A seven-day Helicobacter pylori treatment regimen using clarithromycin, omeprazole and tripotassium dicitrato bismuthate

The present work investigated the magnitude of microsphere-induced acute myotoxicity and determined whether this myotoxicity is related to microsphere size and/or reconstitution solvent. Using a high molecular weight poly(dl-lactide-co-glycolide) copolymer, the myotoxicity of two different size microsphere formulations (3.6 microns and 19 microns) in normal saline or distilled water was quantified using a previously validated isolated rat muscle system. Overall, microspheres were found to be relatively nontoxic compared to known myotoxic agents (e.g., phenytoin) and control muscles. The smaller microspheres were found to be significantly more myotoxic than larger microspheres. Furthermore, the myotoxicity was lower in large microspheres reconstituted with normal saline or normal saline with 0.5% (w/v) carboxymethylcellulose (to prevent aggregation) compared to those reconstituted with distilled water. Smaller microspheres were found to be extremely difficult to inject, due to aggregation, which could not be prevented by the addition of carboxymethylcellulose. This study suggests that larger microspheres are less myotoxic than smaller microspheres.     

In vitro-in vivo myotoxicity of intramuscular liposomal formulations

PURPOSE: The first objective was to study the in vitro myotoxicity of empty liposomes and to examine whether liposome size, charge and fluidity affect of liposomal myotoxicity. The second objective was to investigate the effect of liposomal encapsulation on the in vitro and in vivo myotoxicity of loxapine compared to the loxapine commercial preparation (Loxitane). METHODS: The in vitro myotoxicity of empty liposomes and loxapine liposomes was evaluated by the cumulative efflux of the cytosolic enzyme creatine kinase (CK) from the isolated rat extensor digitorum longus (EDL) muscle over a 2 hour period. In the in vivo studies, the area under plasma CK curve over 12 hours was used to evaluate muscle damage. RESULTS: The in vitro myotoxicity for all empty liposomal formulations was not statistically different from negative controls (untreated control muscles and normal saline injected muscles). However, these empty liposomal formulations were significantly less myotoxic than the positive controls (muscles injected with phenytoin and muscle sliced in half). In vitro-in-vivo studies showed that the liposomal encapsulation of loxapine resulted in significant (P < 0.05) reduction in myotoxicity (80% in vitro and 60% in vivo) compared to the commercially available formulation which contains propylene glycol (70% V/V) and polysorbate 80 (5% W/V) prepared at equal concentration. CONCLUSIONS: Results indicate that empty liposomes do not induce myotoxicity. Furthermore, liposomal size, charge and fluidity do not affect myotoxicity. In addition, in vitro and in vivo studies have demonstrated that liposomal encapsulation of loxapine can reduce myotoxicity compared to a formulation containing organic cosolvents.     

Ability of wedelolactone, heparin, and para-bromophenacyl bromide to antagonize the myotoxic effects of two crotaline venoms and their PLA2 myotoxins

We examined the ability of wedelolactone, heparin and para-bromophenacyl bromide to antagonize the myotoxic activity in mice of venoms from Crotalus viridis viridis and Agkistrodon contortrix laticinctus and two phospholipase A2 myotoxins, CVV myotoxin and ACL myotoxin, isolated from them. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours and histological changes in extensor digitorum longus muscle (EDL) at three hours after injection of the test solution. Both heparin and wedelolactone independently reduced the myotoxic effect of both crude venoms and both myotoxins, but wedelolactone was more effective. Wedelolactone plus heparin reduced the myotoxic effect of CVV myotoxin more than either antagonist alone. The PLA2 inhibitor, para-bromophenacyl bromide (pBPB), reduced the myotoxic effect of both myotoxins more than either wedelolactone or heparin. On the other hand, the myotoxic effect of polylysine was not reduced by either wedelolactone or para-bromophenacyl bromide, but it was reduced by heparin. These results indicate that wedelolactone, para-bromophenacyl bromide and heparin are antagonists of these two phospholipase A2 myotoxins, and that antagonism by the first two compounds may be due to a more specific interaction with these proteins than that by the latter.     

