Antioxidant properties of conjugates of polyethylene glycols containing terpenophenolic fragments

A series of water-soluble conjugates has been synthesized from polyethylene glycols of various lengths and 4-bromomethyl-2,6-diisobornylphenol. The membrane protective and antioxidant activities of the synthesized products were evaluated on the model of the H₂O₂-induced hemolysis of erythrocytes. It was shown that the studied conjugates have a significant antioxidant activity, and a significant membrane protective effect was shown for the conjugates containing 0.2% and 0.8% of the 2,6-diisobornyl-4-methylenephenolic fragments.     

Enhanced transfection rates of small-interfering RNA using dioleylglutamide-based magnetic lipoplexes

Magnetic force-guided delivery (magnetofection) has been studied as a new modality for introducing small-interfering RNA (siRNA) into target cells, but its delivery efficiency needs to be improved. Here, we report that magnetofection of N,N'-dioleylglutamide (DG)-based magnetic lipoplexes can substantially enhance the cellular delivery rates of siRNA. The siRNA was triply complexed with DG-based cationic liposomes and cationic iron-oxide nanoparticles. The formation of siRNA-containing magnetic lipoplexes was confirmed by gel retardation, sizes, and zeta potential values. Fluorescence microscopy and flow cytometry of fluorescent marker-labeled siRNA revealed that the DG-based magnetic lipoplexes conferred a higher cellular delivery rate of siRNA than DG-based lipoplexes or Lipofectamine 2000. In addition to the enhanced delivery of siRNA, the DG-based magnetic lipoplexes showed lack of cytotoxicity. We then tested the application of these magnetic lipoplexes for the cellular delivery of anticancer siRNA. Cancer cell lines magnetofected with DG-based magnetic lipoplexes containing Mcl1-specific siRNA (siMcl1) showed much lower viability than the groups treated with DG-based lipoplexes or Lipofectamine 2000, indicating that our magnetofection strategy conferred greater siMcl1-induced anticancer activity. These results suggest that DG-based magnetic lipoplexes are promising candidates for enhancing the efficiency of magnetic field-guided siRNA delivery.     

Cancer therapy with chemically modified enzymes. I. Antitumor properties of polyethylene glycol-asparaginase conjugates

The covalent attachment of monomethoxypolyethylene glycol (PEG) to asparaginases from Escherichia coli and Vibrio succinogenes by new coupling methodology produced conjugates that are active, stable, without significant immune response, and with greatly extended plasma half-lives in mice. Therapeutic efficacies were greater for the PEG-asparaginases than for the unmodified asparaginases in mice infected with the L5178Y lymphosarcoma or the 6C3HED tumor. Large single doses of native or modified enzymes were more effective against tumors than the same amount of enzyme given in smaller doses over several days.     

Gene transfection activities of amphiphilic steroid-polyamine conjugates

The design and evaluation of a novel potent class of DNA delivery agents based on steroid-polyamine conjugates bearing a flexible linker are reported. The hydrophobic regions are based on steroids, i.e. chlolestane and lithocholic acid motifs. The linker, which couples a hydrophobic steroid and a hydrophilic polyamine, in this study can be regarded as a two-atom extension of the conventional carbamate linker. We found that the gene transfection activity of the steroid-polyamine conjugates is influenced by the polyamine chain length and steroid structure. Molecular modeling of the relevant amphiphilic molecules revealed low-energy structures in which the polyamine chains are folded rather than stretched. This work suggests a significant effect of space-filling, i.e. the shape and orientation of the hydrophilic and hydrophobic regions, upon the efficiency of gene transfection.     

DODAB:monoolein-based lipoplexes as non-viral vectors for transfection of mammalian cells

DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine? LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the relation between structure and activity of MO-based lipoplexes will further strengthen the development of these novel delivery systems.     

Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.

Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was 相似文献   

Synergy between cationic lipid and co-lipid determines the macroscopic structure and transfection activity of lipoplexes

The large number of cytofectin and co-lipid combinations currently used for lipoplex-mediated gene delivery reflects the fact that the optimal cytofectin/co-lipid combination varies with the application. The effects of structural changes in both cytofectin and co-lipid were systematically examined to identify structure–activity relationships. Specifically, alkyl chain length, degree of unsaturation and the head group to which the alkyl side chain was attached were examined to determine their effect on lipoplex structure and biological activity. The macroscopic lipoplex structure was assessed using a dye-binding assay and the biological activity was examined using in vitro transfection in three diverse cell lines. Lipoplexes were formulated in three different vehicles currently in use for in vivo delivery of naked plasmid DNA (pDNA) and lipoplex formulations. The changes in dye accessibility were consistent with structural changes in the lipoplex, which correlated with alterations in the formulation. In contrast, transfection activity of different lipoplexes was cell type and vehicle dependent and did not correlate with dye accessibility. Overall, the results show a correlation between transfection and enhanced membrane fluidity in both the lipoplex and cellular membranes.     

