Expression of exogenous DNA in rat liver cells after liposome-mediated transfection in vivo.

Liposomes containing the expression vector pRSVneo coding for neomycin phosphotransferase-II were used for a gene transfer into rat liver cells in vivo. After intravenous application of liposomes to male Wistar-rats, non-integrated vector DNA was detected by blot-hybridisation in isolated nuclei of hepatocytes. Presence of messenger RNA specific for the transferred construct was demonstrated by Northern analyses of poly(A)+RNA. The biological activity of the neomycin phosphotransferase-II gene and its enzyme could be demonstrated in hepatocytes for at least 7d post injection. Liposome-mediated gene transfer into adult animals is discussed as a model system to study the expression and fate of transferred genes in adult rat hepatocytes under physiological conditions.     

Short,single-stranded oligonucleotides mediate targeted nucleotide conversion using extracts from isolated liver mitochondria

Site-specific single-nucleotide changes in chromosomal DNA of eukaryotic cells have been achieved using chimeric RNA/DNA oligonucleotides (ONs) and short single-stranded (SS) ONs. However, a variety of human diseases originate from single-point mutations in the genome of mitochondrial DNA. We previously demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate targeted single-nucleotide changes using chimeric ONs in vitro. However, different factor(s) and/or mechanism(s) appear to be involved in SS and RNA/DNA ON mediated DNA repair. Because mitochondria are deficient in certain factors involved in nuclear DNA repair pathways, we investigated whether mitochondria possess the enzymatic machinery for SS ON mediated DNA alterations. Using in vitro DNA repair assays based on mutagenized plasmids and a bacterial read-out system, SS ONs were designed to correct the point mutations in the genes encoded by the different plasmids. In this system, protein extracts from purified rat liver mitochondria and nuclei catalyzed similar levels of site-specific nucleotide modifications using SS ONs. Interestingly, extracts isolated from quiescent liver mediated significantly higher conversion rates than those isolated from regenerating liver. The results suggest that mitochondria contain the factors necessary for correction of single-point mutations by SS ONs. In addition, at least some are different than those required for DNA repair by RNA/DNA ONs. Moreover, correction with SS ONs appears to occur one strand at a time suggesting that repair of the DNA substrate involves strand transfer. The ability of unmodified SS ONs to mediate targeted alteration of the mitochondrial genome may provide a new tactic for treatment of certain mitochondrial-based diseases.     

Anti-Influenza Virus Activities of Nicked and Circular Dumbbell RNA/DNA Chimeric Oligonucleotides

Abstract

We have designed a new type of antisense oligonucleotide, containing two hairpin loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem (nicked and circular dumbbell DNA/RNA chimeric oligonucleotides). The reaction of the nicked and circular dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding anti-DNA together with the sense RNA cleavage products. These oligonucleotides were more resistant to exonuclease attack. We also describe the anti-Fluv activities of nicked and circular dumbbell DNMA chimeric oligonucleotides.     

The Use of Transmembrane pH Gradient-Driven Drug Encapsulation in the Pharmacodynamic Evaluation of Liposomal Doxorubicin

Abstract

The toxicity and efficacy properties of doxorubicin entrapped inside liposomes are sensitive to the physical characteristics of the vesicle carrier system. Studies addressing such relationships must use preparation procedures with the ability to independently vary vesicle size, lipid composition and drug to lipid ratio while maintaining high trapping efficiencies. The transmembrane pH gradient-driven encapsulation technique allows such liposomal doxorubicin formulations to be prepared. Pharmacokinetic, toxicology and antitumour studies with these systems have revealed several important relationships between liposome physical properties and biological activity. The acute toxicity of liposomal doxorubicin is related primarily to the ability of the liposomes to retain doxorubicin after administration. Including cholesterol and increasing the degree of acyl chain saturation of the phospholipid component in the liposomes significantly decreases drug leakage in the blood, reduces cardiac tissue accumulation of doxorubicin and results in increased LD₅₀ values. In contrast, the efficacy of liposomal doxorubicin is most influenced by liposome size. Specifically, liposomes with a diameter of approximately 100 nm or less exhibit enhanced circulation lifetimes and antitumour activity. While these relationships appear to be rather straightforward, there exist anomalies which suggest that a more thorough evaluation of liposomal doxorubicin pharmacokinetics may be required in order to fully understand its mechanism of action. A key feature in this regard is the ability to differentiate between non-encapsulated and liposome encapsulated doxorubicin pools in the circulation as well as in tumours and normal tissues. This represents a major challenge that must be addressed if significant advances in the design of more effective liposomal doxorubicin formulations are to be achieved.     

