The adsorption of cellular proteins affects the uptake and cellular distribution of gold nanoparticles

Nanotechnology has been widely used in the field of medicine, and it can significantly improve the bioavailability and the target efficiency of medicines. However, after administration, nanomedicines can adsorb biomolecules that can influence their effects. It was reported that the adsorption of plasma proteins can change the surface properties of nanoparticles. When nanoparticles pass through cells, they may carry some cellular proteins out of cells. Currently, it is unclear whether the adsorbed proteins affect the uptake of nanoparticles in the next cell layer. To simplify this complex biological process, BSA-capped gold nanoparticles were prepared and incubated with Caco-2 cell lysate to simulate conditions of transcytosis through epithelial cells. The surface morphology of nanoparticles was examined by TEM. SRB was used to evaluate the cytotoxicity of the nanoparticles. The uptake and cellular distribution of the nanoparticles were detected by ICP-MS and CLSM. The results suggested that the adsorption of cell proteins could enhance the adhesion and uptake of gold nanoparticles. The gold nanoparticles were mainly located in lysosomes, and there were some Lysate-capped AuNPs in the mitochondria whereas no BSA-capped AuNPs appeared there.     

A combined proteomics and metabolomics approach to assess the effects of gold nanoparticles in vitro

Omics technologies, such as proteomics or metabolomics, have to date been applied in the field of nanomaterial safety assessment to a limited extent. To address this dearth, we developed an integrated approach combining the two techniques to study the effects of two sizes, 5 and 30?nm, of gold nanoparticles (AuNPs) in Caco-2 cells. We observed differences in cells exposed for 72?h to each size of AuNPs: 61 responsive (up/down-regulated) proteins were identified and 35 metabolites in the cell extract were tentatively annotated. Several altered biological pathways were highlighted by integrating the obtained multi-omics data with bioinformatic tools. This provided a unique set of molecular information on the effects of nanomaterials at cellular level. This information was supported by complementary data obtained by immunochemistry, microscopic analysis, and multiplexed assays. A part from increasing our knowledge on how the cellular processes and pathways are affected by nanomaterials (NMs), these findings could be used to identify specific biomarkers of toxicity or to support the safe-by-design concept in the development of new nanomedicines.     

Relevance of multidrug resistance proteins for intestinal drug absorption in vitro and in vivo

Multidrug resistance proteins (p-glycoprotein and mrps) are becoming increasingly important to explain the pharmacokinetics and action of drugs. Located in epithelial and endothelial cells of the gastrointestinal tract, liver, kidney, blood brain barrier, choroid plexus and other organs, they are critical determinants for the movement of a large number of commonly prescribed drugs across cellular barriers. Here we provide a brief overview of the role of multidrug resistance proteins in drug absorption from the gastrointestinal tract. We address the different types of multidrug resistance proteins involved, describe experimental models to study the influence of these proteins on transcellular transport and discuss the impact of multidrug resistance proteins on overall drug bioavailability in vivo.     

Altered subcellular distribution of nucleolar protein fibrillarin by actinomycin D in HEp-2 cells

INTRODUCTION The nucleolus of eukaryotic cells is the site of ri-bosome assembly where synthesis and processing ofthe rRNA precursor molecules (pre-rRNAs) as well astheir coordinate assembly with specific ribosomal andnonribosomal proteins to form preribosomal particlestook place[1]. Besides the small nucleolar RNAs(snRNAs), several proteins, includingfibrillarin andB23,are found in the nucleolus[2]. Fibrillarin (B36, NOP1) is an abundant nucleolarprotein which plays a r…     

Altered subcellular distribution of nucleolar protein fibrillarin by actinomycin D in HEp-2 cells

AIM: To study the effects of actinomycin D on subcellular distribution of nucleolar protein fibrillarin in HEp-2 (human esophageal epithelial type 2) cells, and molecular mechanisms for maintenance of fibrillarin in nucleolus.METHODS: Indirect immunofluorescence assay was employed to investigate subcellular distribution of nucleolarprotein fibrillarin and immunoblotting analysis was used to detect the total cellular amount of fibrillarin. RESULTS:Control cells with no drug treatment showed bright clumpy nucleolar staining, which indicated that fibrillarin decorated the nucleolus only. Treatment with actinomycin D caused dislocation of fibrillarin from nucleoli to nucleoplasm with numerous stained small nucleoplasmic entities. Immunoblotting showed that neither total cellular amount of fibrillarin nor the integrity of fibrillarin was changed upon the treatment. The dislocation of fibrillarin incells treated at a lower concentration (0.05 mg/L) of actinomycin D, was totally reversible after removal of the drug from the medium. However, this reversion was not observed at a high drug concentration (1 mg/L). CONCLUSION:Actinomycin D induced dislocation of fibrillarin from nucleoli to nucleoplasm in HEp-2 cells. The retention of fibrillarin within the nucleolus was related to active RNA synthesis.     

