Galactosyl Ligands Research Articles

A label-free impedimetric cytosensor based on galactosylated gold-nanoisland biointerfaces for the detection of liver cancer cells in whole blood

Circulating tumor cells (CTCs), as potential cancer biomarkers, play a crucial role in cancer diagnosis, prognosis and treatment. However, there are still significant challenges due to their rarity. In this paper, a label-free and ultrasensitive electrochemical impedance cytosensor was developed by a galactosylated gold-nanoisland (Gal-GNI) biointerface for the capture and detection of rare cancer cells (HepG-2) in whole blood samples. By mimic the natural multivalent biorecognition systems, the biointerface was designed to capture the target cancer cells by the specific binding between galactosyl ligands of Gal-GNI and over-expressed asialoglycoprotein receptors (ASGP-R) on the surface of HepG-2. From the results of electrochemical impedance spectroscopy (EIS) and fluorescence spectroscopy, the nanostructured Gal-GNI provides an improved electron-transfer rate, specific detection for HepG-2, and good biocompatibility for viable captured cells. The capture of HepG-2 to the EIS cytosensor increased the electron-transfer resistance, with a good correlation with the logarithm of the concentration from 1.0×102 to 1.0×105cellsmL−1, with a low detection limit of 30cellsmL−1. This cytosensing strategy has good reproducibility with the RSD of 1.9% in blood samples, indicating that it can be used as a potential noninvasive assay tool for the early diagnosis and therapy of tumor.

Spacer Length: A Determining Factor in the Design of Galactosyl Ligands for Hepatoma Cell-Specific Liposomal Gene Delivery.

Use of nucleic acids to treat acquired or inherited hepatic diseases has considerable potential. Although recombinant viruses are popular vectors, interest in cheaper, often less immunogenic, non-viral modalities, is increasing. Thus hepatotropic, galactosylated lipoplexes directed to the hepatic asialoglycoprotein receptor (ASGP-R) are promising candidates. Here we examine the effect that galactosyl ligand spacer length has on the transfection activity of ASGP-Rtargeted lipoplexes in the human hepatoma cell line HepG2. Galactosyl ligands with spacer lengths in the range 2.4-24.1 Å were prepared and formulated into lipoplexes that were characterized by cryo-TEM, band shift, dye displacement and nuclease digestion assays. Cytotoxicity and transfection profiles were determined in liver-derived HepG2 cells and the renal ASGP-R-negative HEK293 line. Lipoplexes, which formed at endpoint +/- charge ratios in the range 1:1-3:1, accorded cargo DNA good protection from serum nuclease digestion and were well-tolerated by both cell lines. Transfection activities in the hepatoma cell line decreased markedly in the presence of a competing ASGP-R cognate ligand and also as the ligand spacer length increased, while activities in HEK293 cells were significantly lower (P <0.05-0.001). Targeted lipoplexes enter HepG2 cells by receptor mediation and the uptake of transfecting complexes and those displaying more rigid short and medium length spacers is more efficient. This observation will inform the design of hepatotropic lipoplexes that are suitable for applications in vivo.

Optimization of lipase-catalyzed synthesis of novel galactosyl ligands for selective targeting of liposomes to the asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is a potential target in the search for hepatic cancer drugs. However, application of ASGPR targeting in the clinic is limited by inefficient synthetic methods for the ligand. In this study, we designed and synthesized a novel galactosylated lipid with a mono-galactoside moiety using a lipase. Then we investigated the optimal reaction conditions and analyzed the targeting ability of liposomes modified with the galactosylated lipid. In an organic phase system, different lipases were used as catalysts to synthesize (5-cholesten-3b-yl) [(4-O-β-D-galactopyranosyl)D-glucitol-6] sebacate (CHS-SE-LA). Variables in enzymatic esterification, such as the type of enzyme and solvent, were explored by single-factor experiments. Optimal reaction conditions were determined through response surface methodology. The (CHS-SE-LA)-incorporated galactosylated liposome containing fluorescent dye was then prepared by thin-film hydration and a HepG2 cell transfection test used to confirm the targeting efficiency of galactosylated liposomes to hepatic cancer cells. The structure of CHS-SE-LA was identified by electrospray ionization or ESI and nuclear magnetic resonance or NMR. Under optimal conditions, the predicted yield of CHS-SE-LA was 94.3%, and the actual experimental value was 95.6 ± 1.35%, n = 3. For HepG2 cells, the cellular fluorescence intensities of liposomes modified with CHS-SE-LA (galactosylated liposomes [GAL-FL]) were as much as 2.6-fold (P < 0.01) the control liposomes (FL). Moreover, the presence of excess galactose significantly inhibited the uptake of GAL-FL suggesting ASGPR mediated uptake. The novel galactosylated ligand was synthesized enzymatically with high efficiency under mild conditions. Liposomes containing CHS-SE-LA have great potential as drug delivery carriers for hepatocyte-selective targeting.

