Glioblastomas exploit truncated O-linked glycans for local and distant immune modulation via the macrophage galactose-type lectin.

Glioblastoma is the most aggressive brain malignancy, for which immunotherapy has failed to prolong survival. Glioblastoma-associated immune infiltrates are dominated by tumor-associated macrophages and microglia (TAMs), which are key mediators of immune suppression and resistance to immunotherapy. We and others demonstrated aberrant expression of glycans in different cancer types. These tumor-associated glycans trigger inhibitory signaling in TAMs through glycan-binding receptors. We investigated the glioblastoma glycocalyx as a tumor-intrinsic immune suppressor. We detected increased expression of both tumor-associated truncated O-linked glycans and their receptor, macrophage galactose-type lectin (MGL), on CD163+ TAMs in glioblastoma patient-derived tumor tissues. In an immunocompetent orthotopic glioma mouse model overexpressing truncated O-linked glycans (MGL ligands), high-dimensional mass cytometry revealed a wide heterogeneity of infiltrating myeloid cells with increased infiltration of PD-L1+ TAMs as well as distant alterations in the bone marrow (BM). Our results demonstrate that glioblastomas exploit cell surface O-linked glycans for local and distant immune modulation.

Label-free cytosensing of cancer cells based on the interaction between protein and an electron-transfer carbohydrate-mimetic peptide.

We used an electron-transfer carbohydrate-mimetic peptide (YYYYC) to construct an electrochemical cytosensing system. Magnetic beads were modified with either asialofetuin (ASF) or soybean agglutinin (SBA) to evaluate the effect on cell sensing. Because SBA binds to the galactose residue that exists at the terminals of the carbohydrate chains in ASF, the target protein was accumulated on the protein magnetic beads. SBA is an example of N-acetylgalactosamine- and galactose-binding proteins that readily combine with YYYYC. When the peptides and protein-immobilized beads competed for a target protein, the peak current of the peptides changed according to the concentration of the protein at the 10-12 M level. Next, human myeloid leukemia cells (K562 cell) were measured using the peptide and the carbohydrate chains on the cell surface that recognize SBA. The electrode response was linear to the number of K562 cells and ranged from 1.0 × 102 to 5.0 × 103 cells mL-1. In addition, detection of a human liver cancer cell (HepG2 cell) was carried out using interactions with the peptide, the ASF receptors in HepG2 cells, and the carbohydrate chains of ASF. The peak currents were proportional and ranged between 5.0 × 101 and 1.5 × 103 cells mL-1. When the values estimated from an electrochemical process were compared with those obtained by ELISA, the results were within the acceptable range of measurement error.

N-linked glycan truncation causes enhanced clearance of plasma-derived von Willebrand factor.

Essentials von Willebrands factor (VWF) glycosylation plays a key role in modulating in vivo clearance. VWF glycoforms were used to examine the role of specific glycan moieties in regulating clearance. Reduction in sialylation resulted in enhanced VWF clearance through asialoglycoprotein receptor. Progressive VWF N-linked glycan trimming resulted in increased macrophage-mediated clearance. Click to hear Dr Denis discuss clearance of von Willebrand factor in a free presentation from the ISTH Academy SUMMARY: Background Enhanced von Willebrand factor (VWF) clearance is important in the etiology of both type 1 and type 2 von Willebrand disease (VWD). In addition, previous studies have demonstrated that VWF glycans play a key role in regulating in vivo clearance. However, the molecular mechanisms underlying VWF clearance remain poorly understood. Objective To define the molecular mechanisms through which VWF N-linked glycan structures influence in vivo clearance. Methods By use of a series of exoglycosidases, different plasma-derived VWF (pd-VWF) glycoforms were generated. In vivo clearance of these glycoforms was then assessed in VWF-/- mice in the presence or absence of inhibitors of asialoglycoprotein receptor (ASGPR), or following clodronate-induced macrophage depletion. Results Reduced amounts of N-linked and O-linked sialylation resulted in enhanced pd-VWF clearance modulated via ASGPR. In addition to this role of terminal sialylation, we further observed that progressive N-linked glycan trimming also resulted in markedly enhanced VWF clearance. Furthermore, these additional N-linked glycan effects on clearance were ASGPR-independent, and instead involved enhanced macrophage clearance that was mediated, at least in part, through LDL receptor-related protein 1. Conclusion The carbohydrate determinants expressed on VWF regulate susceptibility to proteolysis by ADAMTS-13. In addition, our findings now further demonstrate that non-sialic acid carbohydrate determinants expressed on VWF also play an unexpectedly important role in modulating in vivo clearance through both hepatic ASGPR-dependent and macrophage-dependent pathways. In addition, these data further support the hypothesis that variation in VWF glycosylation may be important in the pathophysiology underlying type 1C VWD.

