Lactose Residues Research Articles

Synthesis of lipid mediators based on 1,2-dialkylglycerol and cholesterol for targeted delivery of oligo- and polynucleotides into hepatocytes

The synthesis of cationic glycolipids containing galactose or lactose residue as hepatocyte-targeted markers is described.

Synthesis of Bivalent Lactosides Based on Terephthalamide, N,N′-Diglucosylterephthalamide, and Glycophane Scaffolds and Assessment of Their Inhibitory Capacity on Medically Relevant Lectins

Glycan recognition by lectins initiates clinically relevant processes such as toxin binding or tumor spread. Thus, the development of potent inhibitors has a medical perspective. Toward this goal, we report the synthesis of both rigid and flexible bivalent lactosides on scaffolds that include secondary and tertiary terephthalamides and N,N'-diglucosylterephthalamides. Construction of these compounds involved Schmidt-Michel glycosidation, and amide coupling or Ugi reactions of relevant glycosyl amines in key steps. A glycocluster based on a rigid glycophane was also prepared from coupling of a glucuronic acid derivative and p-xylylenediamine with subsequent ring-closing metathesis. Finally, a more flexible bivalent lactoside was produced from lactosyl azide with use of the copper-catalyzed azide-alkyne cycloaddition. Distances between lactose residues were analyzed computationally as were their orientations for the relatively rigid subset of compounds. Distinct spacing properties were revealed by varying the structure of the scaffold or by varying the location of the lactose residue on the scaffold. To relate these features to bioactivity a plant toxin and human adhesion/growth-regulatory galectins were used as sensors in three types of assay, i.e. measuring agglutination of erythrocytes, binding to glycans of a surface-immobilized glycoprotein, or binding to human cells. Methodologically, the common hemeagglutination assay was found to be considerably less sensitive than both solid-phase and cell assays. The bivalent compounds were less effective at interfering with glycoprotein binding to the plant toxin than to human lectins. Significantly, a constrained compound was identified that displayed selectivity in its inhibitory potency between galectin-3 and its proteolytically processed form. Conversely, compounds with a high degree of flexibility showed notable ability to protect human cells from plant toxin binding. The applied conjugation chemistry thus is compatible with the long-term aim to produce potent and selective lectin inhibitors.

Surface modification of polymer latex particles by AGET ATRP of a styrene derivative bearing a lactose residue

Grafting of a styrene derivative bearing a lactose residue, i.e., N-2-4-(vinylbenzenesulfonamido)ethyl lactobionamide (VBSAELA), onto polymer latex particles was carried out in aqueous media by activator generated electron transfer atom transfer radical polymerization (AGET ATRP). The core polymer latex particles having α-chloroester groups as ATRP-initiating groups were prepared by miniemulsion polymerization of styrene and 2-chloropropionyloxyethyl methacrylate (CPEM) in the presence of a polymerizable surfactant, i.e., N, N-dimethyl- N-dodecyl- N-2-methacryloyloxyethylammonium bromide (C 12Br). AGET ATRP was initiated with tris[(2-pyridylmethyl)amine] copper (II) dichloride and l-ascorbic acid. Dynamic light scattering (DLS) revealed that the P(St-CPEM)- g-P(VBSAELA) particles possess graft layers of 10 nm in thickness on the core particles of 91 nm in diameter. Critical coagulation concentration measurement revealed that the dispersion stability of the particles in water increased due to hydrated P(VBSAELA) shell layers. Adsorption of bovine serum albumin (BSA) onto the particles was considerably suppressed by the hydrated shell layers.

