Figure 1 Diagrams drawn from cryoelectron micrographs of cross-sections through vitrified films of various types of liposomes and DNA-liposome complexes. SUVs condense nucleic acids on the surface and produce ''spaghetti and meatballs'' structures. MLVs appear as ''Swiss rolls'' after mixing with DNA. BIVs are produced using a formulation developed in our laboratory (14). Nucleic acids are efficiently encapsulated between 2 BIVs.

Figure 1 Diagrams drawn from cryoelectron micrographs of cross-sections through vitrified films of various types of liposomes and DNA-liposome complexes. SUVs condense nucleic acids on the surface and produce ''spaghetti and meatballs'' structures. MLVs appear as ''Swiss rolls'' after mixing with DNA. BIVs are produced using a formulation developed in our laboratory (14). Nucleic acids are efficiently encapsulated between 2 BIVs.

they demonstrated efficiency primarily for the transfection of some cell types in culture and not for in vivo delivery. SUVs condense nucleic acids on the surface and form ''spaghetti and meatballs'' structures (21). DNA-liposome complexes made using SUVs produce little or no gene expression upon systemic delivery, although these complexes transfect numerous cell types efficiently in vitro (1,2). Furthermore, SUV liposome-DNA complexes cannot be targeted efficiently. SUV liposome-DNA complexes also have a short half-life within the circulation, generally about 5 to 10 min. Polyethylene glycol (PEG) has been added to liposome formulations to extend their half-life (22-24); however, PEGylation created other problems that have not been resolved. PEG seems to hinder delivery of cationic liposomes into cells due to its steri-cally hindering ionic interactions, and it interferes with optimal condensation of nucleic acids onto the cationic delivery vehicle. Furthermore, extremely long half-life in the circulation (e.g., several days) has caused problems for patients because the bulk of the PEGylated liposomal formulation doxil that encapsulates the cytotoxic agent, doxorubicin, accumulates in the skin, hands, and feet. For example, patients contract mucositis and hand and foot syndrome (25,26) that cause extreme discomfort to the patient. Attempts to add ligands to doxil for delivery to specific cell surface receptors has not resulted in much cell-specific delivery, and the majority of the injected targeted formulation still accumulates in the skin, hands, and feet. Addition of PEG into formulations developed in our laboratory also caused steric hindrance in the bilamellar invaginated structures that did not encapsulate DNA efficiently, and gene expression was substantially diminished.

Some investigators have loaded nucleic acids within SUVs using a variety of methods; however, the bulk of the DNA does not load or stay within the liposomes. Furthermore, most of the processes used for loading nucleic acids within liposomes are extremely time consuming and not cost effective. Therefore, SUVs are not the ideal liposomes for creating non-viral vehicles for targeted delivery.

Complexes made using MLVs appear as ''Swiss rolls'' when viewing cross-sections by cryoelectron microscopy (27). These complexes can become too large for systemic administration or deliver nucleic acids inefficiently into cells due to inability to ''unravel'' at the cell surface. Addition of ligands onto MLV liposome-DNA complexes further aggravates these problems. Therefore, MLVs are not useful for the development of targeted delivery of nucleic acids.

Using a formulation developed in our laboratory, nucleic acids are efficiently encapsulated between 2 BIVs (14). We created these unique structures using 1,2-bis(oleoyloxy)-3-(trimethylammino)propane (DOTAP) and cholesterol (Chol), and a novel formulation procedure. This procedure is different because it includes a brief, low-frequency sonication, followed by manual extrusion through filters of decreasing pore size. The 0.1- and 0.2-um filters used are made of aluminum oxide and not polycarbonate, which is typically used by other protocols. Aluminum oxide membranes contain more pores per surface area, evenly spaced and sized pores, and pores with straight channels. During the manual extrusion process, the liposomes are passed through each of 4 different-size filters only once. This process produces 88% invaginated liposomes. Use of high-frequency sonication and/or mechanical extrusion produces only SUVs.

The BIVs produced condense unusually large amounts of nucleic acids of any size (Fig. 2) or viruses (Fig. 3). Furthermore, addition of other DNA-condensing agents including polymers is not necessary. For example, condensation of plasmid DNA onto polymers first before encapsulation in the BIVs did not increase condensation or subsequent gene expression after transfection in vitro or in vivo. Encapsulation of nucleic acids by these BIVs alone is spontaneous and immediate, and therefore, cost effective, requiring only 1 step of simple mixing. The extruded DOTAP : Chol-nucleic acid complexes are also large enough that they are not cleared rapidly by Kupffer cells in the liver, and yet extravasate across tight barriers, including the endothelial cell barrier of the lungs in a normal mouse, and diffuse through target organs efficiently (15). Our recent work demonstrating efficacy for treatment of nonsmall cell lung cancer (15) showed that only BIV DOTAP : Chol-p53 DNA liposome complexes produced efficacy, and SUV DOTAP:Chol-p53 DNA liposome complexes produced no efficacy. Therefore, the choice of lipids alone is

Assembly of Complexes

Figure 2 Proposed model showing cross-sections of extruded DOTAP:Chol liposomes (BIVs) interacting with nucleic acids. Nucleic acids adsorb onto a BIV via electrostatic interactions. Attraction of a second BIV to this complex results in further charge neutralization. Expanding electrostatic interactions with nucleic acids cause inversion of the larger BIV and total encapsulation of the nucleic acids. Inversion can occur in these liposomes because of their excess surface area, which allows them to accommodate the stress created by the nucleic acid-lipid interactions. Nucleic acid binding reduces the surface area of the outer leaflet of the bilayer and induces the negative curvature due to lipid ordering and reduction of charge repulsion between cationic lipid headgroups. Condensation of the internalized nucleic acid-lipid sandwich expands the space between the bilayers and may induce membrane fusion to generate the apparently closed structures. The enlarged area shows the arrangement of nucleic acids condensed between two 4-nm bilayers of extruded DOTAP:Chol.

not sufficient for optimal DNA delivery, and the morphology of the complexes is essential.


