC-J2 cell monolayers

The aim of our study was to prepare arginine-and ornithine-conjugated Polyamidoamine (PAMAM) dendrimers and study their permeability across IPEC-J2 cell monolayers, a new intestinal cell line model for drug absorption studies. Arginine and ornithine were conjugated to the amine terminals of the PAMAM(G4) dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P(app)) for the PAMAM dendrimers increased by conjugating the dendrimers with both of these polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. Correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of these surface-modified dendrimers was evaluated in IPEC-J2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were insignificantly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer-polyamine conjugates as the carriers for antigen/drug delivery through the oral mucosa.     

Development and validation of a liquid chromatographic method for the simultaneous determination of aniracetam and its related substances in the bulk drug and a tablet formulation

Simultaneous determination of aniracetam and its related impurities (2-pyrrolidinone, p-anisic acid, 4-p-anisamidobutyric acid and (p-anisoyl)-4-methyl-2-pyrrolidinone) was accomplished in the bulk drug and in a tablet formulation using a high performance liquid chromatographic method with UV detection. Separation was achieved on a Hypersil BDS-CN column (150 mm × 4.0 mm, 5 μm) using a gradient elution program with solvent A composed of phosphate buffer (pH 4.0; 0.010 M) and solvent B of acetonitrile-phosphate buffer (pH 4.0; 0.010 M) (90:10, v/v). The flow rate of the mobile phase was 1.0 mL min⁻¹ and the total elution time, including the column re-equilibration, was approximately 20 min. The UV detection wavelength was varied appropriately among 210, 250 and 280 nm. Injection volume was 20 μL and experiments were conducted at ambient temperature. The developed method was validated in terms of system suitability, selectivity, linearity, range, precision, accuracy, limits of detection and quantification for the impurities, short term and long term stability of the analytes in the prepared solutions and robustness, following the ICH guidelines. Therefore, the proposed method was suitable for the simultaneous determination of aniracetam and its studied related impurities.     

On the nanotoxicity of PAMAM dendrimers: Superfect stimulates the EGFR–ERK1/2 signal transduction pathway via an oxidative stress-dependent mechanism in HEK 293 cells

Polyamidoamine (PAMAM) dendrimers are cationic branch-like macromolecules that may serve as drug delivery systems for gene-based therapies such as RNA interference. For their safe use in the clinic, they should ideally only enhance drug delivery to target tissues and exhibit no adverse effects. However, little is known about their toxicological profiles in terms of their interactions with cellular signal transduction pathways such as the epidermal growth factor receptor (EGFR). The EGFR is an important signaling cascade that regulates cell growth, differentiation, migration, survival and apoptosis. Here, we investigated the impact of naked, unmodified Superfect (SF), a commercially available generation 6 PAMAM dendrimer, on the epidermal growth factor receptor (EGFR) tyrosine kinase–extracellular-regulated kinase 1/2 (ERK1/2) signaling pathway in human embryonic kidney (HEK 293) cells. At concentrations routinely used for transfection, SF exhibited time and dose-dependent stimulation of EGFR and ERK1/2 phosphorylation whereas AG1478, a selective EGFR tyrosine kinase antagonist, inhibited EGFR–ERK1/2 signaling. SF-induced phosphorylation of EGFR for 1 h was partly reversible upon removal of the dendrimer and examination of cells 24 later. Co-treatment of SF with epidermal growth factor (EGF) ligand resulted in greater EGFR stimulation than either agent alone implying that the stimulatory effects of SF and the ligand are synergistic. Dendrimer-induced stimulation of EGFR–ERK1/2 signaling could be attenuated by the antioxidants apocynin, catalase and tempol implying that an oxidative stress dependent mechanism was involved. These results show for the first time that PAMAM dendrimers, aside from their ability to improve drug delivery, can modulate the important EGFR–ERK1/2 cellular signal transduction pathway – a novel finding that may have a bearing on their safe application as drug delivery systems.