Liver target delivery of small interfering RNA to the HCV gene by lactosylated cationic liposome

RNA interference has considerable therapeutic potential, particularly for anti-viral therapy. We previously reported that hepatitis C virus (HCV)-directed small interfering RNA (siRNA; siE) efficiently inhibits HCV replication, using HCV replicon cells. To employ the siRNA as a therapeutic strategy, we attempted in vivo silencing of intrahepatic HCV gene expression by siE using a novel cationic liposome. The liposomes consisted of conjugated lactose residues, based on the speculation that lactose residues would effectively deliver siRNA to the liver via a liver specific receptor. The lactosylated cationic liposome 5 (CL-LA5) that contained the most lactose residues introduced the most siRNA into a human hepatoma cell line, which then inhibited replication of HCV replicons. In mice, the siRNA/CL-LA5 complexes accumulated primarily in the liver and were widespread throughout the hepatic parenchymal cells. Moreover, siE/CL-LA5 specifically and dose-dependently suppressed intrahepatic HCV expression in transgenic mice without an interferon response. The present results indicate that the CL-LA5 we developed is a good vehicle to lead siRNA to the liver. Hence, CL-LA5 will be helpful for siRNA therapy targeting liver diseases, especially hepatitis C.

Which mechanism for nuclear import of plasmid DNA complexed with polyethylenimine derivatives?

To investigate the nuclear import mechanism of plasmid/polyethylenimine (PEI) derivative complexes and the putative nuclear targeting of therapeutic genes by the use of oligosaccharides, we have studied the nuclear import of plasmid DNA complexed either with PEI or with lactosylated PEI (Lac-PEI) in cystic fibrosis human airway epithelial cells ( summation operatorCFTE29o- cells). Cells were synchronized by a double-thymidine block protocol and gene transfer efficiency was evaluated: Lac-PEI- and PEI-mediated gene transfer was greatly increased when cells have undergone mitosis during the course of transfection. However, both types of complexes were able to transfect some growth-arrested cells. When the nuclear import of plasmid/Lac-PEI or plasmid/unsubstituted PEI complexes was studied in digitonin-permeabilized cells, the nuclear uptake of both types of complexes did not follow the classic pathway of nuclear localization sequence (NLS)-containing proteins and lactose residues did not act as a nuclear localization signal. Our results show that for complexes made with PEI derivatives, the major route for plasmid DNA nuclear entry is a passive nuclear importation during mitosis when the nuclear membrane temporarily breaks down. However, albeit to a lesser extent as that observed in dividing cells, a plasmid DNA importation also occurs in nondividing cells by a yet unknown mechanism.

Lactosylated Chitosan for DNA Delivery into Hepatocytes: The Effect of Lactosylation on the Physicochemical Properties and Intracellular Trafficking of pDNA/Chitosan Complexes

Chitosan is a useful nonviral vector for gene delivery. To make a pDNA/chitosan complex specific to hepatocytes, lactose-modified chitosan (lac-chitosan) was synthesized. When the percentage of lactose residues substituted was 8%, lac-chitosan showed excellent DNA-binding ability, good protection of DNA from nuclease, and the suppression of self-aggregation and serum-induced aggregation. Although the cellular uptake efficiency of the pDNA/lac-chitosan complex was almost the same as that of the pDNA/chitosan complex, the cell transfection efficiency of the former was greater for HepG2 cells having asialoglycoprotein receptors. Inhibitor of endocytosis such as bafilomycin A1 and nocodazole significantly reduced the transfection efficiency of the pDNA/lac-chitosan complex. Observations with a confocal laser scanning microscope indicated that the pDNA/lac-chitosan complexes traversed endocytic compartments more rapidly than the pDNA/chitosan complex. Furthermore, the pDNA/lac-chitosan complexes were delivered to the late endosome and have the advantage of delivering DNA to the perinuclear region.

A Click Approach to Unprotected Glycodendrimers

Click chemistry in combination with ultrafiltration has allowed the quick, efficient, and reliable multivalent conjugation of unprotected alkyne-derived carbohydrates to three generations of azido-terminated gallic acid−triethylene glycol dendrimers under aqueous conditions. The reported procedure allows the atom economical incorporation of up to 27 unprotected fucose, mannose, and lactose residues, in reproducible high yields (up to 92%), requiring only catalytic amounts of Cu. The completion of the conjugation process was clearly established in all cases by both 1H NMR and MALDI-TOF MS.