Choosing the best cationic lipids and neutral lipids are also essential for producing the optimal in vivo formulation. For example, using our novel manual extrusion procedure does not produce BIVs using the cationic lipid dimethyldioctadecy-lammonium bromide (DDAB). Furthermore, DOTAP is biodegradable, whereas DDAB is not biodegradable. Use of biodegradable lipids is preferred for use in humans. Furthermore, only DOTAP and not DDAB-containing liposomes produced highly efficient gene expression in vivo (14). DDAB did not produce BIVs and was unable to encapsulate nucleic acids. Apparently, DDAB- and DOTAP-containing SUVs produce similar efficiency of gene delivery in vivo; however, these

Assembly of BIV + Adenovirus Complexes

Figure 3 Proposed model showing cross-sections of an extruded DOTAP:Chol liposome (BIV) interacting with adenovirus. Adenovirus interacts with a BIV, causing negative curvature and wrapping around the virus particle.

SUVs are not as efficient as BIV DOTAP:Chol (14). In addition, use of L-a-dioleoyl phosphatidylethanolamine (DOPE) as a neutral lipid creates liposomes that cannot wrap or encapsulate nucleic acids. Several investigators have reported efficient transfection of cells in culture using DOPE in liposomal formulations. However, our data showed that formulations consisting of DOPE were not efficient for producing gene expression in vivo (14).

Investigators must also consider the source and lot of certain lipids purchased from companies. For example, different lots of cholesterol from the same vendor can vary dramatically and will affect the formulation of liposomes. Recently, we are using synthetic cholesterol (Sigma, St. Louis, MO). Synthetic cholesterol, instead of natural cholesterol purified from the wool of sheep, is preferred by the Food and Drug Administration for use in producing therapeutics for injection into humans.

Our BIV formulations are also stable for a few years as liquid suspensions. Freeze-dried formulations that are stable indefinitely even at room temperature can also be made. Stability of liposomes and liposomal complexes is also essential, particularly for the commercial development of human therapeutics.


A common belief is that artificial vehicles must be 100 nm or smaller to be effective for systemic delivery. However, this belief is most likely true only for large, inflexible delivery vehicles. Blood cells are several microns (up to 7000 nm) in size and yet have no difficulty circulating in the blood, includ ing through the smallest capillaries. However, sickle-cell blood cells, which are rigid, do have problems in the circulation. Therefore, we believe that flexibility is a more important issue than small size. In fact, BIV DNA-liposome complexes in the size range of 200 to 450 nm produced the highest levels of gene expression in all tissues after intravenous injection (14). Delivery vehicles, including nonviral vectors and viruses, which are not PEGylated and are smaller than 200 nm, are cleared quickly by the Kupffer cells in the liver. Therefore, increased size of liposomal complexes could extend their circulation time, particularly when combined with injection of high colloidal suspensions. BIVs are able to encapsulate nucleic acids and viruses, apparently due to the presence of cholesterol in the bilayer (Fig. 4). Whereas formulations including DOPE instead of cholesterol could not assemble nucleic acids by a ''wrapping type'' of mechanism (Fig. 5), and produced little gene expression in the lungs and no expression in other tissues after intravenous injections. Because the extruded DOTAP:Chol BIV complexes are flexible and not rigid, are stable in high concentrations of serum, and have extended half-life, they do not have difficulty circulating efficiently in the bloodstream.

We believe that colloidal properties of nucleic acid-lipo-some complexes also determine the levels of gene expression produced after in vivo delivery (14,28). These properties include the DNA:lipid ratio that determines the overall charge density of the complexes and the colloidal suspension that is monitored by its turbidity. Complex size and shape, lipid composition and formulation, and encapsulation efficiency of nucleic acids by the liposomes also contribute to the colloidal properties of the complexes. The colloidal properties affect serum stability, protection from nuclease degradation, blood circulation time, and biodistribution of the complexes.

Our in vivo transfection data showed that an adequate amount of colloids in suspension was required to produce

Figure 4 Cryoelectron micrograph of BIV DOTAP:Chol-DNA liposome complexes. The plasmid DNA is encapsulated between 2 BIVs.
Figure 5 Cryoelectron micrograph of extruded DOTAP:DOPE liposomes complexed to plasmid DNA. Although these liposomes were prepared by the same protocol that produces BIV DOTAP: Chol, these vesicles cannot wrap and encapsulate nucleic acids. The DNA condenses on the surfaces of the liposomes shown.

efficient gene expression in all tissues examined (14). The colloidal suspension is assessed by measurement of ad-sorbance at 400 nm using a spectrophotometer optimized to measure turbidity. Our data showed that transfection efficiency in all tissues corresponded to OD400 of the complexes measured prior to intravenous injection.

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