Association of albumin or protamine to lipoplexes: enhancement of transfection and resistance to serum

BACKGROUND: The successful application of gene therapy depends on the availability of carriers to efficiently deliver genetic material into target cells. Such efficacy is strongly related to key parameters including serum resistance and protection of DNA. METHODS: The complexes were tested in terms of their biological activity, in the absence or presence of serum, by following transfection activity. Interaction with plasma proteins was evaluated by immunoblotting, while cytotoxicity was assessed by the Alamar Blue assay. Extent of DNA protection was determined both by using ethidium bromide intercalation and DNase I digestion assays. RESULTS: Our results show that, depending on the charge ratio and on the lipid composition, albumin and protamine can be used (either individually or co-associated) to generate cationic liposome/DNA complexes fulfilling in vivo requirements, while exhibiting high levels of transfection activity. In the present work a novel cationic lipid was tested. It was demonstrated that 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC):cholesterol (Chol) liposomes constitute a very promising carrier for gene delivery as illustrated by their enhancing effect on transfection, as compared with DOTAP-containing liposomes. Moreover, the biological activity of EPOPC-containing complexes is significantly improved upon association of albumin, even in the presence of 60% serum (namely for the 4/1 lipid/DNA charge ratio). Nevertheless, our studies also show that transfection activity mediated by DOTAP-containing complexes can be significantly enhanced upon pre-condensation of DNA with protamine. CONCLUSIONS: Co-association of HSA and protamine to lipoplexes ensures a high degree of DNA protection and results in high levels of transfection activity even in the presence of serum.     

Peptide-polyethylene glycol conjugates: synthesis and properties of peptides bearing a C-terminal polyethylene glycol chain

The introduction of a polyethylene glycol chain has become a popular tool for increasing water solubility and bioavailability. Our interest in the development of catalytically active peptides and the selective recognition of peptides has led us to investigate strategies to increase the solubility of peptides in organic solvents. Specifically, we became interested in the introduction of solubilizing moieties at the C-terminus of two peptides. Here we present different synthetic strategies for the preparation of peptide-polyethylene glycol conjugates and discuss the effect of the polyethylene glycol chain on the solubility and other properties, such as the catalytic activity of these peptides.     

Efficient nucleic acid transduction with lipoplexes containing novel piperazine- and polyamine-conjugated cholesterol derivatives

To advance the use of cationic lipids for non-viral nucleic acid vector formulation, a panel of novel nitrogen heterocycle cholesteryl derivatives containing a biodegradable carbamate linker was synthesised. Optimally acting piperazine and cyclen compounds had nucleic acid-binding and lipoplex nanoparticle formation properties that were suitable for their use as non-viral vectors. It was found that the lipoplexes formed were capable of efficient non-toxic nucleic acid delivery to cells in culture. The chemical structure of individual cationic lipids, which is likely to influence lipoplex formation, affected efficiency of DNA or RNA transfection. The results indicated that the cyclen containing compound possessing two cholesteryl moieties resulted in efficient siRNA-mediated target gene silencing but was a poor reagent for DNA transfection.     

Synthesis,antimalarial properties and 2D-QSAR studies of novel triazole-quinine conjugates

Click chemistry technique led to novel 1,2,3-triazole-quinine conjugates 8a–g, 10a–o, 11a–h and 13 utilizing benzotriazole-mediated synthetic approach with excellent yields. Some of the synthesized analogs (11a, 11d–h) exhibited antimalarial properties against Plasmodium falciparum strain 3D7 with potency higher than that of quinine (standard reference used) through in vitro standard procedure bio-assay. Statistically significant BMLR-QSAR model describes the bio-properties, validates the observed biological observations and identifies the most important parameters governing bio-activity.     

Synthesis of novel terpenophenol-chlorin conjugates and evaluation of their membranotropic and membrane-protecting properties

A series of conjugates has been synthesized by the reaction of methylpheophorbide a with ortho-alkylaminomethyl derivatives of 2-isobornyl-4-methylphenol; the terpenophenol fragment in the conjugates is attached to the methylpheophorbide a macrocycle by an amide bond formed upon the amidation of the 13(2)-ester group. A scanning electron microscopy study of the surface structure of erythrocytes incubated with these compounds confirmed their ability to interact with the cell membrane. It was found, based on the ability of the conjugates to inhibit the H₂O₂-induced hemolysis of erythrocytes and slow down the accumulation of the secondary lipid peroxidation products, that they possess membrane-protecting and antioxidant properties.     

