还原可降解聚磺酸甜菜碱型纳米水凝胶的制备及载药研究

目的:制备与表征还原可降解的聚磺酸甜菜碱型纳米水凝胶,利用该纳米递药系统包载阿霉素(DOX)并初步评价其抗肿瘤性能。方法:利用回流沉淀聚合的方法合成含二硫键的聚磺酸甜菜碱甲基丙烯酸酯(PSBMA)纳米水凝胶及不含二硫键的PSBMA纳米凝胶(nd-PSBMA);通过粒度仪和透射电镜考察两种纳米水凝胶的粒径、形态以及稳定性;通过考察谷胱甘肽(GSH)对纳米凝胶溶液相对浊度的影响以评价还原环境对两种纳米凝胶的还原可降解性;利用纳米凝胶包载阿霉素(DOX),考察载药凝胶在GSH中的释药行为,并初步评价其对A549肿瘤细胞的杀伤作用。结果:以N, N'-双丙烯酰胱胺为交联剂制备了含二硫键的PSBMA纳米凝胶,其粒径在180～200 nm;同时以N, N'-双丙烯酰胺为交联剂制备了不含二硫键的n-PSBMA纳米凝胶。两种纳米凝胶与小鼠血清共孵育7天水合粒径仍无明显变化,表明磺酸甜菜碱型纳米凝胶具有良好的抗蛋白吸附能力。此外,PSBMA纳米凝胶在GSH溶液中迅速地降解,且降解速度与GSH浓度呈正相关;而nd-PSBMA纳米凝胶在GSH溶液中几乎不降解。载DOX的PSBMA纳米凝胶可在GSH作用下快速的释放药物而载DOX的nd-PSBMA纳米凝胶在GSH作用下缓慢的释放药物;体外细胞实验显示空白纳米凝胶和载药nd-PSBMA对A549细胞无明显毒性作用,但载DOX的PSBMA纳米凝胶可高效地杀死A549肿瘤细胞,其药效与游离DOX相仿。结论:还原可降解的PSBMA纳米水凝胶有望成为智能型控释药物载体。     

Galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers as hepatocyte-targeting gene carriers

As alternatives of viral and cationic lipid gene carriers, cationic polymer-based vectors may provide flexible chemistry for the attachment of targeting moieties. In this report, galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers (galactosylated HPMA-b-GPMA block copolymers, or abbreviated as GHG) were prepared in order to develop hepatocyte targeting gene transfection carriers. The block copolymers were synthesized by aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization of N-2-hydroxypropyl methacrylamide (HPMA) and N-3-aminopropyl methacrylamide (APMA), followed by galactosylation and guanidinylation. The molecular weight of GHG copolymers determined by static light scattering method was in the range from 48?600 to 76?240 g/mol. In addition, the galactose content in the GPMA block in the copolymers was determined to be 6.5-8.0 mol % according to the sulfuric acid method. The GHG copolymers complexed completely with plasmid DNA (pDNA) to show positive zeta-potential values with diameter 100-250 nm from charge ratio of 4, which demonstrated the excellent DNA condensing ability of guanidino groups. Furthermore, the MTT assay data of GHG/pDNA complexes on HepG2 cells and HeLa cells indicated that GHG copolymers had significantly lower cytotoxicity than PEI. In addition, the copolymers with GPMA component from 30.23% showed higher transfection efficiency than PEI at charge ratio of 12 in HepG2 cells. The result revealed that the conjugation of galactose groups in the copolymers brought asialoglycoprotein-receptor (ASGP-R) mediated transfection. The employing of HPMA component decreased the aggregation of protein in transfection presence of serum. The GHG copolymers combined the advantages of galactose moieties, guanidino groups, and HPMA component might show potential in safe hepatocyte targeting gene therapy.     