A glycolipid containing a lactose residue: A novel agent for targeted DNA delivery for the purpose of genetic therapy

A glycolipid containing a lactose residue: A novel agent for targeted DNA delivery for the purpose of genetic therapy

Lactosylated polyethylenimine for gene transfer into airway epithelial cells: role of the sugar moiety in cell delivery and intracellular trafficking of the complexes

As we have previously shown that lactosylated polyethylenimine (PEI) is the most efficient glycosylated PEI for gene transfer into human airway epithelial cells in primary culture, we have studied here the role of the lactose residue in the enhancement of gene transfer efficiency observed with lactosylated PEI as compared with unsubstituted PEI in immortalized (Sigma CFTE29o- cells) and primary human airway epithelial cells. After three transfections of 1 h performed daily, 60% of Sigma CFTE29o- cells were transfected with lactosylated PEI, whereas 25% of cells were transfected with unsubstituted PEI (p < 0.05). Cell viability was 1.8-fold greater with lactosylated PEI as compared with unsubstituted PEI (p < 0.05). As assessed by flow cytometry, the cellular uptake of lactosylated complexes was greater than that of complexes made with unsubstituted PEI (p < 0.05) and involved mostly a receptor-mediated endocytosis. The study of the intracellular trafficking in airway epithelial cells of complexes showed an endosomal and lysosomal accumulation of lactosylated complexes. In the presence of a proton pump inhibitor, the level of lactosylated and unsubstituted PEI-mediated gene expression was reduced more than 20-fold, whereas the cell viability increased to almost 100%. For both complexes, a nuclear localization was observed for less than 5% of intracellular complexes. Our results show that the greater gene transfer efficiency observed for lactosylated complexes may be attributed to a higher amount of lactosylated complexes incorporated by airway epithelial cells and a lower cytotoxicity that might be related to reduced endosomolytic properties. However, the lactose residues substituting the PEI did not promote the entry of the plasmid into the nucleus.

Synthesis of HIV Vaccine Model Based on Lactose-Functionalized Poly(lysine) Dendrimer Scaffold

A series of HIV vaccine models based on lactose-bound poly(lysine) scaffolds was synthesized. A spherical poly(lysine) dendrimer was prepared to bind lactose at the peripheral through reductive amination. Lactose-poly(lysine) dendrimers with the degree of substitution (DS) varying from 5 to 19 were obtained by controlling the feeding ratio of lactose to the poly(lysine) dendrimer. Three succinylated compounds such as succinyl monobenzylate, ethyl-phenylalanyl succinylamide and succinyl-L-alanyl-L-prolyl-L-alanine p-nitroanilide which were regarded as antigen peptide models were bound to the lactose-poly(lysine) dendrimer through esterification between the carboxyl group of the succinyl moiety and the 6-OH group of the lactose residue. The DS of resulting vaccine models was investigated by NMR spectra analyses.

Synthesis and physicochemical characteristics of a liver-targeted conjugate of fluorodeoxyuridine monophosphate with lactosaminated human albumin.

In previous experiments fluorodeoxyuridine monophosphate (FUdRMP) was conjugated with lactosaminated human albumin (L-HSA). Fluorodeoxyuridine (FUdR) is an anticancer agent and L-HSA is a hepatotropic carrier of drugs obtained by the covalent linkage of lactose residues to the albumin molecule. The conjugate was synthesised via the imidazolide of FUdRMP at alkaline pH. Peripheral venous administration of L-HSA-FUdRMP produced enhanced FUdR levels in hepatic blood and might accomplish a non-invasive loco-regional chemotherapy of liver micrometastases. In the present paper some physicochemical characteristics of L-HSA-FUdRMP are reported. Polyacrylamide gel electrophoresis indicated that the coupling reaction did not cause covalent aggregation of the L-HSA molecules. 31P NMR spectra of the conjugate showed that FUdRMP was linked to L-HSA by phosphoamide bonds to lysine and histidine residues, and the area of the peak due to the lysine bond represented more than 80% of the spectrum of L-HSA-FUdRMP. MALDI analysis revealed a partial degradation of the peptide backbone of the conjugate which could not be detected using other methods of analysis. The degradation was not caused by the coupling of lactose molecules to albumin, but rather a consequence of FUdRMP conjugation with L-HSA. This fragmentation was dependent on the pH of the medium used for the FUdRMP coupling reaction. By decreasing the pH to 7.5, conjugates were obtained with a lower drug load but with a substantially reduced fragmentation, which should be preferred for a clinical use of L-HSA-FUdRMP.