Herbal and polymeric approaches for liver-targeting drug delivery: novel strategies and their significance.

The liver is a vital organ present in vertebrates, which performs many functions including detoxification, protein synthesis and production of various bio-chemicals which are very important for digestion. A large number of serious liver disorders affect millions of people worldwide which are very difficult to treat properly despite many efforts. There are several factors which are responsible for liver injuries, include plants (Crotalaria Senecio Heliotropium Symphytum officinale), drugs (analgesic and antibiotics), industrial toxins (mercury and lead), water, alcohol and so on. Herbal medicinal preparations can be used for the treatment of a large number of human liver disorders like cirrhosis, hepatitis, carcinomas, etc. Indian Medicinal Practitioner's Co-operative pharmacy and Stores (IMPCPS) approved herbal-based systems (Unani, Siddha and Ayurveda) for the treatment of various chronic liver disorders. Different types of the receptors are found on the surface of hepatocytes, Kupffer cell, hepatic stellate cell and sinusoidal endothelial cells, etc., which can be used for achieving liver targeting. These receptors bind to different types of ligands (galactosylated, lactobionic acid, asialofetuin, etc.) which can be used in the formulation to achieve targeted delivery of the drug. Various novel particulate approaches (liposomes, niosomes, nanoparticles, micelles, nanosuspensions, etc.) can be used to enhance the targeting efficiency of systems to receptors found on the surface of different cells present in the liver. In this review, we focused on the status of liver targeting via herbal and nanotechnology inspired formulation approaches.

Carbohydrate-based materials for targeted delivery of drugs and genes to the liver.

The insult to liver by toxic materials leads to cirrhosis, hepatitis and cancer. Upon administration, drugs accumulate in liver, which is systemically cleared by reticuloendothelial system. However, specific targeting of drugs to liver is a serious challenge. Specific delivery of molecules to hepatocytes is accomplished by targeting cell surface lectins, asialoglycoprotein receptors. Asialofetuin, N-acetyl glucosamine and galactose are high-affinity ligands of asialoglycoprotein receptors. The bioconjugation of drugs, fluorescent molecules and gene delivery vectors with lectin-targeting agents, and their delivery in liver hepatocytes, is discussed. Mannose and N-acetyl glucosamine conjugates are evaluated for their delivery to hepatic stellate and kupffer cells. The glycosylated gene and drug delivery vectors in clinical trials are outlined.

Noninvasive imaging of sialyltransferase activity in living cells by chemoselective recognition.

To elucidate the biological and pathological functions of sialyltransferases (STs), intracellular ST activity evaluation is necessary. Focusing on the lack of noninvasive methods for obtaining the dynamic activity information, this work designs a sensing platform for in situ FRET imaging of intracellular ST activity and tracing of sialylation process. The system uses tetramethylrhodamine isothiocyanate labeled asialofetuin (TRITC-AF) as a ST substrate and fluorescein isothiocyanate labeled 3-aminophenylboronic acid (FITC-APBA) as the chemoselective recognition probe of sialylation product, both of which are encapsulated in a liposome vesicle for cellular delivery. The recognition of FITC-APBA to sialylated TRITC-AF leads to the FRET signal that is analyzed by FRET efficiency images. This strategy has been used to evaluate the correlation of ST activity with malignancy and cell surface sialylation, and the sialylation inhibition activity of inhibitors. This work provides a powerful noninvasive tool for glycan biosynthesis mechanism research, cancer diagnostics and drug development.

