Cell line-dependent internalization pathways and intracellular trafficking determine transfection efficiency of nanoparticle vectors.

It has been suggested that cell physiology may affect the internalization pathways of non-viral vectors, leading to cell line-dependent transfection efficiency. To verify this hypothesis, fluorescently labeled alginate-chitosan nanoparticle complexes were used as non-viral vectors to transfect 293T, COS7, and CHO cells and to observe the cellular interactions and internalization mechanisms of the complexes in each cell line. 293T cells, which demonstrate the highest transfection efficiency, internalize complexes primarily through clathrin-mediated processes. COS7 cells also demonstrate some internalization of complexes through the clathrin-dependent pathway, explaining the moderate transfection exhibited. In contrast, CHO cells internalize complexes predominantly through caveolin-mediated pathways and are not transfected. Results suggest that following clathrin-mediated endocytosis, complexes are trafficked to the endo-lysosomal pathway, where the proton-sponge effect leads to their release into the cytosol. Contrarily, the absence of trafficking to this pathway following caveolin-mediated endocytosis results in vesicle-entrapped complexes that become transfection-incompetent. These results demonstrate that cell physiology is a critical factor in efficient transfection, and that trafficking to the endo-lysosomal pathway through specific internalization mechanisms is essential for transfection with alginate-chitosan nanoparticle complexes.     

Alteration of hepatic carboxylesterase activity by soman: inhibition in vitro and enhancement in vivo

1. Hydrolysis of the drug esters procaine, chloramphenicol succinate, and prednisolone succinate was studied. Addition of soman to guinea pig liver microsomes caused a dose-dependent inhibition of hydrolysis of all three substrates; at the highest soman concentration (1 microM), ester hydrolysis was totally abolished. 2. Ester hydrolysis was also measured in liver microsomes from guinea pigs pretreated with soman at a low dose (10% of LD50) or at a high dose (90% of LD50) either 1 h or 12 h before killing. Plasma-cholinesterase activity was decreased in all pretreated animals. Liver carboxylesterase activity, measured with the three drug substrates and by hydrolysis of 4-nitrophenyl acetate was increased by all pretreatments. 3. This enhancing effect varies with the substrate and increases with dose of soman. The 12 h pretreatment produced a greater increase in activity than did the 1 h pretreatment. 4. These studies indicate that soman is a potent inhibitor of carboxylesterase activity in vitro but increases the activity of the liver enzyme when administered in vivo.     

Structure-activity relationships for in vivo cadmium mobilization by dithiocarbamates derived from lactose and maltotriose.

The relationships between chemical structure and the relative ability to mobilize cadmium in vivo from its aged renal and hepatic deposits have been examined in a series of newly synthesized dithiocarbamates derived from lactose and maltotriose. The results suggest that, in the selection of hydrophobic groups to counter the hydrophilicity contributed by the disaccharides, aromatic groups provide compounds which have a superior efficacy to compounds containing aliphatic groups. The compounds derived from trisaccharides are much less effective than those derived from disaccharides, suggesting that there is a practical size limit to the hydrophilic groups which can be used in the structures of such compounds. With both di- and trisaccharides, aliphatic derivatives with straight chains containing more than eight carbon atoms tend to be less effective than the ones with seven or fewer carbon atoms in the alkyl chain. The three compounds prepared from lactose which contain a benzyl or a methyl-substituted benzyl group are the most effective compounds reported to date for the reduction of whole-body cadmium levels.     

Optimization of cancer immunotherapy by controlling immune cell trafficking and biodistribution

An immunosurveillance system for tumor-associated antigens (TAAs) plays an important role in the elimination of cancer cells during the initial stage. Although cancer immunotherapy targeting TAAs has progressed steadily with the development of various vaccine strategies, excellent therapeutic efficacy, as evidenced by marked tumor regression and complete response, has not been reported in a clinical setting to date. To improve the therapeutic effects of cancer immunotherapy, we are attempting to establish an innovative concept, the "cell delivery system," capable of better controlling the trafficking and biodistribution of immune cells by applying chemokine-chemokine receptor coupling, which regulates leukocytic migration and infiltration of local sites in the living body. This review introduces our approaches that employ an Arg-Gly-Asp (RGD) fiber-mutant adenovirus vector encoding the chemokine or chemokine receptor gene in cancer immunotherapy.     