Stable and monodisperse lipoplex formulations for gene delivery

A stable single vial lipoplex formulation has been developed that can be stored frozen without losing either biological activity or physical stability. This formulation was identified by systematically controlling several formulation variables and without introducing either stabilizers or surfactants. Analytical assays were used to unambiguously characterize the formulations. The critical formulation parameters were: (1) the size of the cationic liposomes; (2) the rate and method of DNA and cationic liposome mixing; and (3) the ionic strength of the suspending vehicle. The mixing conditions were precisely controlled by using a novel, specially designed continuous flow pumping system in which the DNA and liposome solutions were mixed at the junction of a T-connector. Homogenous cationic liposome preparations were prepared by extrusion in two different size ranges of either 400 or 100 nm. Extruded liposomes produced more monodisperse and physically stable lipoplex formulations than unextruded liposomes, but the formulations prepared with 100 nm liposomes were less active in in vitro transfection assays than either the 400 nm or unextruded liposomes. Low ionic strength and 5% sorbitol were required for the lipoplex formulations to survive freezing and thawing. A frozen lipoplex formulation stored for more than a year maintained its biological activity. These results have broad implications for the pharmaceutical development of lipoplex formulations for gene delivery.     

Cationic liposomes in gene delivery

Cationic liposomes have been extensively explored as gene delivery vector for several reasons. It is because disadvantages of viral vectors include risk of replication, possible immunogenicity, and the difficulty of obtaining a large quantity of viral vectors. Currently, a variety of cationic components for liposome formulations have been developed. The components are broadly divided into two classes based on the chemical structure of hydrophobic moieties: long aliphatic (saturated or unsaturated) hydrocarbons and cholesterol ring. A variety of hydrophilic moieties also include tertiary amines, ammonium salt and spermine. The role of liposomes is to condense DNA to form complexes with high affinity to cell surfaces where possible fusion or destabilization of the membrane and/or endocytosis are involved. However, at present, little structure-activity relationships are known. Some vectors are on clinical trials approved by NIH.     

Biodistribution and gene expression of lipid/plasmid complexes after systemic administration

The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.     

Intratumoral pharmacokinetics and in vivo gene expression of naked plasmid DNA and its cationic liposome complexes after direct gene transfer

The pharmacokinetic properties and gene expression of naked plasmid DNA and its cationic liposome complexes were studied after direct intratumoral injection. Using a Walker 256 tissue-isolated tumor perfusion system, we quantified the recovery of naked plasmid DNA and cationic liposome complexes in the tumor, leakage from the tumor surface, and the venous outflow after intratumoral injection. Approximately 50% of naked plasmid DNA had been eliminated from the tumor 2 h after injection, whereas more than 90% of plasmid DNA was retained in the tumor when it was complexed with cationic liposomes. However, the distribution of these complexes in the tumor was restricted to the tissue surrounding the injection site. Pharmacokinetic analysis of the venous outflow profiles suggested that the rate-limiting process that determines the retention of plasmid DNA in the tumor is transferred from the injection site in the tumor tissue and that complexation with cationic liposomes may retard this process. Furthermore, we examined the gene expression of chloramphenicol acetyltransferase DNA constructs (naked pCMV-CAT) and the corresponding cationic liposome [3-beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol] complexes. A similar level of gene expression was observed in vivo after direct intratumoral injection of naked DNA and its cationic liposome complexes. In both cases, a great variation was observed between tumors, and localization of gene-transduced cells in the tumor tissue was limited to the area in the vicinity of the injection site. Thus, these pharmacokinetic and gene expression studies have demonstrated that cationic liposomes can enhance the retention of injected DNA in the tumor model, whereas cationic liposome complex does not necessarily improve gene expression because of its poor dissemination in this tumor. The present study also suggested that there is a need to control the behavior of the injected naked plasmid DNA and its cationic liposome complexes to ensure better distribution throughout the tumor.     

Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells

The beta-galactosidase reporter gene, either free or complexed with various cationic vectors, was microinjected into mammalian cells. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus. Polyethylenimine and to a lesser extent polylysine, but not cationic lipids, enhance transgene expression when complexes are injected into the cytoplasm. This latter effect was independent of the polymer vector/cDNA ionic charge ratio, suggesting that nucleic acid compaction rather than surface charge was critical for efficient nuclear trafficking. Cell division was not required for nuclear entry. Finally, comparative transfection and microinjection experiments with various cell lines confirm that barriers to gene transfer vary with cell type. We conclude that polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and that transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.     