Chemical Approach to a Whole Body Imaging of Sialo-N-Linked Glycans.

PET and noninvasive fluorescence imaging of the sialo-N-linked glycan derivatives are described. To establish the efficient labeling protocol for N-glycans and/or glycoconjugates, new labeling probes of fluorescence and 68Ga-DOTA, as the positron emission nucleus for PET, through rapid 6π-azaelectrocyclization were designed and synthesized, (E)-ester aldehydes. The high reactivity of these probes enabled the labeling of lysine residues in peptides, proteins, and even amino groups on the cell surfaces at very low concentrations of the target molecules (~10⁻8 M) within a short reaction time (~5 min) to result in "selective" and "non-destructive" labeling of the more accessible amines. The first MicroPET of glycoproteins, 68Ga-DOTA-orosomucoid and asialoorosomucoid, successfully visualized the differences in the circulatory residence of glycoproteins, in the presence or absence of sialic acids. In vivo dynamics of the new N-glycoclusters, prepared by the "self-activating" Huisgen cycloaddition reaction, could also be affected significantly by their partial structures at the non-reducing end, i.e., the presence or absence of sialic acids, and/or sialoside linkages to galactose. Azaelectrocyclization chemistry is also applicable to the engineering of the proteins and/or the cell surfaces by the oligosaccharides; lymphocytes chemically engineered by sialo-N-glycan successfully target the tumor implanted in BALB/C nude mice, detected by noninvasive fluorescence imaging.

Galactose-functionalized multi-responsive nanogels for hepatoma-targeted drug delivery.

We report here a hepatoma-targeting multi-responsive biodegradable crosslinked nanogel, poly(6-O-vinyladipoyl-D-galactose-ss-N-vinylcaprolactam-ss-methacrylic acid) P(ODGal-VCL-MAA), using a combination of enzymatic transesterification and emulsion copolymerization for intracellular drug delivery. The nanogel exhibited redox, pH and temperature-responsive properties, which can be adjusted by varying the monomer feeding ratio. Furthermore, the volume phase transition temperature (VPTT) of the nanogels was close to body temperature and can result in rapid thermal gelation at 37 °C. Scanning electron microscopy also revealed that the P(ODGal-VCL-MAA) nanogel showed uniform spherical monodispersion. With pyrene as a probe, the fluorescence excitation spectra demonstrated nanogel degradation in response to glutathione (GSH). X-ray diffraction (XRD) showed an amorphous property of DOX within the nanogel, which was used in this study as a model anti-cancer drug. Drug-releasing characteristics of the nanogel were examined in vitro. The results showed multi-responsiveness of DOX release by the variation of environmental pH values, temperature or the availability of GSH, a biological reductase. An in vitro cytotoxicity assay showed a higher anti-tumor activity of the galactose-functionalized DOX-loaded nanogels against human hepatoma HepG2 cells, which was, at least in part, due to specific binding between the galactose segments and the asialoglycoprotein receptors (ASGP-Rs) in hepatic cells. Confocal laser scanning microscopy (CLSM) and flow cytometric profiles further confirmed elevated cellular uptake of DOX by the galactose-functionalised nanogels. Thus, we report here a multi-responsive P(ODGal-VCL-MAA) nanogel with a hepatoma-specific targeting ability for anti-cancer drug delivery.

Crosslinking of Cys-mutated human galectin-1 to the model glycoprotein ligands asialofetuin and laminin by using a photoactivatable bifunctional reagent.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. In our previous study, we showed that a human galectin-1 (hGal-1) mutant, in which a cysteine residue was introduced at Lys(28), forms a covalently cross-linked complex with the model glycoprotein ligands asialofetuin and laminin by using the photoactivatable sulfhydryl reagent benzophenone-4-maleimide (BPM). In the present study, we used several hGal-1 mutants in which single cysteine residues were introduced at different positions and examined their ability to form a covalent complex with asialofetuin or laminin by using BPM. We found that the efficiency of formation of the cross-linked products differed depending on the positions of the cysteine introduced and also on the ligand used for crosslinking. Therefore, by using different cysteine hGal-1 mutants, the chances of isolating different ligands for hGal-1 should increase depending on the systems and cells used.