Coexpression of alpha1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae

We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system. Two heterologous genes, gma12(+) encoding alpha1, 2-galactosyltransferase (alpha1,2 GalT) from Schizosaccharomyces pombe and UGT2 encoding UDP- galactose (UDP-Gal) transporter from human, were functionally expressed to examine the intracellular conditions required for galactosylation. Detection by fluorescence labeled alpha-galactose specific lectin revealed that 50% of the cells incorporated galactose to cell surface mannoproteins only when the gma12(+) and hUGT2 genes were coexpressed in galactose media. Integration of both genes in the Delta mnn1 background cells increased galactosylation to 80% of the cells. Correlation between cell surface galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter rather than alpha1,2 GalT played a key role for efficient galactosylation in S.cerevisiae. In addition, this heterologous system enabled us to study the in vivo function of S. pombe alpha1,2 GalT to prove that it transfers galactose to both N - and O -linked oligosaccharides. Structural analysis indicated that this enzyme transfers galactose to O -mannosyl residue attached to polypeptides and produces Galalpha1,2-Man1-O-Ser/Thr structure. Thus, we have successfully generated a system for efficient galactose incorporation which is originally absent in S. cerevisiae, suggesting further possibilities for in vivo glycan remodeling toward therapeutically useful galactose containing heterologous proteins in S. cerevisiae.      

Effect of Environmental Parameters on Glycosylation of Recombinant Immunoglobulin G Produced from Recombinant CHO Cells

Site specific glycosylation of immunoglobulin G (IgG) occurs at Asn297 in the Fc region. The heterogeneous ensemble of glycoform occurs due to the degree of terminal galactosylation and sialylation, and these differences in glycosylation affect both the pharmacokinetic behavior and effector functions of the IgG, such as complementdependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). In this study, the differential glycosylation of IgG was compared and environmental physical and chemical parameters were evaluated in an attempt to promote glycosylation of recombinant antibodies, thereby creating more humanized glycoform antibodies and increasing their in vivo efficacy as therapeutic drugs. It was shown that cells at late stationary growth phase in batch cultures, cells with increased passage number, and the culture conditions of lowered temperature and pH promoted galactosylation and sialylation of antibodies. Galactose, fructose and mannose were found to elicit galactosylation and sialylation when they were used alone as a substitute of glucose. Mannose showed synergistic effects on glycosylation when used with other sugars, such as glucose and galactose. However when fructose was used with other sugars, the degree of galactosylation mechanism appeared to be decreased. These results support understandings of the glycosylation mechanisms in glycoprotein, particularly recombinant antibodies for therapeutics.     

An HPLC method for determination of oridonin in rabbits using isopsoralen as an internal standard and its application to pharmacokinetic studies for oridonin-loaded nanoparticles

A simple and sensitive HPLC method has been developed and validated for the determination of oridonin (ORI) in rabbit plasma. A simple liquid-liquid extraction (LLE) method was applied to extract ORI and the internal standard (IS), isopsoralen, from rabbit plasma. Chromatographic separation of ORI and the IS was achieved with a Kromasil C18 5-mum column (250 mm x 4.6 mm) using methanol-water (50:50, v/v) as mobile phase at a flow rate of 1 mL/min. The ultraviolet (UV) detection wavelength was set at 241 nm. The lower limit of quantification (LLOQ) was 0.02 microg/mL. The calibration curves were linear over a concentration range of 0.02-10 microg/mL. The assay accuracy and precision were within the range of 95.1-113.5% and 5.4-8.6%, respectively. This HPLC method was applied successfully to the pharmacokinetic study of ORI-loaded poly(caprolactone)-poly(ethylene oxide)-poly(caprolactone) copolymer nanoparticles (ORI-PCL-PEO-PCL-NP) in rabbits, given as a single intravenous injection at the dose equivalent to 2mg of ORI/kg, and the pharmacokinetic parameters for ORI were compared with a single intravenous injection of a ORI solution at the same dose.     