Capturing intercellular sugar-mediated ligand-receptor recognitions via a simple yet highly biospecific interfacial system

Intercellular ligand-receptor recognitions are crucial natural interactions that initiate a number of biological and pathological events. We present here the simple construction of a unique class of biomimetic interfaces based on a graphene-mediated self-assembly of glycosyl anthraquinones to a screen-printed electrode for the detection of transmembrane glycoprotein receptors expressed on a hepatoma cell line. We show that an electroactive interface confined with densely clustered galactosyl ligands is able to ingeniously recognize the asialoglycoprotein receptors on live Hep-G2 cells employing simple electrochemical techniques. The only facility used is a personal laptop in connection with a cheap and portable electrochemical workstation.

Evaluating the potential of a new isotope‐labelled glyco‐ligand for estimating the remnant liver function of schistosoma‐infected mice

A new glyco-derivative compound (OCTAM) was developed and labelled with isotope to form (188) Re-OCTAM as a candidate nuclear medicine imaging agent for testing the liver function. We evaluated the potential of isotope-labelled OCTAM for estimating the remnant liver function in vitro and in vivo schistosoma-infected mice. The affinity of OCTAM to liver asialoglycoprotein receptors (ASGPR) was assessed by competitive inhibition assay in vitro. In vivo assessments were performed to score the remnant liver function in mice at different schistosomal infection stages. OCTAM binds specifically to ASGPR and showed competitive inhibition of anti-ASGPR antibody binding to hepatocytes, and was higher than that of other galactosyl ligands. Micro-SPECT/CT images of uninfected mice revealed strong liver uptake. Quantified serial images of mice infected for 9, 12 and 18weeks showed delayed liver uptake, and the retention of uptake was inversely correlated with stage and grade of schistosoma infection. Pathological and biochemical analysis demonstrated that gradually accumulating liver injury caused by infection significantly influenced uptake of (188) Re-OCTAM. Hepatic ASGPR expression diminished only in the chronic infection stage. This study demonstrated that the isotope-labelled OCTAM could accumulate in the liver, might have potential as an imaging agent for in vivo hepatic function evaluation of schistosomiasis.

Trivalent galactosyl-functionalized mesoporous silica nanoparticles as a target-specific delivery system for boron neutron capture therapy

A multi-functional mesoporous silica nanoparticle (MSN)-based boron neutron capture therapy (BNCT) agent, designated as T-Gal-B-Cy3@MSN, was synthesized with hydrophobic mesopores for incorporating a large amount of o-carborane (almost 60% (w/w) boron atoms per MSN), and the amines on the external surface were conjugated with trivalent galactosyl ligands and fluorescent dyes for cell targeting and imaging, respectively. The polar and hydrophilic galactosyl ligands enhance the water dispersibility of the BNCT agent and inhibit the possible leakage of o-carborane loaded in the MSN. Confocal microscopic images showed that T-Gal-B-Cy3@MSNs were endocytosed by cells and were then released from lysosomes into the cytoplasm of cells. Moreover, in comparison with the commonly used clinical BNCT agent, sodium borocaptate (BSH), T-Gal-B-Cy3@MSN provides a higher delivery efficiency (over 40-50 fold) of boron atoms and a better effect of BNCT in neutron irradiation experiments. MTT assays show a very low cytotoxicity for T-Gal-B-Cy3@MSN over a 2 h incubation time. The results are promising for the design of multifunctional MSNs as potential BNCT agents for clinical use.

