Injection of DNA into liposomes by bacteriophage lambda

Small unilamellar vesicles (75-100 nm diameter) and large liposomes (greater than 1 micron in diameter) were prepared containing the lamB protein, an outer membrane protein of Escherichia coli and Shigella which serves as the receptor for bacteriophage lambda. Bacteriophage were observed to bind to these liposomes and vesicles by their tails and in most cases the heads of the bound bacteriophage appeared empty or partially empty of DNA. The lambda DNA was usually only partially ejected from the bacteriophage head when small unilamellar liposomes were used, presumably because the vesicles are too small to contain all the DNA. The partially ejected DNA was not susceptible to DNase unless the vesicle bilayer was first disrupted suggesting that DNA injection of phage DNA into the vesicle had occurred. After disruption of these vesicles on electron microscope grids, the bacteriophage are seen to have partially empty heads and a small mass of DNA associated with their tails. Using larger liposomes prepared by the fusion of lamB bearing vesicles with polyethylene glycol and n-hexyl bromide, the heads of most of the bound bacteriophage appeared to be completely empty of DNA. Disruption of these preparations on electron microscope grids revealed circular arrays of empty-headed bacteriophage surrounding DNA which had apparently been contained within the intact liposomes. These results indicate that high molecular weight DNA can be entrapped within liposomes with high efficiency by ejection from bacteriophage lambda. The possible use of these DNA-containing liposomes to facilitate gene transfer in eukaryotic cells is discussed.     

Protein-mediated DNA transfer into liposomes

The transfer of a foreign genome into a bacterium by means of phage infection is a very efficient but poorly understood process. To analyse the mechanism of phage DNA transfer at a molecular level, we have reconstituted FhuA, the receptor for phage T5 in the outer membrane of Escherichia coli, into unilamellar vesicles made of natural phospholipids. Cryoelectron microscopy studies showed that the binding of the phage to FhuA triggered the transfer of its double-stranded DNA (121000 bp) into the proteoliposomes. DNA was entrapped within vesicles with a diameter ranging from 70 to 150 nm. The DNA appeared to be densely packed, but its presence did not alter the morphology of the liposomes, suggesting no DNA-lipid interactions. These liposomes represent an attractive model system for studying the mechanisms of DNA transport and condensation. They may also serve as alternative vehicles for the transfer of foreign genes into eukaryotic cells.     

Entrapment of high-molecular-mass DNA molecules in liposomes for the genetic transformation of animal cells.

We modified the Ca/EDTA procedure for the production of liposomes [Papahadjopoulos, Vail, Jacobson & Poste (1975) Biochim. Biophys. Acta 394, 483-491] to entrap intact DNA molecules of very high molecular mass into large unilamellar phospholipid vesicles. The use of DNA-protein complexes and phage particles instead of naked linear DNA increases the efficiency of entrapment and protects the integrity of DNA molecules. We investigated the interaction of mammalian cells with liposome-encapsulated recombinant lambda bacteriophages carrying marker genes. The liposomes bind surprisingly fast to the cellular surface and are taken up by the cells. A significant proportion of the encapsulated DNA is transported to and soon located in or around the nuclei. Experiments prove that these liposomes can be used for the genetic transformation of mammalian cells.     

Targeting of lactosylceramide-containing liposomes to hepatocytes in vivo

Incorporation of 8 mol% lactosylceramide in small unilamellar vesicles consisting of cholesterol, dimyristoylphosphatidylcholine and phosphatidylserine in a molar ratio of 5:4:1 and containing [³H]inulin as an aqueous-space marker resulted in a 3-fold decreased half-life of the vesicles in blood and a corresponding increase in liver uptake after intracardial injection into rats. The increase in liver uptake was mostly accounted for by an enhanced uptake in the parenchymal cells, while the uptake by the non-parenchymal cells was only slightly increased. The uptake of both the control and the glycolipid-containing vesicles by the non-parenchymal cell fraction could be attributed completely to the Kupffer cells; no radioactivity was found in the endothelial cells. The effect of lactosylceramide on liver uptake and blood disappearance of the liposomes was effectively counteracted by desialylated fetuin, injected shortly before the liposome dose. This observation supports the notion that a galactose-specific receptor is involved in the liver uptake of lactosylceramide liposomes.     