Galactosylation variations in marketed therapeutic antibodies

There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary (CHO) cells or mouse myeloma cells as the expression system. Because CHO and mouse myeloma cells are mammalian cells, rIgGs produced in these cell lines are typically N-glycosylated at the conserved asparagine (Asn) residues in the CH2 domain of the Fc, which is also the case with serum IgGs. The Fc glycans present in these rIgGs are for the most part complex biantennary oligosaccharides with heterogeneity associated with the presence or the absence of several different terminal sugars. The major Fc glycans of rIgGs contain 0 or 1 or 2 (G0, G1 and G2, respectively) terminal galactose residues as non-reducing termini and their relative proportions may vary depending on the cell culture conditions in which they were expressed. Since glycosylation is strongly associated with antibody effector functions and terminal galactosylation may affect some of those functions, a panel of commercially available therapeutic rIgGs expressed in CHO cells and mouse myeloma cells were examined for their galactosylation patterns. The results suggest that the rIgGs expressed in CHO cells are generally less galactosylated compared to the rIgGs expressed in mouse myeloma cells. Accordingly, rIgGs produced in CHO cells tend to contain higher levels of G0 glycans compared with rIgGs produced in mouse myeloma cell lines. Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies. Nevertheless, galactosylation may have an effect on the mechanisms of action of some therapeutic antibodies (e.g., effector pathways) and hence further studies to assess effects on product efficacy may be warranted for such antibodies. For antibodies that do not require effector functions for biological activity, however, setting a narrow specification range for galactose content may be unnecessary.     

Galactosylation variations in marketed therapeutic antibodies

There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary (CHO) cells or mouse myeloma cells as the expression system. Because CHO and mouse myeloma cells are mammalian cells, rIgGs produced in these cell lines are typically N-glycosylated at the conserved asparagine (Asn) residues in the CH2 domain of the Fc, which is also the case with serum IgGs. The Fc glycans present in these rIgGs are for the most part complex biantennary oligosaccharides with heterogeneity associated with the presence or the absence of several different terminal sugars. The major Fc glycans of rIgGs contain 0 or 1 or 2 (G0, G1 and G2, respectively) terminal galactose residues as non-reducing termini and their relative proportions may vary depending on the cell culture conditions in which they were expressed. Since glycosylation is strongly associated with antibody effector functions and terminal galactosylation may affect some of those functions, a panel of commercially available therapeutic rIgGs expressed in CHO cells and mouse myeloma cells were examined for their galactosylation patterns. The results suggest that the rIgGs expressed in CHO cells are generally less galactosylated compared to the rIgGs expressed in mouse myeloma cells. Accordingly, rIgGs produced in CHO cells tend to contain higher levels of G0 glycans compared with rIgGs produced in mouse myeloma cell lines. Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies. Nevertheless, galactosylation may have an effect on the mechanisms of action of some therapeutic antibodies (e.g., effector pathways) and hence further studies to assess effects on product efficacy may be warranted for such antibodies. For antibodies that do not require effector functions for biological activity, however, setting a narrow specification range for galactose content may be unnecessary.     

Characteristics of the killing mechanism of human natural killer cells against hepatocellular carcinoma cell lines HepG2 and Hep3B

Purpose Unlike normal hepatocytes, most hepatocellular carcinomas (HCCs) are quite resistant to death receptor-mediated apoptosis when the cell surface death receptor is cross linked with either agonistic antibodies or soluble death ligand proteins in vitro. The resistance might play an essential role in the escape from the host immune surveillance; however, it has not been directly demonstrated that HCCs are actually resistant to natural killer (NK) cell-mediated death. Therefore, this study investigated the molecular mechanism of NK cell-mediated cytotoxicity against the HCCs, HepG2, and Hep3B, using two distinct cytotoxic assays: a 4-h ⁵¹Cr-release assay and a 2-h [³H] thymidine release assay which selectively measures the extent of necrotic and apoptotic target cell death, respectively.Methods Most of the target cells exhibited marked morphologic changes when they were co-incubated with the NK cells, and the NK cytotoxicity against these HCCs was comparable to that against K562, a NK-sensitive leukemia cell line, when the cytotoxicity was assessed by a 4-h ⁵¹Cr release assay.Results The NK cells also induced significant apoptotic cell death in the Hep3B targets, but not in the HepG2 targets, when the cytotoxicity was assessed by a 2-h [³H]-thymidine release assay. In agreement with these results, procaspase-3 was activated in the Hep3B targets, but not in the HepG2 targets. Interestingly, mildly fixed NK cells had no detectable activity in the 4-h ⁵¹Cr release assay against both HepG2 and Hep3B targets, while they were similarly effective as the untreated NK cells in the 2-h [³H]-thymidine release assay, suggesting that the level of apoptotic cell death of the Hep3B targets is granule independent and might be primarily mediated by the death ligands of the NK cells.Conclusion This study found that a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor interaction is involved in the NK cell-mediated apoptotic death of the Hep3B targets, but a Fas/Fas ligand (FasL) interaction is not.     