Chemically defined sialoside scaffolds for investigation of multivalent interactions with sialic acid binding proteins.

Four glycodendrons and a glycocluster were synthesized from carbohydrate building blocks to form paucivalent (di- to tetravalent) structures of controlled scaffold architectures. Enzymatic sialylation of the functionalized cluster and dendrons, terminated in lactose residues, generated a library of paucivalent synthetic sialosides displaying sialic acids with different dispositions. These newly constructed bioactive sialic acid-based structures were differentially recognized by sialoadhesin, a mammalian macrophage sialic acid binding protein. The binding of the sialosides to sialoadhesin was evaluated by an enzyme-linked immunosorbant assay to investigate the complementarity of scaffold structure and binding to sialoadhesin. Modulating the interaction between sialoadhesin and its sialic acid ligands has important implications in immunobiology.

Synthesis of poly(L-lactide) end-capped with lactose residue.

The synthesis of poly(L-lactide) (polyLA) end-capped with lactose residue was studied from the standpoint of development of a new bioabsorbable material. After the hydroxyl group of t-butoxycarbonyl(Boc)-aminoethanol was converted to Boc-aminoethanol-OK by using potassium/naphthalene, L-lactide was polymerized in tetrahydrofuran using Boc-aminoethanol-OK as an initiator at room temperature to prepare polyLA-NHBoc. Subsequently, the removal of the Boc group in terminal Boc-aminoethanol residue was performed by treatment of formic acid to obtain the amino group end-capped polyLA (polyLA-NH(2)) as a reactive polyLA derivative. The coupling reactions of lactose with polyLA-NH(2) were investigated by two methods; the synthetic method through reductive amination of lactose with polyLA-NH(2) in the presence of sodium cyanoborohydride as a reducing agent did not give high degree of substitution of end-capped lactose residue per polyLA molecule, whereas the synthetic method through the ester interchange reaction of lactonolactone with polyLA-NH(2) gave Lac-polyLA perfectly end-capped with lactose residue.

Thermodynamic binding studies of cell surface carbohydrate epitopes to galectins-1, -3, and -7: Evidence for differential binding specificities

Binding of a series of sialylated and non-sialylated cell surface carbohydrates to bovine heart galectin-1, recombinant murine galectin-3, and recombinant human galectin-7 was investigated by isothermal titration microcalori metry (ITC) and hemagglutination inhibition measurements. Galectin-7 shows nearly equal affinities for lactose and Galbeta(1–4)GlcNAc (LacNAc-II). Galectin-7, however, displays six- and 11-fold weaker affinity for LacNAc-II compared with galectins-1 and -3, respectively. The affinity of galectin-7 for LacNAc-II containing oligosaccharides is also weaker than the other two galectins. ITC measurements show that all three galectins bind to di- and trimeric oligomers of LacNAc-II, which are epitopes found in poly-N-acetyllactosamine chains of glycoprotein receptors, with affinity constants similar to that of LacNAc-II. The binding valencies of the di- and trimeric LacNAc-II oligomers were observed to be one from ITC measurements, indicating formation of 1:1 complexes with all three galectins. Thus, galectins-1, -3, and -7 all possess binding sites that primarily accommodate one LacNAc-II moiety per monomer of protein. Sialylated oligosaccharides show different specificities for the three galectins. While 2,3-sialyl LacNAc-II binds to all three galectins, 2,6-sialyl LacNAc-II fails to bind to any of the galectins; 2,6-sialylated diLacNAc binds well to galectin-3 and galectin-7, but only weakly to galectin-1. Similar results are obtained with 2,6-sialyl lacto-N-neo-tetraose, which has a reducing end lactose moiety. Thus, unlike galectin-1, which predominantly recognizes non-reducing terminal LacNAc-II residues in oligosaccharides, galectins-3 and -7 recognize both non-reducing terminal LacNAc-II residues as well as internal LacNAc-II and lactose residues in sialylated and non-sialylated oligosaccharides.Key words: isothermal titration microcalorimetry, galectins, binding specificities, lectins, carbohydrates.