Human serum albumin enhances DNA transfection by lipoplexes and confers resistance to inhibition by serum

Cationic liposome-DNA complexes ('lipoplexes') are used as gene delivery vehicles and may overcome some of the limitations of viral vectors for gene therapy applications. The interaction of highly positively charged lipoplexes with biological macromolecules in blood and tissues is one of the drawbacks of this system. We examined whether coating cationic liposomes with human serum albumin (HSA) could generate complexes that maintained transfection activity. The association of HSA with liposomes composed of 1, 2-dioleoyl-3-(trimethylammonium) propane and dioleoylphosphatidylethanolamine, and subsequent complexation with the plasmid pCMVluc greatly increased luciferase expression in epithelial and lymphocytic cell lines above that obtained with plain lipoplexes. The percentage of cells transfected also increased by an order of magnitude. The zeta potential of the ternary complexes was lower than that of the lipoplexes. Transfection activity by HSA-lipoplexes was not inhibited by up to 30% serum. The combined use of HSA and a pH-sensitive peptide resulted in significant gene expression in human primary macrophages. HSA-lipoplexes mediated significantly higher gene expression than plain lipoplexes or naked DNA in the lungs and spleen of mice. Our results indicate that negatively charged HSA-lipoplexes can facilitate efficient transfection of cultured cells, and that they may overcome some of the problems associated with the use of highly positively charged complexes for gene delivery in vivo.     

Calcium enhances the transfection potency of stabilized plasmid-lipid particles

Previous work from this laboratory has shown that plasmid DNA can be encapsulated in small (70-nm-diameter) stabilized plasmid-lipid particles (SPLP) that consist of a single plasmid encapsulated within a bilayer lipid vesicle. SPLP preferentially transfect tumor tissue following intravenous administration. Although the levels of transgene expression in vivo are greater for SPLP than can be achieved with naked DNA or complexes, they are lower than may be required for therapeutic benefit. In the present work we examine whether Ca2+ can enhance the transfection potency of SPLP. It is shown that Ca2+ can enhance SPLP transfection potency in bovine hamster kidney cells by 60- to 100-fold when treated in serum containing medium and an additional 60-fold when serum is absent for the initial 10 min of the transfection period. When cells are treated with SPLP in the presence of Ca2+, there is a fivefold increase in intact plasmid in the cell. It is also shown that this Ca2+ effect involves the formation of calcium phosphate precipitates; however, these precipitates are not directly associated with the SPLP plasmid DNA. The ability of calcium phosphate to facilitate delivery of other macromolecules without direct association is also demonstrated by the release of large-molecular-weight dextrans from endosomal/lysosomal compartments in the presence of calcium phosphate. Finally, it is shown that, unlike naked DNA, SPLP transfection potency in the presence of calcium phosphate is not affected by nuclease activity.     

Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles.     

Biofilm inhibition and drug-eluting properties of novel DMAEMA-modified polyethylene and silicone rubber surfaces

Poly(2-(dimethylaminoethyl) methacrylate) (pDMAEMA) was grafted to low density polyethylene (LDPE) and silicone rubber (SR) in order to make them less susceptible to microbial biofilm formation. The direct grafting of DMAEMA using γ-rays was an efficient and fast procedure for obtaining modified materials, which could be quaternized in a second step using methyl iodide. Raman spectroscopy showed that the grafting occurred only at the surface of the LDPE, but both at the surface and in the bulk of the SR. Consequently, the grafted chains caused changes in the surface-related features of the LDPE (water contact angle and viscoelastic behavior in the dry state) and in the bulk-related properties of the SR (swelling and viscoelasticity in the swollen state). The microbiological assays revealed that the grafted DMAEMA reduced Candida albicans biofilm formation (almost no biofilm on SR), while the quaternized surfaces inhibited C. albicans and Staphylococcus aureus biofilm by more than 99% compared to pristine materials. Modified LDPE and SR were capable of holding considerable amounts of nalidixic acid, an anionic antimicrobial drug, and sustained the release for several hours. In addition, the grafted materials were cytocompatible (fibroblast cell survival?>?70%). In conclusion, these materials have the ability to inhibit microbial biofilm formation and at the same time act as drug-eluting systems, and for that reason may hold great promise for anti-biofouling applications.     

Catalytic activity of administered gulonolactone oxidase polyethylene glycol conjugates

Scurvy in guinea pigs provides a convenient model of inborn metabolic disease for the investigation of enzyme therapy protocols. Gulonolactone oxidase, the enzyme in ascorbic acid biosynthesis that is missing from the scurvy-prone species, was modified by attachment of polyethylene glycol. The catalytic properties of this enzyme were affected little by the modification. Intravenous injection of this modified form of the enzyme elicited ascorbic acid synthesis in a dose-dependent manner. The modified enzyme was stabilized to incubation at 37 degrees C but was not protected from inactivation by trypsin. The circulating half-life of enzyme activity was not prolonged by this modification. Further, attachment of polyethylene glycol did neither abolish the enzyme's ability to react with preformed antibodies nor eliminate its immunogenicity.