Hollow chitosan/poly(acrylic acid) nanospheres as drug carriers

The preparation, in-vitro release, in-vitro cytotoxicity, and in-vivo drug delivery of doxorubicin (DOX)-loaded chitosan (CS)-poly(acrylic acid) (PAA) hollow nanospheres were investigated. The loading was done by dissolving a certain amount of DOX in non-cross-linked CS-PAA nanospheres aqueous solution followed by cross-linking chitosan with glutaraldehyde. The drug-loading content was up to 4.3% and the size of drug-loaded hollow nanospheres, determined by dynamic light scattering, was 118 nm. The nanospheres showed a continuous release of the entrapped DOX up to 10 days in vitro and showed comparable in-vitro cytotoxicity against HepG2 cells compared to the free DOX. In-vivo DOX delivery of DOX-loaded CS-PAA nanospheres showed that DOX concentration in blood can be maintained for a longer period than free DOX solution, and the DOX concentration in mice liver can be maintained constantly at relatively high level. The interesting feature of DOX-loaded CS-PAA hollow nanopspheres is that the loaded DOX can be delivered into the mice brain. The confocal laser scanning microscopy analysis reveals that fluorescein isothiocyanate (FITC)-labeled CS-PAA can deposit in different organs including liver, spleen, and brain.     

Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.     

Co-delivery of Vorinostat and Etoposide <Emphasis Type="Italic">Via</Emphasis> Disulfide Cross-Linked Biodegradable Polymeric Nanogels: Synthesis,Characterization, Biodegradation,and Anticancer Activity

Treatment regimens for cancer patients using single chemotherapeutic agents often lead to undesirable toxicity, drug resistance, reduced uptake etc. Combination of two or more drugs is therefore becoming an imperative strategy to overcome these limitations. A step forward can be taken through delivery of the drugs used in combination via nanoparticles. Co-administration of chemotherapeutic drugs encapsulated in nanoparticles has been shown to result in synergistic effects and enhanced therapeutic efficacy. In present study, we explored the combination treatment of histone deacetylase inhibitor vorinostat (VOR) and topoisomerase II inhibitor etoposide (ETOP). The concurrent combination treatment of VOR and ETOP resulted in synergistic effect on human cervical HeLa cancer cells. VOR and ETOP were encapsulated into poly(ethylene glycol) monomethacrylate (POEOMA)-based disulfide cross-linked nanogels. The nanogels were synthesized using atom transfer radical polymerization (ATRP) via cyclohexane/water inverse mini-emulsion and were degradable in presence of intracellular glutathione (GSH) concentration. Both the drugs were loaded into the nanogels by physical encapsulation method and characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), dynamic light scattering (DLS), and differential scanning calorimetry (DSC). Both VOR- and ETOP-loaded nanogels showed sustained release profile. Furthermore, combination treatment drugs encapsulated of POEOMA nanogel demonstrated enhanced synergistic cytotoxic effect compared with combination of free drugs. Enhanced synergistic cell killing efficiency of drug-loaded POEOMA nanogels was due to increased apoptosis via caspase 3/7 activation. Therefore, combination of VOR- and ETOP-loaded PEG-based biodegradable nanogels may provide a promising therapy with enhanced anticancer effect.     

Increased oxidative stress and cytotoxicity by hydrogen sulfide in HepG2 cells overexpressing cytochrome P450 2E1