Efficiency of cationic lipid-mediated transfection of polarized and differentiated airway epithelial cells in vitro and in vivo

Systematic analysis of a large number of different cationic lipids has led to the identification of novel structures (GL-67) and formulations of cationic lipid:plasmid DNA (pDNA) complexes that facilitate high levels of gene expression in lungs of mice. However, despite significant improvement in gene transfer activity, we show here that the efficiency of GL-67-mediated gene transduction of intact airway epithelia is still relatively low. Administration of GL-67:pCF1-CFTR (encoding the cystic fibrosis transmembrane conductance regulator) complexes into the nasal epithelium of cystic fibrosis (CF) transgenic mice resulted only in marginal correction of the ion transport defects. Measurements of nasal potential differences (PD) showed no correction of the sodium (Na+) transport defect, and only partial restitution of the chloride (Cl-) transport defect was achieved in a small proportion of the animals after perfusion of the nasal epithelium with the complexes. Furthermore, in contrast to results obtained following instillation of GL-67:pDNA complexes into the lungs of mice, perfusion of GL-67:pDNA into the nasal epithelium resulted only in a moderate enhancement of gene transduction activity relative to that attained with naked pDNA alone. To determine the basis for this low efficiency of transfection, a series of studies was conducted to identify some of the barriers governing cationic lipid-mediated gene transfer to the airway epithelium. We show here that the transfection activity of GL-67 was affected by the polarization, differentiation, and proliferative state of the cells. Diminished transfection activity was observed with nonmitotic, highly polarized and differentiated airway epithelial cells. This observed reduction in gene expression with nonmitotic cells was determined to be due in part to inefficient nuclear translocation of the pDNA from the cytoplasm. Together these data indicate that much improvement in the ability of cationic lipids to transfect polarized and differentiated airway epithelial cells is a necessary prerequisite for effective cationic lipid-mediated gene therapy of airway diseases such as CF.     

Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice

This study sought to determine whether angiogenic blood vessels in disease models preferentially bind and internalize cationic liposomes injected intravenously. Angiogenesis was examined in pancreatic islet cell tumors of RIP-Tag2 transgenic mice and chronic airway inflammation in Mycoplasma pulmonis-infected C3H/HeNCr mice. For comparison, physiological angiogenesis was examined in normal mouse ovaries. We found that endothelial cells in all models avidly bound and internalized fluorescently labeled cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]/cholesterol or dimethyldioctadecyl ammonium bromide [DDAB]/cholesterol) or liposome-DNA complexes. Confocal microscopic measurements showed that angiogenic endothelial cells averaged 15-33-fold more uptake than corresponding normal endothelial cells. Cationic liposome-DNA complexes were also avidly taken up, but anionic, neutral, or sterically stabilized neutral liposomes were not. Electron microscopic analysis showed that 32% of gold-labeled liposomes associated with tumor endothelial cells were adherent to the luminal surface, 53% were internalized into endosomes and multivesicular bodies, and 15% were extravascular 20 min after injection. Our findings indicate that angiogenic endothelial cells in these models avidly bind and internalize cationic liposomes and liposome-DNA complexes but not other types of liposomes. This preferential uptake raises the possibility of using cationic liposomes to target diagnostic or therapeutic agents selectively to angiogenic blood vessels in tumors and sites of chronic inflammation.     

Target specific optimization of cationic lipid-based systems for pulmonary gene therapy

PURPOSE: Cationic lipids are capable of transferring foreign genes to the pulmonary epithelium in vivo. It is becoming increasingly clear that factors other than lipid molecular structure also influence efficiency of delivery using cationic lipid systems. This study is aimed at evaluating the effect of formulation variables such as cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and plasmid topology on transgene expression in the lung. METHODS: The effect of varying the surface and colloidal properties of cationic lipid-based gene delivery systems was assessed by intratracheal instillation into rats. An expression plasmid encoding chloramphenicol acetyl transferase (CAT) was used to measure transgene expression. RESULTS: Cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and topology of the plasmid, were found to significantly affect transgene expression. Complexation with lipids was found to have a protective effect on DNA integrity in bronchoalveolar lavage fluid (BALF). DNA complexed with lipid showed enhanced persistence in rat lungs as measured by quantitative polymerase chain reaction. CONCLUSIONS: Fluorescence microscopy analysis indicated that the instilled formulation reaches the lower airways and alveolar region. Data also suggests cationic lipid-mediated gene expression is primarily localized in the lung parenchyma and not infiltrating cells isolated from the BALF.     