Bifunctional bacterial magnetic nanoparticles for tumor targeting

Bifunctional bacterial magnetic nanoparticles (BBMPs), which present both magnetic drug targeting and tumor bio-targeting properties, have been developed by chemically coupling both doxorubicin and a galactosyl ligand on to the membrane surface of the bacterial magnetic nanoparticles (BMPs). The BBMP product has a high drug load ratio and magnetic respondence, and exhibits a narrow size distribution and is sensitive to pH to enable drug release. In comparison to doxorubicin-coupled BMPs, without modification with a galactosyl ligand, BBMPs present a higher uptake by the target asialoglycoprotein receptor (ASGP-R) expressed by HepG2 cells and display stronger cytotoxicity.

Synthesis of bivalent neogalactolipids via modified Staudinger reaction

The synthesis of bivalent galactose-containing neutral lipids for the development of a targeted gene delivery system to the hepatocytes has been described. For introducing galactosyl ligands into the lipid molecule, a convenient approach based on modified Staudinger reaction was used.

Galactose Encapsulated Multifunctional Nanoparticle for HepG2 Cell Internalization

AbstractThe fluorescent dye Cy3 and galactose derivatives are covalently assembled with different ratios on the surfaces of magnetic nanoparticles (MNPs) to produce multifunctional HepG2 cancer cell–targeting agents and the effect of ligand spatial orientation on the MNP surface is investigated on targeting specificity. By using a mixture of bis‐N‐hydroxysuccinimide ester (a bifunctional linker) and OSu‐activated Cy3 (w/w = 30:1), stable and quantifiable fluorescent MNPs (Cy3@MNPs) are synthesized that could be subsequently loaded with galactosyl ligands. A mono‐antennary and two different tri‐antennary galactosyl ligands are individually immobilized on Cy3@MNPs, and the uptake efficiencies of the resulting galactosyl Cy3@MNPs by HepG2 and HeLa cells are investigated using confocal microscopy. The confocal images show that galactosyl Cy3@MNPs are sprayed over cytoplasm of the HepG2 cells, indicating that the MNP uptake occurs via receptor‐mediated endocytosis that is followed by release from endosomes. The results also reveal that the ligand spatial orientation affects the efficiency of the receptor‐mediated endocytosis and one of the tri‐antennary galactosyl ligands shows the best uptake efficiency owing to the optimal spatial presentation of the galactosyl moieties. Overall, it is shown that the MNP is a good ligand carrier and that, when pre‐assembled, the multivalent ligand structure enhances the interactions between the surface ligands of the MNPs and receptors of HepG2 cells. Additionally, the galactosyl Cy3@MNPs are not cytotoxic, indicating that they may potentially be used for in vivo applications.

Galactosylated solid lipid nanoparticles with cucurbitacin B improves the liver targetability

This study intended to prepare liver-targeting solid lipid nanoparticles (SLNs) with a hepatoprotective drug, cucurbitacin B (Cuc B), using a galactosylated lipid, N-hexadecyl lactobionamide (N-HLBA). The galactosyl-lipid N-HLBA was prepared via the lactone form intermediates of lactobionic acid and synthesized by anchoring galactose to hexadecylamine lipid. The Cuc B-loaded galactosylated and conventional SLNs were successfully prepared by a high-pressure homogenization method. The two SLNs showed similar physical and pharmaceutical properties, including: the particle size measured by laser diffraction was 135 nm for galactosylated SLN (GalSLN) and 123 nm for conventional SLNs (CSLN); zeta potentials were −31.6 mV (GalSLN) and −34.3 mV(CSLN); in vitro release behavior of the two SLNs was similar, and both showed the biphasic drug release pattern with burst release at the initial stage and prolonged release afterwards. In contrast, the two SLNs demonstrated a marked difference in in vitro cellular cytotoxicity and in vivo tissue distribution performances. The IC50 values of Cuc B in the two SLNs were by far lower than those of Cuc B solution and further Cuc B-GalSLN had about half the IC50 value of Cuc B-CSLN. These results indicated that the encapsulation of Cuc B in SLNs resulted in the enhancement of cytotoxic activity, and galactosyl ligand could further enhance the cellular accumulation and cytotoxicity of Cuc B. The weighted-average overall drug targeting efficiency (Te) was used to evaluate the liver targetability. Cuc B-GalSLN gave a relatively high (Te)liver value of 63.6%, ∼ 2.5-times greater than that of Cuc B-CSLN (25.3%) and Cuc B solution (23.8%). In summary, the incorporation of N-HLBA into SLNs significantly enhanced the liver targetability of Cuc B-loaded SLNs and GalSLN had a great potential as a drug delivery carrier for improved liver targetability.