Liposome-entrapped plasmid DNA: characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.     

Fluorescence measurement of the kinetics of DNA injection by bacteriophage lambda into liposomes

Bacteriophage lambda attaches to Gram-negative bacteria using the outer membrane protein LamB as its receptor. Subsequently, DNA is injected by the bacteriophage into the host cell for replication and expression. The mechanism of DNA injection, however, is poorly understood. In order to begin to characterize DNA injection, a quantitative kinetic assay to detect injection into reconstituted LamB liposomes is described. The technique involves monitoring the increase in fluorescence of liposome-encapsulated ethidium bromide, which occurs as DNA enters the aqueous compartment of the vesicles. The data indicate that injection is several times faster than indicated by earlier studies and is complete within 1 min. Such assays which allow direct observation of this process are necessary first steps toward a mechanistic understanding.     

Transformation of competent Bacillus subtilis cells by chromosomal and plasmid DNA incorporated in liposomes

Transformation with chromosomal and plasmid DNAs comprised in liposomes of different compositions was studied on competent cells of Bacillus subtilis. Transformation with chromosomal DNA comprised in liposomes appeared to constitute 1.1 to 1.5% of the control, and transformation with plasmid DNA in liposomes reaches 8 to 11%, as compared to the control. It has been revealed that absorbtion of chromosomal or plasmid DNA comprised in liposomes by competent cells is 1-2 orders higher than that of chromosomal or plasmid DNAs which are not contained in liposomes. Besides, chromosomal DNA in liposomes was found to be transferred to competent cells in the double-stranded form, while during common transformation without liposomes, the DNA transferred is single-stranded.     

Intracellular fate of liposome-encapsulated DNA in mouse liver. Analysis using electron microscope autoradiography and subcellular fractionation

Transient expression of liposome-encapsulated DNA in liver after intravenous injection to rats and mice has raised questions concerning the intracellular fate of this DNA. Electron microscope autoradiography shows that at 10 min after injection the highest concentration of liposomal DNA which is taken up by the liver is associated with lysosomes and vesicles. The proportion of DNA associated with the mitochondria steadily increases for 1 h after injection, up to 48% of the exogenous DNA found in the tissue. Part of this DNA follows the subcellular fractionation profile of the mitochondrial matrix marker, malate dehydrogenase. In contrast, 14% of the liposomal DNA taken up by the liver is found in the nuclei at 3 min after injection, and this percentage decreases over a period of 1 h. These results permit us to establish the distribution of liposome-encapsulated DNA among subcellular organelles in liver at different times after injection.     

Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA

A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.     

Expression of hepatitis B virus surface antigen in adult rat liver. Co-introduction of DNA and nuclear protein by a simplified liposome method.

We established a simple and efficient method for gene transfer in vitro (to cultured cells) and in vivo (to an adult organ) using liposomes. Plasmid DNA and proteins were efficiently co-encapsulated in liposomes by agitation and sonication, and were co-introduced into cells by hemagglutinating virus of Japan (HVJ)-mediated membrane fusion. Introduction of the Escherichia coli beta-galactosidase gene with non-histone chromosomal protein high mobility group 1 (HMG1) into LLCMK2 cells resulted in about 3 times higher beta-galactosidase activity than that on introduction of the gene alone. Two days after injection of HVJ-liposomes containing the beta-galactosidase gene and HMG1 under the perisplanchnic membrane of adult rat liver, hepatic cells near the injection site were found by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to have beta-galactosidase activity. After similar injection of HVJ-liposomes containing the hepatitis B virus surface antigen (HBsAg) gene and HMG1, HBsAg was detected in the serum for 9 days with a maximum of 25-45 ng/ml on day 2 after the injection.     

Detection of localization and internalization of membrane DNA in the Tetrahymena by the lactoferrin--colloidal gold technique

It is known that lactoferrin binds to DNA. In the present study a lactoferrin-gold complex bound initially to the ciliary membrane, then appeared in coated pits and intracytoplasmic vesicles. On treatment for 1 h, the gold particles could be detected near to the nucleus. The lactoferrin-gold complex neither bound to the surface, nor appeared in the cytoplasm of those cells which had been exposed to DNAse for pretreatment.     

Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA.

A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.     

Homologous recombination of exogenous DNA fragments with genomic DNA in somatic cells of mice.

We compared liposomes and empty viral capsids for their use as vehicles for DNA transfer into cells and animals. DNA binding capacity was high for liposomes, but DNase I protection of DNA bound to liposomes was only moderate in comparison to DNA incorporated into viral capsids. Cellular uptake of radiolabeled and physiologically active DNA was also compared. For animal studies we chose an endogenous retroposon as target gene. To identify recombinational events we replaced a part of this gene with an artificial sequence not present in the mouse genome. The recombination rate for DNA fragments transfected in Polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DNA transfer methods, mediated by positively charged liposomes (DOTMA) and by empty Polyoma viral capsids.     

Use of liposomes, viral capsids, and nanoparticles as DNA carriers

We tested a variety of liposomes for parameters such as DNA binding capacity and DNase I protection of incorporated and attached DNA to elucidate their use as vehicles for DNA transfer into cells and animals. The results were compared to other potential DNA vehicles, empty viral capsids, and nanoparticles. Maximal binding capacity was achieved for positively charged nanoparticles, DNase I protection was observed for most preparations with neosome preparations being least efficient. The uptake of radiolabeled DNA by cells in culture was determined for cationic and nonionic surfactant vesicles, viral capsids, and nanoparticles. Cellular DNA uptake was best for dioleoyl-derived positively charged liposomes (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride; DOTMA) and the DNA could be shown to be physiologically active. The recombination rate for DNA fragments transfected in polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DOTMA and polyoma-mediated DNA transfer.     

Function of coated membranes and multivesicular bodies during membrane regulation in stimulated exocrine pancreas cells

Summary Stimulation of secretion by pilocarpine results in a 70% loss of zymogen granules from pancreatic acinar cell during the first hr after injection of the drug. In previous work (Geuze and Poort, 1973), we found that the amount of membrane stored in the surface of the microvilli and of the numerous infoldings present in highly stimulated cells, increases during the first 2 hr and then decreases again during the 3rd hr after stimulation, concurrently with maximal endocytosis of sorbitol-[su14C].Further observations on the fine structure of stimulated cells at various time intervals after injection of pilocarpine showed that during the first hr numerous smooth vesicles and multivesicular bodies (mvb's) appear in the apical cytoplasm, while the number of coated vesicles and their relative total volume increase significantly 3 hr after stimulation.By infusion of ferritin in the pancreatic duct system in vivo and application of cytochemical techniques (osmium impregnation, electron microscope autoradiography and acid phosphatase cytochemistry) it could be established that after stimulated exocytotic secretion, redundant apical cell membrane is withdrawn by at least two routes: 1) During the initial rapid increase of the amount of apical cell membrane, withdrawal is accomplished by interiorization of luminal invaginations into smooth endocytotic vesicles, which in turn give rise to mvb's by infolding and subsequent fission of their limiting membrane. 2) Once the bulk of stored secretion granules has been discharged, endocytotic coated vesicles become gradually more prominent as carriers for redundant cell membrane. The contents of endocytotic structures ultimately become incorporated in residual bodies, suggesting lysosomal degradation of cell membrane prior to eventual reutilization.Coated vesicles also originate by pinching off from mature Golgi cisternae and condensing vacuoles. A possible function of the coated membranes in the concentration of exportable protein within forming secretory granules is discussed.     

Preclinical Studies with Doxorubicin Encapsulated in Polyethyleneglycol-Coated Liposomes

Abstract

Doxorubicin (DOX) has been encapsulated with high efficiency in the water phase of small-sized lipid vesicles. Plasma-induced drug leakage from these vesicles is minimal when hydrogenated phosphatidylcholine is present as the main component. A prolonged circulation time of liposome-encapsulated DOX is observed in animal models when a small fraction of polyethyleneglycol-derivatized phospholipid (PEG) is present in the liposome bilayer. Using these PEG-coated liposomes, we found that the concentration of DOX in tumor implants of the mouse M-109 carcinoma is significantly enhanced by liposome delivery. The antitumor activity of liposome-encapsulated DOX in a lung metastases model of the M-109 carcinoma is superior to that of free DOX. The minimal lethal dose of DOX to tumor-free mice was substantially increased by encapsulation in PEG-coated liposomes, indicating that toxicity is reduced. We also found that the vesicant of DOX after intradermal injection is prevented by liposome encapsulation. These preclinical observations, suggesting that encapsulation of DOX in PEG-coated liposomes may lead to a significant improvement of the therapeutic index of DOX, have led to the initiation of clinical trials in cancer patients.     