The role of HNF-1alpha in controlling hepatic catalase activity

Mice deficient in hepatocyte nuclear factor 1alpha (HNF-1alpha) develop Laron dwarfism and non-insulin-dependent diabetes mellitus (Lee et al., 1998). Oxidative stress was present in the diabetic HNF-1alpha-null mice. To understand the mechanism underlying the oxidative stress in HNF-1alpha-null mice, we examined whether HNF-1alpha deficiency affects the integrity of the cellular defense system against oxidative stress. The glutathione level and activities of superoxide dismutase and glutathione reductase in liver and other tissues examined were not affected by HNF-1alpha deficiency. However, activities of cytosolic glutathione peroxidase and catalase, two enzymes responsible for detoxification of hydrogen peroxide within cells, were reduced specifically in liver of HNF-1alpha-null mice. The mRNA and protein levels of hepatic catalase in HNF-1alpha-null mice did not differ from those in normal mice. The loss of hepatic catalase activity in HNF-1alpha-null mice is probably caused by an insufficient heme pool in liver cells, because the mRNA level of ferrochelatase, the enzyme that catalyzes the last step of heme biosynthesis, was significantly reduced in liver, and the daily hemin treatment restored partial catalase activity in liver of HNF-1alpha-null mice. Furthermore, our results of cell transfection and luciferase reporter assay indicated that the mouse ferrochelatase promoter could be trans-activated directly by HNF-1alpha.     

Inhibition of hepatic mixed function oxidase activity by propyl gallate

Propyl gallate was found to inhibit microsomal benzpyrene hydroxylase activity and demethylase activity with ethylmorphine, aminopyrine or benzphetamine as a substrate. The extent of inhibition with different substrates varied with the age and diet of the animals. The benzpyrene hydroxylase activity of the microsomes of the 3-methylcholanthrene-treated rats was shown to be less susceptible to propyl gallate inhibition. Propyl gallate does not inhibit the NADPH-dependent reduction of cytochrome P-450; therefore, the site of inhibition is not on NADPH-cytochrome c reductase as suggested previously. Propyl gallate interacts with cytochrome P-450 to produce a positive absorption peak around 420 nm, and it may also interfere with the binding of a type I substrate, benzphetamine. It inhibits ethylmorphine demethylation by a noncompetitive mechanism and aminopyrine demethylation by a mixed mechanism. The mode of propyl gallate inhibition and the implications of these observations are discussed.     

Effect of chlormethiazole on hepatic monooxygenases activity in vivo

Summary Chlormethiazole is a strong inhibitor of cytochrome P-450-dependent monooxygenases in isolated human liver microsomes. To assess its effect in vivo, we measured the pharmacokinetic parameters of antipyrine (1.2 g orally) and tolbutamide (0.5 g i. v.) before and after administration of chlormethiazole 314 mg b. d. for 2 days to 8 healthy volunteers.The elimination of neither substance was affected, indicating that chlormethiazole did not inhibit in vivo the cytochrome P-450 isozymes responsible for the elimination of antipyrine and tolbutamide.     

Nonviral gene transfection nanoparticles: function and applications in the brain

In vivo transfer and expression of foreign genes allows for the elucidation of functions of genes in living organisms and generation of disease models in animals that more closely resemble the etiology of human diseases. Gene therapy holds promise for the cure of a number of diseases at the fundamental level. Synthetic "nonviral" materials are fast gaining popularity as safe and efficient vectors for delivering genes to target organs. Not only can nanoparticles function as efficient gene carriers, they also can simultaneously carry diagnostic probes for direct "real-time" visualization of gene transfer and downstream processes. This review has focused on the central nervous system (CNS) as the target for nonviral gene transfer, with special emphasis on organically modified silica (ORMOSIL) nanoparticles developed in our laboratory. These nanoparticles have shown robust gene transfer efficiency in brain cells in vivo and allowed to investigate mechanisms that control neurogenesis as well as neurodegenerative disorders.     

Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thiono-sulfur-containing compounds.

Ten thiono-sulfur-containing compounds of varying structure were administered by intraperitoneal injection to untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated adult male rats. Six hr later, the concentration of hepatic cytochrome P-450 and the ability of the hepatic microsomes to metabolize benzphetamine were examined. In the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated groups, two, four and four compounds, respectively, significantly decreased the concentration of cytochrome P-450 in the hepatic microsomes. A similar effect on benzphetamine metabolism was also seen. When examined 48 hr after the administration of the ten thiono-sulfurcontaining compounds, four, five and seven of the compounds decreased both the levels of hepatic cytochrome P-450 and the rate of benzphetamine metabolism in the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated animals respectively. Eight of the thiono-sulfur-containing compounds were incubated in the presence of NADPH with hepatic microsomes isolated from untreated, phenobarbital-pretreated or 3-methylcholanthrene-pretreated animals. All of the compounds examined significantly decreased the concentration of cytochrome P-450 in the microsomes from each treatment group. Similar reductions in benzphetamine metabolism were also seen. When these same compounds were incubated with microsomes in the absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen. When the oxygen analogs of six of the thiono-sulfur compounds were administered in vivo or incubated with hepatic microsomes either in the presence or absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen.     