Anticancer double lipid prodrugs: liposomal preparation and characterization

The escape of encapsulated anticancer drugs from liposomes by passive diffusion often leads to suboptimal drug concentrations in the cancer tissue, therefore calling for effective trigger mechanisms to release the drug at the target. We investigated mixtures of lipid components that not only form stable liposomes, but also can be turned into active drugs by secretory phospholipase A? (sPLA?), an enzyme that is upregulated in various cancer cells, without the necessity for conventional liposome drug loading. The liposomes are composed of a novel lipid-based retinoid prodrug premixed with saturated phospholipids. The prodrug is found to be miscible with phospholipids, and the lipid mixtures are shown to form liposomes with the desired size distribution. The preparation procedure, phase behavior, and physicochemical properties of the formed liposomes are described as a function of lipid composition. We show that the premixing of the prodrug with phospholipids can be used to modify the physicochemical properties of liposomal formulations. The results should prove useful for further exploration of the potential for using these novel lipid prodrugs in liposomal formulations for cancer treatment.     

The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2′-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.     

Properties of circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters", containing two alkyl loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA) in the double helical stem. The reaction of nicked (NDRDON) and circular (CDRDON) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 35mer RNA, which gave 35mer RNA cleavage products by treatment with RNaseH. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide(anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide.     

Characteristics of DNA isolated from a complex form of DNA-polymerase alpha from the rat liver

A complex from of DNA polymerase alpha was isolated from the nuclear membrane of hepatocytes. DNA fragments were shown to be among components of the complex under study. In this paper we present evidence that DNA from the alpha-polymerase complex from quiescent hepatocytes (DNA-G) differs in its nucleotide composition from its counterpart (DNA-S) isolated from hepatocytes synthesizing DNA. As judged by dot hybridization, DNA-G0 does not contain nucleotide sequences which are complementary to ribosomal or messenger RNA, whereas the abovementioned sequences are present in DNA-S. At the same time DNA-G0 is found to contain sequences which are homologous to both SV40 DNA and yeast TRPI-ARS1 DNA. The difference in nucleotide sequences between DNA-G0 and DNA-S indicates that in the process of replication DNA is being stretched across the multienzyme complex located on the nuclear membrane.     

Oligonucleotides encapsulated in pH sensitive liposomes are efficient toward Friend retrovirus.

Retroviruses present multiple RNA targets for antisense oligonucleotides. An oligodesoxyribonucleotide (15 mer) complementary to the region of the initiation codon AUG of the env gene mRNA of Friend retrovirus was an inhibitor of the translation of Env protein in vitro. No effect was observed on cells infected with Friend retrovirus. We observed that these oligomers were rapidly degraded in cellular medium. After encapsulation into liposomes, they inhibited the spreading of the virus for chronic or de novo infection. We have compared the efficiency of two compositions of liposomes: pH sensitive and non pH sensitive formulations. Oligomers encapsulated in pH sensitive liposomes were more active that those encapsulated in non pH sensitive liposomes. pH sensitive liposomes could allow to avoid degradation of oligomers by lysosomes.     

Inhibition of DNA polymerization and DNA transcription to RNA by seminal plasma peptides

An oligopeptide fraction purified from the extracellular compartment of bull semen and strongly interacting with DNA was shown to hinder mononucleotide polymerizations to DNA and RNA in vitro. The fraction, collectively called seminal plasma inhibitor, was active in the endogenous DNA and RNA polymerase reactions of the nuclei from rat hepatocytes and in the analogous nucleotide polymerizations catalyzed by purified enzymes of bacterial origin. The type of the induced inhibition was studied using the RNA polymerase from Escherichia coli as a representative nucleotidyl transferase. In the enzymatic polycondensation of mononucleotides, the seminal plasma inhibitor appeared to exert its effect mainly by a competitive inhibition for the utilization of DNA templates without specificity with respect to the source and the base sequence of DNA. Concavities of the plots of V0/Vi versus the amounts of inhibitor in the nucleotide polymerizing reactions and of the Dixon plots in the assays of RNA polymerase from E. coli suggested that the isolated oligopeptide fraction contained more than one active molecular species with differential effects at low and high doses. Preliminary results on the microheterogeneity of the seminal plasma inhibitor supported this contention.     