A new triantennary galactose-targeted PEGylated gene carrier, characterization of its complex with DNA, and transfection of hepatoma cells.

Nonviral gene vectors remain inefficient in vivo largely because of their rapid clearance from the circulation and also their nonspecific association with the extracellular matrix. To overcome such drawbacks, cationic lipoplexes are now frequently coated with hydrophilic polymers such as PEGs to reduce nonspecific interactions, and ligands are also linked to their surface to obtain cell-specific gene transfer. In view of the development of vectors for systemic gene delivery, we have designed and studied lipoplexes that carry a triantennary galactosyl ligand attached to the distal end of a (PEG)45-conjugated lipid. We incorporated this targeted PEGylated lipid into lipoplexes using two strategies of formulation, i.e., using either preformed micelles or liposomes. We demonstrated that the incorporation of PEG chains stabilized lipoplexes and masked, but only partially, the positive charges exposed on the surface of the particles. We have also shown that incorporation into lipoplexes of a lipidated PEG chain, bearing a ligand at its distal end, yielded particles that exhibited an accessible ligand throughout the whole range of cationic lipid to DNA ratios. We obtained a targeted transfection in HepG2 cells with one of the formulations. Our results strengthen the validity of using a ligand carried by a long PEG spacer arm for targeted gene transfer.

SYNTHESIS OF NEW GALACTOSYL AND LACTOSYL CARBAMATE-CONTAINING GLYCOLIPIDS

An efficient synthesis of mono-, di- and tetrasaccharides linked to a lipid has been developed. Galactose or lactose was covalently coupled to a glycyldioctadecylamide, via well-defined chemical steps, in both an α and β anomeric configuration. The multiantennary galactosyl ligands were obtained using 1,3-diamino-2-propanol as a scaffold.

Synthesis of bifunctional cationic compound for gene delivery

Bifunctional cationic compound carrying trivalent galactosides as the cell targeting ligand and DAB-dendr-(NH2)8 (generation 2.0) as the DNA binding domain was synthesized for gene delivery to hepatocytes. DAB-dendr-(NH2)4 (generation 1.0) conjugated with a hydrocarbon chain was used as a scaffold for the attachment of three galactosides, while the other hydrocarbon end was linked with DAB-dendr-(NH2)8 (generation 2.0) through a carbamate bond. This design provided an effective entry for the synthesis of a polyamine compound having the cell targeting galactosyl ligand. Preliminary in vitro transfection results demonstrated that the bifunctional cationic compound could effectively deliver the gene into HepG2 cells.

Syntheses of Novel Galoctosyl Ligands for Liposomes and the Influence of the Spacer on Accumulation in the Rat Liver.

We modified the surface of liposomes with galactosyl ligands. At first we determined whether or not the galactosyl moiety was exposed on the liposomes. We then investigated the effect of the ligands on the hepatic accumulation of liposomes in rats. We introduced an oligoethylene glycol moiety as a spacer. Among the various ligands tested, those with a tri- or tetraethylene glycol moiety as a spacer caused the greatest accumulation of liposomes in the liver. Liposomes bearing ligands with a tri- or tetraethylene glycol moiety as a spacer, were aggregated by Ricinus communis agglutinin. On the other hand, those modified with ligands with a mono- or diethylene glycol spacer did not clearly agglutinate. These results show the importance of a spacer between the homing device and the ligand anchor.