Preparation of Self-Organized Micro-Patterned Polymer Films Having Cell Adhesive Ligands

This article describes novel three methods for micro-patterning of cell adhesive ligands by using the self-organized honeycomb-patterned structure formed by the simple cast method. A first method is a direct preparation of a patterned film by casting an amphiphilic polymer containing lactose residue which is one of cell adhesive ligands. A benzene solution of the amphiphilic polymer was cast at high humidity on a glass substrate. Atomic force microscopy (AFM) observation of the film showed that a honeycomb pattern with microporousness with as large as micrometer size in diameter was formed. The film was immersed into an aqueous fluorescence-labeled lectin solution to investigate the distribution of lactoses on the patterned film. Consistence of a fluorescence image of the lectin bound film with the honeycomb pattern showed that the lactose residues were existed not at the holes but at the rims of the honeycomb-patterned film. A second method is to immobilize gelatin, which is one also one of cell adhesive ligands, on the honeycomb-patterned film by chemical reaction. A honeycomb-patterned film was prepared from chloroform solution of an amphiphilic polymer containing reactive succinimide ester groups, and then the film was immersed into an aqueous fluorescence-labeled gelatin solution to introduce gelatin on the film surface. Immobilization of gelatin onto honeycomb-patterned film was confirmed by the fluorescence microscope. A third method is another way to introduce gelatin onto the honeycomb film by the specific avidin-biotin interaction. A honeycomb-patterned film was prepared from amphiphilic polymer containing biotin residues and dodecyl groups, and then the film was immersed into a avidin solution and a biotinylated fluorescence labeled gelatin solution successively. By the fluorescence microscopic observation of the film, gelatin was confirmed to be immobilized at the rims of the honeycomb pattern via the avidin-biotin interaction. Cell culture was performed on the gelatin immobilized patterned film prepared by second method. Bioactivity of gelatin immobilized honeycomb-patterned film was confirmed by adhesion of cell onto the film.

Nuclear translocation of lactosylated poly-L-lysine/cDNA complex in cystic fibrosis airway epithelial cells.

Poly-l-lysine, with 40% of its amino groups substituted with lactose, is an effective vector to transfer the CFTR gene into CF airway epithelial cells and correct the chloride channel dysfunction. The intracellular fate of the lactosylated poly-l-lysine/cDNA complex was studied using confocal microscopy. In the presence of chloroquine the complex remained intact during internalization, intracellular transport, and, most importantly, transport into the nucleus. When cells were transfected in the presence of agents that enhance transfection efficiency such as E5CA peptide, a fusogenic peptide, or glycerol a similar fate of the lactosylated poly-l-lysine/cDNA complex was seen. However, when these agents were omitted from the transfection medium, the complex remained in the perinuclear region. Uncomplexed lactosylated poly-l-lysine reached the nucleus efficiently. In contrast mannosylated poly-l-lysine or unsubstituted poly-l-lysine complexed to plasmid did not. Therefore the nuclear accumulation of the complex may be attributed to the substitution of poly-l-lysine with lactose. It is hypothesized that the lactose residues provide for nuclear localization by means of targeting a potential lectin-like protein with galactose/lactose specificity. This mechanism may be responsible for the nuclear internalization of the complex.