Folate and TAT Peptide Co-Modified Liposomes Exhibit Receptor-Dependent Highly Efficient Intracellular Transport of Payload In Vitro and In Vivo

Purpose

Using different chain lengths of PEG as linkers to develop a novel folate (FA) and TAT peptide co-modified doxorubicin (DOX)-loaded liposome (FA/TAT-LP-DOX) and evaluate its potential for tumor targeted intracellular drug delivery.

Methods

FA/TAT-LP-DOX was prepared by pH gradient method and post-insertion method and the optimal ligand density was screened by MTT assay. In vitro evaluation was systematically performed through cytotoxicity assay, cellular uptake studies, subcellular localization and cellular uptake mechanism in folate receptor (FR) over-expressing KB tumor cells. In vivo tumor targeted delivery of FA/TAT-LP-DOX was also studied by in vivo fluorescence imaging in a murine KB xenograft model.

Results

The particle size and zeta potential determination indicated that FA and TAT were successfully inserted into the liposome and cationic TAT peptide was completely shielded. With the optimal ligand density (5% of FA and 2.5% TAT), the FA/TAT-LP-DOX exhibited improved cytotoxity and cellular uptake efficiency compared with its single-ligand counterparts (FA-LP-DOX and PEG/TAT-LP-DOX). Competitive inhibition and uptake mechanism experiments revealed that FA and TAT peptide played a synergistic effect in facilitating intracellular transport of the liposome, and association between FA and FA receptors activated this transport process. In vivo imaging further demonstrated the superiority of FA/TAT-LP in tumor targeting and accumulation.

Conclusions

Folate and TAT peptide co-modified liposome using different chain lengths of PEG as linkers may provide a useful strategy for specific and efficient intracellular drug delivery.     

Biochemical and molecular pharmacological aspects of transporters as determinants of drug disposition

Membrane transporters are integral membrane proteins typically having 12 transmembrane domains. Most of the SLC family transporters consist of 300-800 amino acid residues with a molecular mass of 40-90 kDa, while the corresponding values of ABC family transporters are 1,200-1,500 residues and 140-180 kDa, respectively. Each transporter has a characteristic tissue distribution and subcellular localization. I have isolated cDNAs of various transporters, including oligopeptide transporter PEPT1, monocarboxylic acid transporter MCT1 and organic cation/carnitine transporters (OCTNs), and determined their tissue distribution and subcellular localization. I have also determined the absolute expression levels of transporters to evaluate their relative contributions to drug transport in various tissues. It is important to note that expression levels of transporters can be changed under various physiological conditions and by administration of drugs. Changes in expression level, subcellular localization and functional properties can all be involved in inter-individual differences in drug pharmacokinetics. Transporters are among the key determinants of drug disposition.     

Overcoming cellular multidrug resistance using classical nanomedicine formulations

Over the past few decades, many different types of nanomedicines have been evaluated, both in vitro and in vivo. In general, nanomedicines are designed to improve the in vivo properties of low-molecular-weight (chemo-) therapeutic drugs, i.e. their biodistribution and the target site accumulation, and to thereby improve the balance between their efficacy and toxicity. A significant number of studies have also addressed the in vitro properties of nanomedicines, showing e.g. their ability to overcome cellular multidrug resistance (MDR). Particularly promising results in this regard have been reported for 'pharmacologically active' carrier materials, such as Pluronics, which are able to directly inhibit drug efflux pumps and other cellular detoxification mechanisms. In the present report, we have set out to evaluate the ability of classical (and pharmacologically inactive) carrier materials to overcome MDR. To this end, four different drug-sensitive and drug-resistant cancer cell lines were treated with increasing concentrations of free doxorubicin, of polymer-bound doxorubicin, of micellar doxorubicin and of liposomal doxorubicin, and resistance indices (IC(50) in resistant cells/IC(50) in sensitive cells) were determined. In addition, the cellular uptake of the four formulations was evaluated using fluorescence microscopy. It was found that the carrier materials did manage to overcome MDR to some extent, but that the overall benefit was quite small; only for polymer-bound doxorubicin in A431 cells, a significant (4-fold) reduction in the resistance index was observed. These findings indicate that the ability of classical nanomedicines to overcome cellular MDR should not be overestimated.     