Label-free detection of glycoproteins by the lectin biosensor down to attomolar level using gold nanoparticles.

We present here an ultrasensitive electrochemical biosensor based on a lectin biorecognition capable to detect concentrations of glycoproteins down to attomolar (aM) level by investigation of changes in the charge transfer resistance (Rct) using electrochemical impedance spectroscopy (EIS). On polycrystalline gold modified by an aminoalkanethiol linker layer, gold nanoparticles were attached. A Sambucus nigra agglutinin was covalently immobilised on a mixed self-assembled monolayer formed on gold nanoparticles and finally, the biosensor surface was blocked by poly(vinyl alcohol). The lectin biosensor was applied for detection of sialic acid containing glycoproteins fetuin and asialofetuin. Building of a biosensing interface was carefully characterised by a broad range of techniques such as electrochemistry, EIS, atomic force microscopy, scanning electron microscopy and surface plasmon resonance with the best performance of the biosensor achieved by application of HS-(CH2)11-NH2 linker and gold nanoparticles with a diameter of 20 nm. The lectin biosensor responded to an addition of fetuin (8.7% of sialic acid) with sensitivity of (338 ± 11) Ω decade(-1) and to asialofetuin (≤ 0.5% of sialic acid) with sensitivity of (109 ± 10) Ω decade(-1) with a blank experiment with oxidised asialofetuin (without recognisable sialic acid) revealing sensitivity of detection of (79 ± 13) Ω decade(-1). These results suggest the lectin biosensor responded to changes in the glycan amount in a quantitative way with a successful validation by a lectin microarray. Such a biosensor device has a great potential to be employed in early biomedical diagnostics of diseases such as arthritis or cancer, which are connected to aberrant glycosylation of protein biomarkers in biological fluids.

Fluorescent SNAP-tag galectin fusion proteins as novel tools in glycobiology.

Galectins,ß-galactoside binding proteins, function in several physiological and pathological processes. The further evaluation of these processes as well as possible applications of galectins in diagnosis and therapy has raised high scientific interest. Therefore, easy and reliable test systems are necessary. Here we present the simple and cost-efficient production of recombinant human galectins as fusion proteins with SNAP-tag and fluorescent proteins. These constructs show binding specificities and oligomerisation properties generally comparable to recombinant galectins. Their direct fluorescence signal was utilised by ELISA-type assay and flow cytometry analysis with human and ovine mesenchymal stem cells (MSC). Flow cytometry demonstrated glycan mediated binding of His6-SNAP-YFP-Gal- 3 to both MSC types, which was specifically inhibited by lactose. Moreover, directed immobilisation by SNAP-tag technology onto benzylguanine- activated sepharose was utilised to prepare galectin affinity columns for glycoprotein analysis and purification. The SNAPtag directed coupling yielded up to three-fold higher binding capacities for the glycoprotein standard asialofetuin compared to nondirected coupled galectin suggesting improved functionality following directed coupling.

Dual-labeled glycoclusters: synthesis and their application in monitoring lectin-mediated endocytosis.

To date, many efforts have been made to detect lectins in cells by using single imaging techniques. However, only a few dual-labeled glycan-based probes, which integrate advantageous features of two imaging methods to enhance the visualization of biological processes associated with lectins in cells, have been reported. Herein we describe the synthesis of dual fluorescence and magnetic resonance imaging agent conjugated neoglycopeptides and their application in the simultaneous imaging of lectins in mammalian cells. The dual-labeled neoglycopeptides bind to lectins on cell surfaces and subsequently enter the cells via lectin-mediated endocytosis. The results of these efforts show that the novel dual-labeled neoglycopeptides are effective fluorescence and MR imaging agents for monitoring biological processes associated with lectins.

Hoxc8 downregulates Mgl1 tumor suppressor gene expression and reduces its concomitant function on cell adhesion.