Liposome Internalization by Isolated Rat Hepatocytes

Abstract

We studied the in vitro interaction and the endocytic process of peroxidase-loaded liposomes with isolated rat hepatocytes maintained in suspension culture. We report morphological (both at light and electron microscopy) and biochemical evidence that cationic egg PC (egg phosphatidylcholine)-liposomes are taken up by isolated rat hepatocytes via an endocytic pathway. The incubation of 2.5 mM liposomal lipids for at least 4 hours was not cytotoxic to the cells. The uptake of peroxidase-fluoresceine isothiocyanate conjugate-liposomes was not distributed homogeneously among the hepatocyte population. However the hepatocytes which have apparently internalized greater amount of probe were not damaged since cell shape and integrity of the membrane are retained as evaluated by conventional light microscopy. Therefore the fusion between liposomes and hepatocytes seems to be dependent on the viability of the isolated hepatocytes.     

Mitochondria isolated from liver contain the essential factors required for RNA/DNA oligonucleotide-targeted gene repair

Chimeric RNA/DNA oligonucleotides (ONs) have been used successfully for site-specific modifications of episomal and chromosomal DNA in eukaryotic cells. We explored the possibility of applying this technique to mitochondrial DNA, as single-nucleotide defects in this genome are associated with a series of human diseases. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeric ON-mediated DNA alterations. We utilized an in vitro DNA repair assay and an Escherichia coli readout system with mutagenized plasmids carrying point mutations in antibiotic resistance genes. RNA/DNA ONs were designed to correct the defects and restore kanamycin and tetracyclin resistance. Using this system, we demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate precise single-nucleotide changes. Interestingly, the frequency of gene conversion was similar in both mitochondrial and nuclear extracts, as well as from quiescent and regenerating liver. The results indicate that mitochondria contain the machinery required for repair of genomic single-point mutations, and suggest that RNA/DNA ONs may provide a novel approach to the treatment of certain mitochondrial-based diseases.     

Chitosan-reduced gold nanoparticles: a novel carrier for the preparation of spray-dried liposomes for topical delivery

Exposure of skin to various chemical and physical agents results in excessive stress to the outermost cell layer of the skin, causing different degenerative effects that can be minimized by using antioxidant formulations. The major challenge, in this regard, is to develop a formulation, which can prevent photodegradation of the actives, thus allowing a significant amount to be deposited at the site. In recent decades, liposomal formulations have been extensively employed to overcome the barrier properties of the skin and photodegradation of actives. In the present study, chitosan-reduced gold nanoparticles were investigated for its potential as a carrier to prepare liposomes by a spray-drying method. Liposomes so obtained were characterized for phospholipid recovery, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, particle size, zeta potential, encapsulation efficiency, and deposition of drug and gold nanoparticles in the rat skin. Further, a liposomal gel formulation was prepared using Carbopol? 980 NF (Noveon Systems, Kochi, India) and evaluated for drug deposition in the skin. Antioxidant activity of vitamin C encapsulated in gold liposomes was determined on a human leukemia (HL-60) cell line. The use of gold nanoparticles as a carrier showed improved phospholipid recovery and thus overcomes the liposome scalability problem. DRIFT spectra confirmed the presence of phospholipid in the formulation. Liposomal gel showed improved drug deposition, as compared to control and marketed preparations. A more interesting contribution of the chitosan-reduced gold nanoparticles was an enhanced antioxidant activity seen in case of the vitamin C-loaded gold liposomal formulation. Liposomal formulation was found to be stable for 3 months at 30°C and 65% relative humidity.     

Efficient Encapsulation of Plasmid DNA in Anionic Liposomes by a Freeze/Thaw-Extrusion Procedure

Abstract

In this study we investigated whether intact plasmid DNA can be efficiently encapsulated in anionic liposomes prepared by freeze/thaw and extrusion techniques. There is controversy about this method of DNA encapsulation, especially as to whether DNA remains intact and retains its biological activity during extrusion. A solution containing supercoiled plasmid pCMVβ (7164 base pairs) was added to dry lipid films, and after freezing and thawing, the suspension was extruded through a filter with 0.2 μm pores. About 20% of the DNA became encapsulated, as evidenced by protection from degradation by endonuclease added externally. Plasmid isolated from the liposomes was structurally intact, and had essentially the same transfection activity as untreated DNA. These results show that plasmid DNA can be reliably and efficiently encapsulated in anionic liposomes by freeze/thaw and extrusion.     