Mechanism of alpha-synuclein oligomerization and membrane interaction: theoretical approach to unstructured proteins studies

Misfolding and oligomerization of unstructured proteins is involved in the pathogenesis of Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, and other neurodegenerative disorders. Elucidation of possible conformations of these proteins and their interactions with the membrane is necessary to understand the molecular mechanisms of neurodegeneration. We developed a strategy that makes it possible to elucidate the molecular mechanisms of alpha-synuclein aggregation-a key molecular event in the pathogenesis of PD. This strategy can be also useful for the study of other unstructured proteins involved in neurodegeneration. The results of these theoretical studies have been confirmed with biochemical and electrophysiological studies. Our studies provide insights into the molecular mechanism for PD initiation and progression, and provide a useful paradigm for identifying possible therapeutic interventions through computational modeling.     

Role of SNARE's in the GLUT4 translocation response to insulin in adipose cells and muscle

Insulin stimulates glucose transport in skeletal muscle, heart, and adipose tissue by promoting the appearance of GLUT4, the major glucose transporter isoform present in these tissues, on the cell surface. This is achieved by differentially modulating GLUT4 exocytosis and endocytosis, between a specialized intracellular compartment and the plasma membrane. Ligands which activate the heterotrimeric GTP-binding proteins Gs and Gi appear to modulate insulin-stimulated glucose transport through effects on the fusion of docked GLUT4-containing vesicles with the plasma membrane. In insulin resistance states, reduced cellular GLUT4 levels in adipose cells fully account for the decreased glucose transport response to insulin in these cells. In contrast, although insulin-stimulated GLUT4 translocation is also impaired in muscle, total cellular levels of GLUT4 are not altered. The defect in muscle has been attributed to a GLUT4 trafficking problem and thus studies of this mechanism could provide clues as to the nature of the impairment. The movement of GLUT4-containing vesicles from an intracellular storage site to the plasma membrane and the fusion of docked GLUT4-containing vesicles with the plasma membrane are conceptually similar to some secretory processes. A general hypothesis called the SNARE hypothesis (soluble NSF attachment protein receptors where NSF stands for N-ethylmaleimide-sensitive fusion protein) postulates that the specificity of secretory vesicle targeting is generated by complexes that form between membrane proteins on the transport vesicle (v-SNARE's) and membrane proteins located on the target membrane (t-SNARE's). Several v- and t-SNARE's have been identified in adipose cells and muscle. VAMP2 and VAMP3/cellubrevin (v-SNARE's) have been shown to interact with the t-SNARE's syntaxin 4 and SNAP-23. The cytosolic protein NSF has the characteristic of binding to the v-/t-SNARE complex through its interaction with alpha-SNAP, another soluble factor. Furthermore, recent studies have demonstrated that VAMP2/3, syntaxin 4, SNAP-23, and NSF are functionally involved in insulin-stimulated GLUT4 translocation in adipose cells and thus are likely to be involved in the Gs- and Gi-mediated modulation of the glucose transport response to insulin as well. This review summarizes recent advances on the normal mechanism of GLUT4 translocation and discusses how this process could be affected in insulin resistant states such as type II diabetes.     

Entry of aminoglycosides into renal tubular epithelial cells via endocytosis-dependent and endocytosis-independent pathways

Aminoglycoside antibiotics such as gentamicin and amikacin are well recognized as a clinically important antibiotic class because of their reliable efficacy and low cost. However, the clinical use of aminoglycosides is limited by their nephrotoxicity and ototoxicity. Nephrotoxicity is induced mainly due to high accumulation of the antibiotics in renal proximal tubular cells. Therefore, a lot of studies on characterization of the renal transport system for aminoglycosides so far reported involved various in-vivo and in-vitro techniques. Early studies revealed that aminoglycosides are taken up through adsorptive endocytosis in renal epithelial cells. Subsequently, it was found that megalin, a multiligand endocytic receptor abundantly expressed on the apical side of renal proximal tubular cells, can bind aminoglycosides and that megalin-mediated endocytosis plays a crucial role in renal accumulation of aminoglycosides. Therefore, megalin has been suggested to be a promising molecular target for the prevention of aminoglycoside-induced nephrotoxicity. On the other hand, recently, some reports have indicated that aminoglycosides are transported via a pathway that does not require endocytosis, such as non-selective cation channel-mediated entry, in cultured renal tubular cells as well as cochlear outer hair cells. In this commentary article, we review the cellular transport of aminoglycosides in renal epithelial cells, focusing on endocytosis-dependent and -independent pathways.     