Formation of transmembrane channels in liposomes during injection of lambda DNA

Bacteriophage lambda binds to unilamellar liposomes bearing its receptor protein, LamB, and the lambda DNA can be injected into the internal aqueous space. During this process, transmembrane channels are formed in the liposomes which permit the entry and escape of small molecules, but not proteins. The channels are stable and persist after DNA injection.     

Glycophorin as a receptor for Escherichia coli alpha-hemolysin in erythrocytes

Escherichia coli alpha-hemolysin (HlyA) can lyse both red blood cells (RBC) and liposomes. However, the cells are lysed at HlyA concentrations 1-2 orders of magnitude lower than liposomes (large unilamellar vesicles). Treatment of RBC with trypsin, but not with chymotrypsin, reduces the sensitivity of RBC toward HlyA to the level of the liposomes. Since glycophorin, one of the main proteins in the RBC surface, can be hydrolyzed by trypsin much more readily than by chymotrypsin, the possibility was tested of a specific binding of HlyA to glycophorin. With this purpose, a number of experiments were performed. (a) HlyA was preincubated with purified glycophorin, after which it was found to be inactive against both RBC and liposomes. (b) Treatment of RBC with an anti-glycophorin antibody protected the cells against HlyA lysis. (c) Immobilized HlyA was able to bind glycophorin present in a detergent lysate of RBC ghosts. (d) Incorporation of glycophorin into pure phosphatidylcholine liposomes increased notoriously the sensitivity of the vesicles toward HlyA. (e) Treatment of the glycophorin-containing liposomes with trypsin reverted the vesicles to their original low sensitivity. The above results are interpreted in terms of glycophorin acting as a receptor for HlyA in RBC. The binding constant of HlyA for glycophorin was estimated, in RBC at sublytic HlyA concentrations, to be 1.5 x 10(-9) m.     

Uptake and utilization of free fatty acids supplied by liposomes to mammary tumor cells in culture.

Unesterified oleic acid was co-dispersed with various phospholipids in Ca-Mg-free saline and sonicated. Most of the fatty acid co-eluted with phospholipid vesicles when the sonicate was chromatographed on a column of Sepharose 4B. Cells from mouse mammary tumors, maintained in primary monolayer culture, were incubated with these phospholipid-oleic acid vesicles and optimal conditions for the uptake of oleic acid were established. When phosphatidylcholine and phosphatidylserine were used to make the vesicles, the oleic acid was taken up by cells more efficiently than was oleic acid complexed with albumin. The liposome-supplied unesterified oleic acid was rapidly incorporated into other cellular compounds, especially triglycerides and phospholipids. Growth of cells was measured after a 2-h pulse with the liposomes. The amount of DNA in treated cultures was 50% greater than in control cultures by 3 days after treatment. The magnitude of the growth response was sensitive to liposome dosage and to cell density in the treated cultures. This survey suggests that liposomes can be employed to insert materials directly into epithelial cells via their apical surface, that oleic acid so supplied is readily metabolized, and that solutes from liposomes can participate in functioning of the cells.     

In situ activation of murine macrophages by liposomes containing lymphokines

Murine alveolar and peritoneal macrophages harvested after injection of lymphokines encapsulated within multilamellar phospholipid vesicles (liposomes) were tumoricidal in vitro. The state and degree of activation depended on the route of liposome administration. Activation of peritoneal macrophages was achieved by intraperitoneal injection of liposomes and alveolar macrophages were activated by injecting liposomes intravenously but not intraperitoneally. The in vivo rendering of macrophages with tumoricidal properties might be useful toward destruction of tumor cells in vivo.