Drug-induced interstitial lung disease

Cationic liposomes provide a means to introduce genes into cells both ex vivo and in vivo. In the past few years their use has been described in several tissues, e.g. lungs, liver, endothelium, brain. In this study we evaluated a commercially available poly-cationic liposome formulation in delivering a reporter gene into cultured myogenic cell lines from mouse and rat, and primary fetal human myoblasts. We also examined the effect of serum on liposome-mediated transfection and designed a new procedure to enhance transfection efficiency, based on the pre-condensation of plasmid DNA with polylysine. Polylysine pre-condensation was particularly effective when transfecting the cells in the presence of serum, a finding that could be significant for in vivo transfections.     

NMR characterization of a single-cysteine mutant of Escherichia coli thioredoxin and a covalent thioredoxin-peptide complex

Cationic liposomes are used as the carriers of polyanionic genes for combating against hereditary diseases in gene therapy. Studies directed to careful biophysical characterizations of the cationic liposomes commonly used in gene delivery have just begun. Herein, we report on a novel liposomal exo-surface bound indazolization reaction of an amphiphilic arenediazonium salt as evidence for the existence of remarkably alkaline exo-surface of cationic liposomes commonly used in gene transfection. Our results demonstrate that formation of 5-hexadecyl-7-methylindazole in thermal indazolization of 2,6-dimethyl-4-hexadecylbenzenediazonium tetrafluoroborate bound to liposome surface is a strong indication for the existence of significantly high exo-surface pH for cationic liposomes commonly used in gene delivery. The present method can be used in determining the relative exo-surface basicities of various cationic liposomes used in gene transfection and subsequently to find any possible correlation between the transfection efficiencies of these liposomes and their exo-surface basicities.     

Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers

Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.     

Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung

Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) that peaked at days 1-2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids.     

Monomolecular collapse of plasmid DNA into stable virus-like particles

Cationic lipids are being widely used for cell transfection in vitro. The lipid/DNA complexes, however, tend to aggregate into large and polydisperse particle mixtures; this hampers their use in vivo. Cationic detergents, on the contrary, do not mediate cell transfection per se, yet are capable of condensing individual DNA molecules into discrete entities. We have taken (only) the interesting features of both types of amphiphiles for the two-step formation of stable core particles reminiscent of viruses. Individual anionic plasmid molecules were cooperatively collapsed with a carefully tailored cationic cysteine-based detergent. The resulting 23-nm particles were then simply "frozen" by spontaneous aerobic dimerization of the cysteine-detergent into a cystine-lipid on the template DNA. The population of spherical particles is monodisperse and stable over days, in physiological conditions. Together with a negative surface potential, these properties should ensure good tissue dissemination and escape from the blood stream after i.v. injection.     

Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations

The application of cationic liposome reagents has advanced DNA and mRNA transfection research in vitro, and data are accumulating which show their utility for in vivo gene transfer. However, chemical structure-activity data leading to a better mechanistic understanding of their biological activity is still limited. Most of the cationic lipid reagents in use today for this application are formulated as liposomes containing two lipid species, a cationic amphiphile and a neutral phospholipid, typically dioleoylphosphatidylethanolamine (DOPE). The studies reported here examine the effects of some systematic chemical structural changes in both of these lipid components. Cationic and neutral phospholipids were formulated together as large multilamellar vesicles (MLV) or small sonicated unilamellar vesicles (SUV) in water, and each formulation was assayed quantitatively in 96-well microtiter plates under 64 different assay conditions using COS.7 cells and an RSV-beta-galactosidase expression plasmid. The cationic lipid molecules used for these studies were derived from a novel series of 2,3-dialkyloxypropyl quaternary ammonium compounds containing a hydroxyalkyl moiety on the quaternary amine. A homologous series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl chain lengths on the quaternary amine were synthesized, formulated with 50 mol % DOPE, and assayed for transfection activity. The order of efficacy was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the quaternary amine. A homologous series of hydroxyethyl quaternary ammonium derivatives with different alkyl chain substitutions were synthesized, formulated with 50 mol % DOPE, and assayed in the transfection assay. The order of transfection efficacy was dimyristyl (di-C14:0) > dioleyl (di-C18:1) > dipalmityl (di-C16:0) > disteryl (di-C18:0). The addition of 100 microM chloroquine in the transfection experiment enhanced the activity of the dioleyl compound by 4-fold and decreased the activity of the dimyristyl compound by 70%. For each of the compounds and formulations examined in this report, large multilamellar vesicles (MLV; diameter 300-700 nm) were more active than small unilamellar vesicles (SUV; diameter 50-100 nm). The neutral phospholipid requirements for transfection activity in COS.7 cells with these cationic lipid molecules were examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Plasmid DNA is protected against ultrasonic cavitation-induced damage when complexed to cationic liposomes