Pharmacophotonics: utilizing multi-photon microscopy to quantify drug delivery and intracellular trafficking in the kidney

The recent introduction of multi-photon microscopy coupled with advances in optics, computer sciences and the available fluorophores used to label molecules of interest have empowered investigators to study the dynamic events within the functioning kidney at cellular and subcellular levels. This emerging technique, with improved spatial and temporal resolution and sensitivity, enables investigators to follow the cell specific uptake of large and small molecules, determine the mode of cellular uptake, intracellular trafficking and drug metabolism in complex heterogeneous organs such as the kidney over time. Repeat determinations over seconds to hours to days allow for multiple observations within the same animal, thereby minimizing animal use and inter-animal variability. This can be particularly useful for preclinical studies. Furthermore, the ability to obtain volumetric data (3-D) makes quantitative 4-D (time) analysis possible. Finally, up to three fluorophores can be visualized simultaneously allowing for three different or interactive processes to be observed and resolved.     

Cannabinoid Receptor 1 trafficking and the role of the intracellular pool: Implications for therapeutics

Cannabinoid Receptor 1 (CB₁), an abundant G-protein coupled receptor in the CNS, is currently of significant interest as a therapeutic target. Although the cellular control of receptor trafficking is intimately linked with drug effects, CB₁ trafficking is poorly defined in the current literature and conflicting evidence exists as to whether CB₁ should be classified as a recycling, degrading or “dual-fate” receptor. Of particular interest is the widely noted intracellular pool which has been speculated to form part of a constitutive internalization and recycling pathway. This study performs a detailed quantification of CB₁ trafficking in four cell lines, one of which expresses CB₁ endogenously. We demonstrate that, contrary to previous reports, CB₁ does not recycle following constitutive or agonist-induced internalization but instead exhibits a primarily degradative phenotype. Further, our data suggest that the intracellular pool does not contribute to cell surface re-population. These findings have significant implications for the interpretation of CB₁ biochemical studies and the design and application of cannabinoid therapeutics.     

A comparison of the inhibitory activity of PDE4 inhibitors on leukocyte PDE4 activity in vitro and eosinophil trafficking in vivo.

1. Phosphodiesterase (PDE) 4 inhibitors have been shown to inhibit eosinophil PDE4 activity in vitro and accumulation of eosinophils in experimental airways inflammation. However, direct effects on eosinophil trafficking have not been studied in detail and it is not known if activity in vitro translates into efficacy in vivo. In the present study, we compared the activity of five PDE4 inhibitors in vitro and against trafficking of (111)In-eosinophils in cutaneous inflammation in the guinea-pig. 2. The rank order of potency for inhibition of PDE4 activity in guinea-pig eosinophil, neutrophil and macrophage, and human neutrophil lysates was RP73401 > SB207499 >CDP840 > rolipram > LAS31025. On TNFalpha production by human PBMC, all inhibitors with the exception of rolipram showed potency similar to their effect on neutrophil lysates. 3. In a brain cerebellum binding assay, the rank order of potency at displacing [3H]-rolipram was RP73401 > rolipram > SB207499 > CDP840 > LAS30125. 4. Trafficking of (111)In-eosinophils to skin sites injected with PAF, ZAP or antigen in sensitized sites was inhibited by oral administration of all PDE4 inhibitors. The rank order of potency was RP73401 = rolipram > LAS31025 > SB207499 > CDP840. 5. With the exception was RP73401, which was the most potent compound in all assays, there was no clear relationship between activity of PDE4 inhibitors in vitro and capacity to inhibit eosinophil trafficking in vivo. Thus, we conclude that in vitro activity of PDE4 inhibitors does not predict in vivo efficacy in an experimental model of eosinophil trafficking.     

Evaluation of the intracellular trafficking of siRNAs in A375 cells by confocal laser scanning microscopy

Investigation intracellular trafficking of siRNAs following their delivery to cells is of great interest to elucidate dynamics of siRNA in cytoplasm. In this study, we present a novel confocal laser scanning microscopy (CLSM) method to evaluate a novel delivery system of 3′-peptide-siRNA therapeutic, which was named 3′-pAs-siRNA/CLD. This method could not only calculate the content of the intracellular 3′-peptide-siRNA, but also quantify its co-localization with cellular substructure. We observed that 3′-pAs-siRNA/CLD, which provided the better antitumor capability, also had a better cell uptake, endosome escape and a longer retention time in A375. This novel strategy was proved to be efficient, quantified and visualized, thus making the dynamics research of siRNA in cytoplasm clear and simplified.     