Hoxc8 is a homeobox gene family member, which is essential for growth and differentiation. Mgl1, a mouse homologue of the Drosophila tumor suppressor gene lgl, was previously identified as a possible target of Hoxc8. However, the biological effects and underlying molecular mechanism of Hoxc8 regulation on Mgl1 has not been fully established. The endogenous expression patterns of Hoxc8 were inversely correlated with those of Mgl1 in different types of cells and tissues. Here we showed that Hoxc8 overexpression downregulated the Mgl1 mRNA expression. Characterization of the ~2 kb Mgl1 promoter region revealed that the upstream sequence contains several putative Hox core binding sites and chromatin immunoprecipitation assay confirmed that Hoxc8 directly binds to the 5' upstream region of Mgl1. The promoter activity of this region was diminished by Hoxc8 expression but resumed by knockdown of Hoxc8 using siRNA against Hoxc8. Functional study of Mgl1 in C3H10T1/2 cells revealed a significant reduction in cell adhesion upon expression of Hoxc8. Taken together, our data suggest that Hoxc8 downregulates Mgl1 expression via direct binding to the promoter region, which in turn reduces cell adhesion and concomitant cell migration.

Bioconjugation using mutant glycosyltransferases for the site-specific labeling of biomolecules with sugars carrying chemical handles.

This chapter presents a technique that employs mutant glycosyltransferase enzymes for the site-specific bioconjugation of biomolecules via a glycan moiety to facilitate the development of a targeted drug delivery system. The target specificity of this methodology is based on unique sugar residues that are present on glycoproteins or engineered glycopeptides. The glycosyltransferases used in this approach have been manipulated in a way that confers the ability to transfer a modified sugar residue with a chemical handle to a sugar moiety of the glycoprotein or to a polypeptide tag of an engineered nonglycoprotein. The availability of the modified sugar moiety thus makes it possible to link cargo molecules at specific sites. The cargo may be comprised of, for example, biotin or fluorescent tags for detection, imaging agents for magnetic resonance imaging (MRI), or cytotoxic drugs for cancer therapy.

Cross-link formation between mutant galectins of Caenorhabditis elegans with a substituted cysteine residue and asialofetuin via a photoactivatable bifunctional reagent.

LEC-1 is the first tandem repeat-type galectin isolated from an animal system; this galectin has two carbohydrate recognition domains in a single polypeptide chain. Because its two lectin domains have different sugar-binding profiles, these domains are thought to interact with different carbohydrate ligands. In our previous study, we showed that a mutant of LEC-1 in which a cysteine residue was introduced at a unique position in the N-terminal lectin domain (Nh) can be cross-linked with a model glycoprotein ligand, bovine asialofetuin, by using a bifunctional photoactivatable cross-linking reagent, benzophenone-4-maleimide. In the present work, we applied the same procedure to the C-terminal lectin domain (Ch) of LEC-1. Cross-linked products were formed in the cases of two mutants in which a cysteine residue was introduced at Lys¹77 and Ser²68, respectively. This method is very useful for capturing and assigning endogenous ligand glycoconjugates with relatively low affinities to each carbohydrate recognition domain of the whole tandem repeat-type galectin molecule.

Cyclic neoglycodecapeptides: how to increase their inhibitory activity and selectivity on lectin/toxin binding to a glycoprotein and cells.

Protein (lectin/toxin)-glycan interaction can be clinically harmful so that the design of inhibitors has become an aim. Cyclic decapeptides are suited as rigid carriers for carbohydrate derivatives. We herein document the bioactivity of sugar headgroups covalently attached to this carrier for the cases of five proteins, i.e. a potent biohazardous plant agglutinin, a leguminous model lectin and three adhesion/growth-regulatory human lectins. They represent the different types of topological organization within the galectin family. The relative inhibitory activities of glycoclusters with the three ligands (galactose, lactose and the disaccharide of the Thomsen-Friedenreich antigen) reflected the affinity of free carbohydrates, hereby excluding an impairment of binding activity by chemical derivatization and conjugation. Headgroup tailoring is thus one route to optimize activity and selectivity of cyclopeptide-based glycoclusters. The increase of ligand density from tetra- to hexadecavalency added a second route. The plant toxin and tandem-repeat-type galectin-4 were especially sensitive to this parameter change. Strategically combining solid-phase assays for screening with analysis of lectin binding to cells in different systems revealed efficient inhibition by distinct glycoclusters, thereby protecting cells from lectin association. Cyclic neoglycodecapeptides thus warrant further study as lectin-directed pharmaceuticals.

Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro.

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.

Serum-resistant lipoplexes in the presence of asialofetuin.

Vectors proposed for gene delivery generally fall into two categories: viral and nonviral. They differ primarily in their assembling process. A viral vector is assembled in a cell, whereas a nonviral vector is constructed in a test tube. While vectors based on viral-based delivery systems are related to safety concerns, immune response, and formulation issues, the problem of nonviral ones is related to their low efficiency for encapsulating large DNA molecules, which has been an important technical obstacle to their utilization. Moreover, for most nonviral vectors, high efficiency in vitro transfection correlates with a global excess of cationic charges. This excess can in vivo facilitate nonspecific interactions with many undesired elements such as extracellular matrix and negatively charged serum components. Scientists have been using liposomes for gene delivery since the late 1970s. However, it was only after the introduction of cationic liposomes, which were shown to complex DNA and form the termed "lipoplexes," which offered some promise for an easy and efficient liposomal gene delivery. In this protocol, we describe the preparation of serum-resistant lipoplexes in the presence of the ligand asialofetuin (AF), in order to design efficient gene therapy carriers to deliver genes to the liver. It is also interesting to note, that although most of the current protocols imply covalent binding of the ligand, our complexes have been formulated by simple mixing of the three components in a studied and established order of addition. Lipoplexes containing the optimal amount of AF (1 microg/microg DNA) showed 16-fold higher transfection activity in HepG2 cells than nontargeted (plain) complexes.

Crosslinking of N-acetyllactosamine-containing glycoproteins to galectin-1 with an introduced cysteine using a photoactivatable sulfhydryl reagent.

Relatively weak interactions between galectins and their potential ligands can hinder identification of physiological lectin ligands using conventional methods such as affinity purification. We have employed a combination of cysteine mutagenesis with chemical crosslinking using a photoactivatable sulfhydryl reagent benzophenone-4-maleimide to obtain a covalent complex between human galectin-1 and the model glycoprotein ligands asialofetuin and laminin which contain an N-acetyllactosamine structure. A crosslinked product was obtained only when galectin-1 with an introduced cysteine interacted with these glycoproteins via their carbohydrate moiety. This procedure should be useful for the detection of important, and as yet unidentified, ligands for galectins which cannot be currently detected because of their relatively weak interaction.

In vivo targeted gene delivery by cationic nanoparticles for treatment of hepatocellular carcinoma.

BACKGROUND: Transgene expression in vivo for therapeutic purposes will require methods that allow for efficient gene transfer into cells. Although current vector technologies are being improved, the development of novel vector systems with improved targeting specificity, higher transduction efficiencies and improved safety is necessary. METHODS: Asialoglycoprotein receptor-targeted cationic nanoparticles for interleukin (IL)-12 encapsulation (NP1) or adsorption (NP2) have been formulated by blending poly(D,L-lactic-co-glycolic) acid (PLGA) (50 : 50) with the cationic lipid 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and the ligand asialofetuin (AF), by using a modified solvent evaporation process. RESULTS: We present a novel targeted lipopolymeric vector, which improves significantly the levels of luciferase gene expression in the liver upon i.v. administration. Targeted-NP2 particles showed a five- and 12-fold higher transfection activity in the liver compared to non-targeted (plain) complexes or naked pCMV DNA, respectively. On the other hand, BNL tumor-bearing animals treated with AF-NP1 containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice, without signs of recurrence. High levels of IL-12 and interferon-gamma were detected in the sera of treated animals. Mice survival also improved considerably. Tumor treatment with AF-NP2 formulations lead only to a retardation in the tumor growth. CONCLUSIONS: In the present study, we have developed an efficient targeted non-viral vector for IL-12 gene transfer in hepatocellular carcinoma in vivo, by employing non-toxic cationic PLGA/DOTAP/AF nanoparticles. These results demonstrate for the first time that this cationic system could be used successfully and safely for delivery of therapeutic genes with antitumor activity into liver tumors with targeting specificity.