Intranasal delivery of biologics to the central nervous system

Treatment of central nervous system (CNS) diseases is very difficult due to the blood-brain barrier's (BBB) ability to severely restrict entry of all but small, non-polar compounds. Intranasal administration is a non-invasive method of drug delivery which may bypass the BBB to allow therapeutic substances direct access to the CNS. Intranasal delivery of large molecular weight biologics such as proteins, gene vectors, and stem cells is a potentially useful strategy to treat a variety of diseases/disorders of the CNS including stroke, Parkinson's disease, multiple sclerosis, Alzheimer's disease, epilepsy, and psychiatric disorders. Here we give an overview of relevant nasal anatomy and physiology and discuss the pathways and mechanisms likely involved in drug transport from the nasal epithelium to the CNS. Finally we review both pre-clinical and clinical studies involving intranasal delivery of biologics to the CNS.     

Deguelin represses both the expression of nucleophosmin and some nucleoporins: Nup88 and Nup214 in Jurkat cells

Since the first report about cytoplasmic nucleophosmin (NPM) in acute myelogenous leukemia with a normal karyotype was announced, the shuttling activity of NPM and its proper subcellular localization have drawn many attentions. Mechanisms that regulate nucleocytoplasmic transport of proteins may provide novel opportunities for drug development. Here we show that, in Jurkat cells, strong fluorescence density of NPM prevails in the nucleus, while, some key nucleoporins: Nup88 and Nup214 localize mainly in the cytoplasm. Deguelin, a natural occurring rotenoid, presents powerful anti-leukemia effects through proliferation inhibition and apoptosis induction in Jurkat cells. Deguelin downregulates the expression of NPM, Nup88 and Nup214 in a dose-dependent manner and reverts the localization of Nup88 and Nup214 to nuclear rim. These results suggest that deguelin exhibit its strong anti-leukemia effects might through the regulation of some nucleoporins, thus influence the subsequent abnormal expressions or localizations of some key proteins involved in proliferation and/or apoptosis, such as: NPM.     

Particle stimulation dephosphorylates glutathione S-transferase π1 of epithelial cells

Environmental pollutant exposure is associated with adverse respiratory outcomes. The phosphorylation of enzymes activates or deactivates many cellular processes and is related to the development of lung diseases such as asthma and chronic obstructive pulmonary disease. However, little is known about protein phosphorylation of bronchial epithelial cells in response to airborne particulates. Herein, we screened differentially phosphorylated proteins in TiO?-treated epithelial cells and validated the change in GSTP1 protein phosphorylation. Two-dimensional electrophoresis was adopted for differential display proteomics of TiO?-treated BEAS-2B cell lysates. Phosphoproteins were screened using Pro-Q? Diamond phosphoprotein gel stain and identified by MALDI-TOF/TOF analysis. Immunoprecipitation and immunoblotting were performed for quantitative measurement of GSTP1 phosphorylation in cell lysates. Normalized relative intensities of nine phosphorylated proteins increased after TiO? treatment, whereas those of 12 proteins decreased in the BEAS-2B cell lysates. From gene ontology and pathway analysis, proteins involved in signal transduction were commonly identified, followed by cytoskeletal proteins, proteins from oxidation and antioxidation pathways, proteins catalyzing reductions, and those involved in cellular process, transport, and modification. Immunoblotting with anti-GSTP1 antibody demonstrated no change in GSTP1 protein levels in the lysates of BEAS-2B cells after treatment with TiO? particles; blotting with anti-phosphoserine and anti-phosphotyrosine antibodies showed dose-dependent decreases in phosphoserine and phosphotyrosine proteins. Stimulation with particulates phosphorylated and dephosphorylated several proteins in epithelial cells, and serine and tyrosine protein phosphorylation of GSTP1 decreased. These data indicate that airborne particles affect the pattern of phosphorylation of proteins involved in defense or apoptosis of respiratory epithelium.     