The hydrophobic character of peanut (Arachis hypogaea) isoagglutinins.

Peanut seed lectin (PNA) is widely used to identify tumor-specific antigens on the eukaryotic cell surface. In this work PNA was purified by affinity chromatography, using a column containing glutaraldehyde-treated human erythrocytes, whereas PNA isoforms were purified by hydrophobic interaction chromatography using Phenyl-Sepharose. The affinity-purified PNA and its isoforms consist of four equal subunits of 24.5 kDa each, all of which agglutinated human sialidase-treated erythrocytes equally well; however, differences in their relative thermostabilities and sugar specificities for lactose were observed. Fractions PNA-I and PNA-II possess higher affinity for lactose residues than the more hydrophobic isoforms III and IV. These findings suggest that the differences observed in PNA isoagglutinins are due to hydrophobic regions of the protein that influence the three-dimensional organization of the molecule as well as its thermal stability and sugar specificity.

Glycation of bovine β‐Lactoglobulin: effect on the protein structure

Summary Since temperature and water activity are among the most important parameters that affect the Maillard reaction, the glycation sites in pure, native bovine β‐lactoglobulin were determined after a mild heat treatment (60 °C) in an aqueous solution and after a treatment under a restricted water environment (50 °C, 65% relative humidity). In both systems, the results obtained underlined the structural heterogeneity of β‐lactoglobulin (β‐LG) glycoforms with respect to the number of lactose residues linked per protein molecule and to the binding sites involved. Subsequently, the effect of the glycation conditions on both the association behaviour and the conformational changes of the glycated β‐LG were characterised by proteolytic susceptibility, binding of the fluorescent probe 8‐anilino‐1‐naphtalene‐sulfonic acid, SDS‐PAGE and size exclusion chromatography. The results showed that dry‐way glycation did not significantly alter the native‐like behaviour of the protein while the treatment in solution led to important structural changes. These changes resulted in a specific denatured β‐LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation to form high molecular weight species, via non–covalent interactions. The use of monoclonal antibodies with defined epitopes, raised against native β‐LG, confirmed that the protein conformation was much more modified when glycation was performed in a solution while the structural changes induced during dry‐way treatment were limited to the AB loop region of the protein.

Glycoconjugates of amino acids. Preparation throughN-alkylation of amino acids withN-chloroacetyl-β-glycopyranosylamines

Monoalkylation of amino acids of different structural types withN-chloroacetyl-glycosylamines was shown to be applicable for the preparation of glycoconjugates containing β-d-galactose,N-acetyl-β-d-glucosamine, β-d-mannose, and lactose residues. The glycoconjugates were synthesized from amino acids with secondary (sarcosine,l-proline) or primary (l-2- and 4-aminobutyric acids,l-tryptophan) amino groups as well as from various amino dicarboxylic acids (N-methyl-dl-aspartic,dl-aspartic,l-glutamic, anddl-2-aminoadipic acids). The derivatives obtained may be of interest for glycotargeting of physiologically active compounds of this series.

Formation of stable covalent dimer explains the high solubility at pH 4.6 of lactose-beta-lactoglobulin conjugates heated near neutral pH.

The solubility of lactose-beta-lactoglobulin conjugates at pH 4.6, after heating near neutral pH in phosphate buffer/0.116 M NaCI, was investigated by size exclusion chromatography and compared with unmodified protein. Heated conjugates in the temperature range 65-90 degrees C showed greater solubility at pH 4.6. The proportion of soluble protein increased with the number of bound lactose molecules. Total solubility was obtained for conjugates with nine lactose residues attached per monomer of beta-lactoglobulin. The protective effect of bound sugar toward precipitation was associated with the formation of soluble disulfide cross-linked dimers, highly accessible to trypsin digestion. These results suggested that bound lactose, through steric hindrance and high surface hydrophilicity, prevents the thiol-disulfide exchange reactions of the polymerization-aggregation process of lactose-beta-lactoglobulin conjugates.