Peptides with indirect in vivo activity against an intracellular pathogen: selective lysis of infected macrophages

Abstract: A collection of natural peptides, simplified analogs of natural peptides, de novo amphipathic peptides and de novo amphipathic peptides composed of 50–80% α,α‐dialkylated glycines (α,α‐Dags) were synthesized on solid‐phase resin as the C‐terminus amides using N‐α‐fluorenylmethyloxycarbonyl protection. The synthesis of the peptides rich in α,α‐Dags used acid fluoride coupling methods. The peptides show antimicrobial activity against Escherichia coli and Staphylococcus aureus but no direct antimicrobial activity against Brucella abortus at 100 µm in vitro. However, in vivo treatment with several of these peptides results in significant reductions of B. abortus in chronically infected immune BALB/c mice relative to infected control animals. The chronically infected mice were susceptible to peptide toxicity at much lower peptide doses than control animals. The highest nonlethal dose for infected mice was only 25 µg for melittin, whereas 500 µg doses were nonlethal for many of the other peptides. Several of the α,α‐Dag‐rich peptides selectively destroy B. abortus‐infected murine macrophages in vitro. Thus, these peptides apparently reduce the bacterial load in vivo by destroying a portion of the infected macrophages and exposing the sequestered bacteria to the immune response in the mice.     

Dietary ascorbic acid and hepatic mixed function oxidase activity in the guinea pig

Studies were carried out to characterize the response of hepatic mixed function oxidase (MFO) activity to chronic ascorbic acid deficiency and excessive ascorbic acid intake in the guinea pig. When guinea pigs were fed excessive ascorbic acid, there was a small increase in hepatic cytochrome P-450 which was unaccompanied by any alteration in drug-metabolizing enzyme activity. Similarly, induction of MFO activity by phenobarbital was not modified by excessive ascorbic acid administration. Chronic ascorbic acid deficiency resulted in depressed metabolism of aniline, aminopyrine, ethoxycoumarin and benzphetamine, but not of ethylmorphine, in comparison with animals fed diets containing control and/or excessive amounts of ascorbic acid. In contrast to the metabolism of all drugs studied, the 7 alpha-hydroxylation of cholesterol was depressed by both inadequate and excessive vitamin C intake, demonstrating the unique sensitivity of cholesterol 7 alpha-hydroxylase to dietary ascorbate.     

Effect of acetone administered in vivo upon hepatic microsomal drug metabolizing activity in the rat

The effects of acetone administered to female rats in vivo, upon the metabolism of drugs by the hepatic microsomal subcellular fraction have been studied. There is a rapid increase, maximal at 0·5 hr in aniline p-hydroxylation of 69 per cent, and an inhibition of aminopyrine N-demethylation of 61 per cent. Thereafter levels return to control values. There is a slower increase in aniline p-hydroxylation of 168 per cent, maximal at 48 hr. whilst aminopyrine N-demethylation is unaltered. Cycloheximide has no effect upon the changes in activity after 0·5 hr but blocks the increase in aniline p-hydroxylation 48 hr after the administration of acetone. It is suggested that acetone may contribute to the changes in drug metabolizing activity found in diabetic animals.     

Perinatal development of hepatic microsomal mixed function oxidase activity in swine

Hepatic microsomal cytochrome P-450, cytochrome b₅, NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase levels were measured in fetal (107-days gestation), newborn and 1-, 2-, 3-, 4- and 6-week-old swine. Cytochrome P-450 levels and NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase activities increased in near parallel with ethylmorphine demethylase (V_max) activity between the first and the sixth postnatal week. The activities or levels of all parameters measured appeared to plateau between the fourth and sixth week post-partum. The only qualitative change observed after 1 week of age was a slight increase in the K_m for ethylmorphine demethylation. NADPH-cytochrome c reductase activity of fetal liver was relatively high, being approximately 40 per cent of the values attained at 6 weeks of age. This was in contrast to very low levels of NADPH-cytochrome P-450 reductase activity and cytochrome P-450 content of fetal liver. Clearly the activity of the flavoprotein NADPH-cytochrome c reductase does not limit the rate of reduction of cytochrome P-450 in the microsomal fraction of fetal liver. The possibility that cytochrome P-450 exists in a different form, or ratio of forms, in fetal liver could not be ascertained from carbon monoxide (CO) or ethylisocyanide (EtCN) difference spectra of fetal microsomal preparations. However, the dithionite difference CO spectra of cytochrome P-450 did not change with age.