Cationic liposomes bound to plasmid DNA are currently used for in vitro and in vivo gene therapy applications, but such complexes readily form large, heterogeneous aggregates that are not appropriate for pharmaceutical development. More importantly, size heterogeneity makes studies focused on optimizing gene transfer to cells difficult to conduct or understand. For this reason we have evaluated the effect of microprobe sonication on these complexes in an effort to achieve process-controlled size homogeneity. Complexes were prepared using a 7.2 kb reporter plasmid and the following liposomal lipid combinations: DDAB/DOPE (50:50 mol %), DDAB/DOPE/PEG-PE (50:45:5 mol %), DDAB/EPC (50:50 mol %), DDAB/EPC/PEG-PE (50:45:5, 50:40:10, 50:35:15 mol %), DODAC/DOPE (50:50 mol %), and DODAC/EPC (50:50 mol %) (DDAB, dimethyldioctadecylammonium bromide; DOPE, dioleoylphosphatidylethanolamine; PEG-PE, monomethoxypolyethylene glycol2000 succinate- distearoylphosphatidylethanolamine; EPC, egg phosphatidylcholine; DODAC, dioleoyldimethylammonium chloride). The influence of complex composition and lipid:DNA ratio was evaluated. Particle size was determined before and after complexation and again after sonication using the quasi-elastic light scattering technique. DNA integrity was assessed via agarose gel electrophoresis. Finally, gene transfection was evaluated using CHO cells that were transfected in vitro with sonicated and unsonicated complexes. It is established in this study that size reduction can occur, but this is dependent on cationic and neutral lipid composition and, in some cases, lipid:DNA ratio. Surprisingly, the process of sonication leaves a significant percentage of the plasmid DNA intact and capable of in vitro transfection. This study shows that plasmid DNA can be protected from damage due to sonication by liposome complex formation. This may indicate that more common pharmaceutical methods for size reduction which subject particles to mechanical stress may be applicable in preparation of liposome/DNA formulations for in vivo application.     

The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes

Four cationic polymers used to deliver DNA into cultured cells: polylysine, intact polyamidoamine dendrimer, fractured polyamidoamine dendrimer and polyethylenimine, are examined for their ability to interact with DNA. Complexes between the polymers and DNA were examined using electron microscopy. Similar toroidal structures with diameters of 55 +/- 12 nm were formed from all of the cationic polymers with DNA. The DNA complexes of the fractured dendrimer and polyethylenimine were observed as single, distinct units; their apparent diameters in solution as measured by dynamic light scattering ranged from 90 to 130 nm. The DNA complexes of polylysine and intact dendrimer generally appeared as clusters in electron micrographs; their diameters in solution were larger than 1000 nm, which suggests that their toroidal complexes aggregate in solution. The cationic polymers bind to DNA in a stoichiometry that is nearly 1:1 in primary amines to DNA phosphates. The apparent binding of all cationic polymers to DNA decreases linearly with increasing ionic strength, up to 0.8 M NaCl. Thus, at the concentrations studied, these polymers interact electrostatically with DNA forming a unit structure with toroidal morphology; the extent of aggregation of the unit structures in solution depends upon the characteristics of the individual polymer.     

Direct gene transfer to the respiratory tract of mice with pure plasmid and lipid-formulated DNA

Direct gene transfer into the respiratory system could be carried out for either therapeutic or immunization purposes. Here we demonstrate that cells in the lung can take up and express plasmid DNA encoding a luciferase reporter gene whether it is administered in naked form or formulated with cationic liposomes. Depending on the lipid used, the transfection efficiency with liposome-formulated DNA may be higher, the same as, or less than that with pure plasmid DNA. Tetramethyltetraalkylspermine analogs with alkyl groups of 16 or 18 carbons and DMRIE/cholesterol formulations proved particularly effective. Similar results for reporter gene expression in the lung were obtained whether the DNA (naked or lipid formulated) was administered by indirect, noninvasive intranasal delivery (inhaled or instilled) or by invasive, direct intratracheal delivery (injected or via a cannula). Reporter gene expression peaks around 4 days, then falls off dramatically by 9 days. The dose-response is linear, at least up to 100 microg plasmid DNA, suggesting better transfection efficiencies might be realized if there was not a volume limitation. For a given dose of DNA, the best results are obtained when the DNA is mixed with the minimum amount of lipid that can complex it completely. These results are discussed in the context of direct gene transfer for either gene therapy or delivery of a mucosal DNA vaccine.