Transporter-mediated uptake into cellular compartments

Active transport across biological membranes represents a critical step in the disposition of many drugs. It is now well-established that different efflux and uptake transporters such as P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) or organic anion transporting polypeptides (OATPs) are involved in the overall disposition and efficacy of numerous compounds. These proteins are mainly expressed at physiological sites of drug absorption and elimination, thus leading to diminished absorption and/or increased transporter-facilitated excretion. Moreover, drug transporters are known to be of protective significance in blood–organ barriers. On the contrary, only little is known about the relevance of transporter function on drug levels within tissues and cellular compartments, i.e. the site of action for many substances. Moreover, the pharmacokinetic processing inside the cell is characterized by uptake, metabolism and elimination. It is gradually being recognized that active uptake and/or efflux transporters may modify target concentrations at the subcellular receptor sites which in turn may have an influence on drug effects. This review will summarize current knowledge about the impact of transporter proteins on drug availability within pharmacologically relevant cellular compartments and tissues as hepatocytes, enterocytes, different blood cell types, brain, and the heart with emphasis on the potential clinical significance of these transporters.     

Transporter-mediated uptake into cellular compartments

Active transport across biological membranes represents a critical step in the disposition of many drugs. It is now well-established that different efflux and uptake transporters such as P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) or organic anion transporting polypeptides (OATPs) are involved in the overall disposition and efficacy of numerous compounds. These proteins are mainly expressed at physiological sites of drug absorption and elimination, thus leading to diminished absorption and/or increased transporter-facilitated excretion. Moreover, drug transporters are known to be of protective significance in blood-organ barriers. On the contrary, only little is known about the relevance of transporter function on drug levels within tissues and cellular compartments, i.e. the site of action for many substances. Moreover, the pharmacokinetic processing inside the cell is characterized by uptake, metabolism and elimination. It is gradually being recognized that active uptake and/or efflux transporters may modify target concentrations at the subcellular receptor sites which in turn may have an influence on drug effects. This review will summarize current knowledge about the impact of transporter proteins on drug availability within pharmacologically relevant cellular compartments and tissues as hepatocytes, enterocytes, different blood cell types, brain, and the heart with emphasis on the potential clinical significance of these transporters.     

肾小管上皮细胞特异性转运马兜铃酸机制研究进展

因应用不当或饮食污染摄入马兜铃酸I所引起的、以进展性间质纤维化为特征的急慢性肾炎称为马兜铃酸肾病或巴尔干地方性肾病。马兜铃酸I特异性损伤近端小管,而对肾脏其他组织细胞未表现出明显的直接损伤作用。因此,近曲小管上皮细胞通过有机阴离子蛋白1和3特异性摄入马兜铃酸I,是马兜铃酸I发挥特异性肾毒性作用的关键。近年来对肾小管摄取马兜铃酸I的机制已明确,但参与其在肾小管上皮细胞顶膜侧转运的蛋白鲜有报道。通过综述马兜铃酸I在肾小管的消除机制,以期为预防和治疗马兜铃酸肾病提供新靶点。     

A proteomic approach to investigate AuNPs effects in Balb/3T3 cells

Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell–nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study the effects of AuNPs. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. Of interest was the difference observed in the protein pattern expression of cells exposed to AuNPs. It was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15 nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, and inflammatory response. Moreover, SPR analysis showed an increase of ECM proteins biosynthesis in cells exposed to AuNPs. We observed by TEM analysis that NPs are internalized and confined mainly in autophagosomes. Endoplasmic reticulum stressed and modification at mitochondrial level occurred. This study aims to improve existing knowledge necessary for a correct assessment of the balance between AuNPs potential adverse and beneficial effects and might have important implications for biomedical applications (e.g. nanomedicine).     

Lipophilicity in trans-bilayer transport and subcellular pharmacokinetics

The aim of subcellular pharmacokinetics in drug design is to model drug disposition and response as a function of the properties of drugs and biosystems involved and the observation time. Biosystems are represented by systems of alternating membranes and aqueous phases that differ in acidity and contain low-molecular cell constituents, enzymes and other proteins. The resulting disposition models are combined with linear free-energy assumptions, drug/receptor binding kinetics and relationships between receptor binding and response to produce model-based quantitativestructure–(time–)activity relationships, QS(T)AR. This review summarizes the present status of subcellular pharmacokinetics with emphasis on passive trans-bilayer transport. In particular, mechanisms of transport are analyzed with respect to amphiphilicity and lipophilicity. The overall rate of transport is strongly governed by amphiphilicity, the tendency of drug molecules to adsorb to the bilayer/water interface. Depending on amphiphilicity, the time needed for a drug to cross a single bilayer ranges from seconds to days. The main advantage of the subcellular pharmacokinetic approach is that the resulting models, once calibrated for a given biosystem, provide a detailed recipe for tailoring the drug properties to ensure optimum disposition. This revised version was published online in August 2006 with corrections to the Cover Date.