Relationship between the Anchor Structure of the Galactosyl Ligand for Liposome Modification and Accumulation in the Liver.

Liposomes which have been modified with (8-hexadecanoylamido-3,6-dioxaoctyl)-beta-D-galactose (Gal-t-pa), a straight chain palmitoyl derivative, and are composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (CH), and dicetyl phosphate (DCP) at a ratio of 10:10:1, showed the same accumulation in the liver as the control liposome. Also, liposomes which have been modified with [8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl]-beta-D-gal actoside (Gal-t-psa) showed remarkable accumulation in the liver. The accumulation of liposomes modified with galactose derivatives in the rat liver differed markedly according to the anchor structure. To clarify the cause of this finding, we produced [3H]inulin entrapped [14C]Gal-t-pa modified double label liposomes and evaluated changes in their rat plasma concentration, distribution in the organs, and the in vitro interaction with rat plasma. [14C]Gal-t-pa on the liposome surface bound to serum albumin and was released, resulting in no accumulation in the liver. In addition, sialic acid palmitoyl derivatives and glucuronic acid palmitoyl derivatives behaved similarly. As with the galactose derivatives, they also bound to serum albumin, being released from liposomes. These results suggest that adequate attention should be paid to the anchor structure of the ligand, in order to incorporate a recognition element into liposomes for transport to cells.

Syntheses of novel galactosyl ligands for liposomes and their accumulation in the rat liver.

The effects of liposomes, bearing galactosyl ligands on their surface, on their clearance from circulation and on tissue distribution were studied in rats. The ligands were obtained through coupling 2,3,4,6-tetraacetyl-(2-[2-(2-aminoethoxy)ethoxy]ethyl)-beta- D-galactoside p-toluenesulfonate with hydrophobic anchors, followed by deprotection. We introduced mid-chain or long, branched or non-branched alipathic chains, and cholesterol as anchors. Among various anchors tested, that with a dihexadecyl branch caused the greatest accumulation of liposomes in the liver. On the other hand, liposome bearing these ligands were aggregated by Ricinus communis agglutin. This showed that these ligands were similarly incorporated to liposomes. These results show the importance of the balance between lipophilicity and the structure of ligands.

Influence of the galactosyl ligand structure on the interaction of galactosylated liposomes with mouse peritoneal macrophages

Liposomes bearing at their surface mono- and triantennary galactosyl ligands were prepared and their interaction with the galactose receptor of mouse peritoneal macrophages studied. Triantennary structures were synthesized by coupling derivatives of 1-thio-beta-D-galactose to the amino groups of lysyl-lysine dipeptide. Galactosylated liposomes were obtained either by synthesis of neo-galactolipids followed by their incorporation into the vesicles or by neo-galactosylation of performed liposomes by reaction between thiol-functionalized galactosyl ligands and vesicles bearing maleimido groups. The interaction of the galactosylated liposomes with the macrophage lectin was remarkably sensitive to the topology of the ligands, i.e., a spacer-arm length about 3 nm was necessary and, in contrast to results obtained with the galactose receptor of other cells, the triantennary structure did not provide additional binding. Related to the strategy of drug delivery with targeted liposomes, these results indicate that lectins from different cells might possibly be distinguished by using multiantennary ligands having optimal geometries.

Studies on lectins: XXXIX. S-glycosyl polyacrylamide gels for affinity chromatography of lectins

Hydrophilic water-insoluble gels suitable for affinity chromatography of lectins have been prepared by copolymerization of acrylamide, N, N′-methylene bisacrylamide and alkenyl 1-thioglycosides. Water-soluble copolymers of analogous type have been obtained by omitting the cross-linking agent, N, N′-methylene bisacrylamide. In affinity chromatography of the Ricinus communis lectin it could be shown that the capacity for the lectin of the water insoluble copolymers was more than four times higher in copolymers having the S-β- D- galactosyl ligand attached through a methylene bridge than in derivatives with a nonamethylene spacer. None of the insoluble S-β- D- glycosyl copolymers prepared could be shown usable as affinity adsorbent for glycosidases though the corresponding soluble copolymers inhibited the activity of the enzymes.