The main objectives of this work were to evaluate the effects of hydrogen sulfide on oxidative stress and cytotoxicity parameters in HepG2 cells and to assess the extent to which cytochrome P450 2E1 (CYP2E1) activity modulates the effects of hydrogen sulfide on oxidative stress and cytotoxicity. Sodium hydrosulfide (NaHS) caused time- and concentration-dependent cytotoxicity in both non-P450-expressing HepG2 cells (C34 cells) and CYP2E1-overexpressing HepG2 cells (E47 cells); however, NaHS-dependent cytotoxicity was higher in E47 than C34 cells. Cytotoxicity by NaHS in C34 and E47 cells was mainly necrotic in nature and associated with an early decrease in mitochondrial membrane potential. NaHS caused increased oxidation of lipophilic (C11-BODIPY^581/591) and hydrophilic (DCFH-DA) probes only in E47 cells, at a time point prior to overt cytotoxicity. Trolox, an amphipathic antioxidant, partially inhibited both the cytotoxicity and the increased oxidative stress detected in E47 cells exposed to NaHS. Cell-permeable iron chelators and CYP2E1 inhibitors significantly inhibited the oxidation of C11-BODIPY^581/591 in E47 cells in the presence of NaHS. NaHS produced lipid peroxidation and cytotoxicity in E47 cells supplemented with a representative polyunsaturated fatty acid (docosahexaenoic acid) but not in C34 cells; these effects were inhibited by α-tocopherol, a lipophilic antioxidant. These data suggest that CYP2E1 enhances H₂S-dependent cytotoxicity in HepG2 cells through the generation of iron-dependent oxidative stress and lipid peroxidation.     

Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells.

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.     

Morin hydrate: a potential antioxidant in minimizing the free-radicals-mediated damage to cardiovascular cells by anti-tumor drugs

The co-incubation of morin hydrate with either doxorubicin or mitomycin C could minimize the toxicity of these anti-tumor drugs on cardiovascular cells, such as red blood cells, human umbilical vein endothelial cells (ECV304) and primary mouse cardiomyocytes, whereas morin hydrate did not lower the cytotoxicity of the drugs on human hepatocellular carcinoma cells (HepG2). Morin hydrate may not exert its antioxidant effect by enhancing the antioxidant enzymatic activity because it did not cause any induction on the mRNA levels of manganese superoxide dismutase expression in ECV304 cells and HepG2 cells.     

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.     

Self-organized nanogels responding to tumor extracellular pH: pH-dependent drug release and in vitro cytotoxicity against MCF-7 cells

The principal objective of this study was to fabricate doxorubicin-loaded self-organized nanogels composed of hydrophobized pullulan (PUL)-Nalpha-Boc-L-histidine (bHis) conjugates. Their responses to tumor extracellular pH (pHe) were determined, and they were also evaluated with regard to their anticancer efficacy against breast cancer cell lines (MCF-7). bHis was grafted to a PUL-deoxycholic acid (DO) conjugate (PUL-DO) via an ester linkage. PUL-DO/bHis conjugates with two different degrees of bHis substitutions (PUL-DO/bHis36 and PUL-DO/bHis78) were synthesized. PUL-DO/bHis nanogels formed via dialysis at a pH of 8.5 evidenced larger particle sizes (<140 nm) and lower critical aggregation concentrations (CACs) than did the PUL-DO nanogels (90 nm). The pH-dependent CAC of PUL-DO/bHis78 changed dramatically, from 1.2 microg/mL at pH 8.5, to 10 at 7.0, and to 660 at 6.2. A similar tendency in pH-dependent size was also noted. The ionization of the imidazole ring in bHis is principally responsible for pH dependency. The bHis moieties function as a switching tool responding to external pH. Doxorubicin (DOX)-loaded nanogels were assessed for pH-dependent releasing kinetics. The release rate of DOX from the PUL-DO/bHis78 nanogels increased significantly with reductions in pH. This resulted in increased cytotoxicity (30% cell viability at a dose of 10 microg/mL DOX equivalent) against sensitive MCF-7 cells at a pH of 6.8 and low cytotoxicity at pH 7.4 (65% cell viability at an identical dose). The results show that PUL-DO/bHis nanogels may potentially be employed as anti-tumor drug carriers.     

Synthesis of galactosyl compounds for targeted gene delivery

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.     