Chloroplast lysates support directed mutagenesis via modified DNA and chimeric RNA/DNA oligonucleotides

Chimeric RNA/DNA and modified DNA oligonucleotides have been shown to direct gene-conversion events in vitro through a process involving proteins from several DNA-repair pathways. Recent experiments have extended the utility of these molecules to plants, and we previously demonstrated that plant cell-free extracts are competent to support oligonucleotide-directed genetic repair. Using this system, we are studying Arabidopsis DNA-repair mutants and the role of plant proteins in the DNA-repair process. Here we describe a method for investigating mechanisms of plastid DNA-repair pathways. Using a genetic readout system in bacteria and chimeric or modified DNA oligonucleotides designed to direct the conversion of mutations in antibiotic resistance genes, we have developed an assay for genetic repair of mutations in a spinach chloroplast lysate system. We report genetic repair of point and frameshift mutations directed by both types of modified oligonucleotides. This system enables the mechanistic study of plastid gene repair and facilitates the direct comparison between plant nuclear and organelle DNA-repair pathways.     

The role of beta2-glycoprotein I in liposome-hepatocyte interaction

Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The approximately 55-kDa protein was identified as beta2-glycoprotein I (beta2GPI) by Western blotting. Despite the high affinity of beta2GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, beta2GP1 inhibits, rather than enhances, liposome uptake by liver cells.     

3'-5' Exonucleolytic activity of DNA polymerases: structural features that allow kinetic discrimination between ribo- and deoxyribonucleotide residues

We have determined rates for the excision of nucleotides from the 3' termini of chimeric DNA-RNA oligonucleotides using the Klenow fragment (KF) and two other DNA polymerases, from phages T4 and T7. For these studies, we synthesized DNA-RNA chimeric oligonucleotides with RNA residues in defined positions. When a ribonucleotide residue was placed at the 3' terminus, all three DNA polymerases removed it at the same rate as they did for substrates composed solely of deoxynucleotide residues. There was a decrease in the excision rate, however, when a ribonucleotide residue was located at the second or third position from the 3' terminus. When both the second and third positions were occupied by ribonucleotide residues, the excision rate for the 3' terminal nucleotide was reduced even further and was almost identical to the rate observed when the DNA polymerases encountered single-stranded RNA. The magnitude of the effect of ribonucleotide residues on the excision rate was lower when Mn(2+) replaced Mg(2+) as the essential divalent cation. Two KF mutations, Y423A and N420A, selectively affected the excision rates for the chimeric substrates. Specifically, Y423A totally abolished the rate reduction when there was a single ribonucleotide residue immediately preceding the 3' terminus, whereas N420A diminished, but did not eliminate, the rate reduction relative to that of wild-type KF when the single ribonucleotide residue occupied either the second or third position from the 3' terminus. These results are consistent with the structure of a KF-ss DNA complex from which it can be deduced, by modeling, that a 2' OH group on the second sugar from the 3' terminus would sterically clash with the Tyr 423 side chain, and a 2' OH group on the third sugar would clash with the side chain of Asn 420. The corresponding mutations in T4 DNA polymerase did not affect the rate of hydrolysis of the chimeric oligonucleotides. Thus, there appears to be a major difference in the kinetic behavior of KF and T4 DNA polymerase with respect to the exonuclease reaction. These results are discussed with respect to their possible biological relevance to DNA replication.     

Cytosolic delivery of macromolecules: I. Synthesis and characterization of pH-sensitive acyloxyalkylimidazoles

A series of 1-(acyloxyalkyl)imidazoles (AAI) were synthesized by nucleophilic substitution of chloroalkyl esters of fatty acids with imidazole. The former was prepared from fatty acid chloride and an aldehyde. When incorporated into liposomes, these lipids show an apparent pK(a) value ranging from 5.12 for 1-(palmitoyloxymethyl)imidazole (PMI) to 5.29 for 1-[(alpha-myristoyloxy)ethyl]imidazole (alpha-MEI) as determined by a fluorescence assay. When the imidazole moiety was protonated, the lipids were surface-active, as demonstrated by hemolytic activity towards red blood cells. As expected, AAI were hydrolyzed in serum as well as in cell homogenate. They were significantly less toxic than biochemically stable N-dodecylimidazole (NDI) towards Chinese hamster ovary (CHO) and RAW 264.7 (RAW) cells as determined by MTT assay. When fed to RAW cells, fluorescein-labeled oligonucleotides encapsulated in liposomes containing 20 mol% 1-(stearoyloxymethyl)imidazole (SMI) resulted in punctate as well as partially diffuse fluorescence. In a functional assay involving down-regulation of luciferase in CV-1 cells, neutral liposomes containing imidazole lipids showed suboptimal delivery of antisense phosphorothioate oligomers. Taken together, the results suggest that AAI are of potential use in developing nontoxic, pH-sensitive liposomes. However, these liposomal formulations need to be optimized to achieve higher concentrations of pH-sensitive detergents within the endosome to facilitate efficient cytosolic release of liposome-entrapped contents.