Chemical modification of an ecotropic murine leukemia virus results in redirection of its target cell specificity

An ecotropic virus was chemically modified in order to determine whether its target cell specificity could be altered. We hypothesized that chemical coupling of galactose residues to a virus might permit specific infection of hepatocytes mediated by asialoglycoprotein receptors unique to these cells. To test this hypothesis, we took advantage of the fact that: 1) artificial asialoglycoproteins can be created by chemical coupling of lactose to proteins; and 2) viruses that are ecotropic have a narrow species specificity. An ecotropic, rodent-specific, replication-defective murine leukemia virus containing the gene for beta-galactosidase was chemically modified with lactose to contain 5.9 mumol of lactose per mg of viral RNA. Modified and unmodified viruses were incubated for 5 days with HepG2, a human hepatoma line that possesses asialoglycoprotein receptors, and SK Hep1, a human cell line that does not. As expected from the ecotropism, unmodified virus did not produce beta-galactosidase activity in either cell type. Modified virus did not produce beta-galactosidase activity in SK Hep1 cells. However, modified virus did produce beta-galactosidase activity, 71.2 units/mg of cell protein, in the human receptor (+) HepG2 cells. Interestingly, modification of the virus also resulted in decreased enzyme activity in previously susceptible host rodent cells. Competition with modified virus by an excess of an asialoglycoprotein completely prevented development of enzymatic activity in HepG2 cells. Histochemical treatment of cells with 5-bromo-4-chloro-3-indoyl beta-D-galactoside to detect in situ beta-galactosidase activity demonstrated that only HepG2 cells treated with modified virus were positive and that 36% of these cells were stained after 5 days. These data indicate that chemical modification of a virus can result in a redirection of the infectivity of the virus toward hepatocyte-derived cells mediated by the presence of asialoglycoprotein receptors.     

In vitro cytotoxic potential of Solanum nigrum against human cancer cell lines

Plants have natural products which use to possess antiproliferative potential against many cancers. In the present study, six isolated fractions (ethyl acetate, petroleum ether, chloroform, n-butanol, ethanol and aqueous) from Solanum nigrum were evaluated for their cytotoxic effect on different cell lines. Hepatic carcinoma cell line (HepG2), cervical cancer cell line (HeLa) and baby hamster kidney (BHK) used as normal non-cancerous cells were evaluated for cytotoxicity against isolated fractions. Cell viability assay was performed to evaluate the cytotoxicity of all fractions on different cell lines followed by the lactate dehydrogenase and vascular endothelial growth factor assays of most active fraction among all screened for cytotoxic analysis. HPLC analysis of most active fractions against cytotoxicity was performed to check the biological activity of compounds. Results displayed the potent cytotoxic activity of ethyl acetate fraction of S. nigrum against HepG2 cells with IC₅₀ value of 7.89 μg/ml. Other fractions exhibited potent anticancer activity against HepG2 cells followed by HeLa cells. Fractions in our study showed no cytotoxicity in BHK cells. Cytotoxic activity observed in our current study exposed high antiproliferative potential and activity of ethyl acetate fraction against HepG2 cells. The results demonstrated that S. nigrum fractions exhibited anticancer activity against hepatic and cervical cancer cell lines with non-toxic effect in normal cells. These results reveal significant potential of S. nigrum for the therapeutic of cancers across the globe in future.     

Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation

The occurrence of N-linked oligosaccharides lacking galactose is significantly higher than normal in serum IgG of patients with rheumatoid arthritis (RA) in whom rheumatoid factor (RF), an autoantibody against autologous IgG, has been detected. In the present study, IgGs with and without RF activity (IgGRF and non-RF IgG, respectively) were prepared from sera of RA patients, and their oligosaccharide structures were characterized in order to investigate the relationship between RF activity and glycosylation. Three IgGRF fractions and a non-RF IgG fraction were obtained based on their ability to bind to an IgG-Sepharose column. The specific RF activity, as measured by immunoassays, was highest in the IgGRF fraction, which bound most avidly to the IgG-Sepharose. When the oligosaccharides were released by hydrazinolysis, and analyzed by MALDI-TOF mass spectrometry and HPLC, in combination with sequential exoglycosidase treatment, all the IgG samples were found to contain a series of biantennary complex-type oligosaccharides. The incidence of galactose-free oligosaccharides was significantly higher in both IgGRFs and non-RF IgG from RA patients compared with IgG from healthy individuals. In all IgGRFs, the levels of sialylation and galactosylation were lower than those in non-RF IgG from RA patients; the sialylation of non-RF IgG was the same as that of IgG from healthy individuals. In addition, the decreases in galactosylation and sialylation of oligosaccharides in IgGRF correlated well with the increase in RF activity. These findings could contribute to our understanding of the mechanisms of IgG-IgG complex formation and the pathogenicity of these complexes in RA patients.