Liposomal nanomedicines: an emerging field.

Liposomal nanoparticles (LNs) encapsulating therapeutic agents, or liposomal nanomedicines (LNMs), represent one of the most advanced classes of drug delivery systems, with several currently on the market and many more in clinical trials. During the past 20 years, a variety of techniques have been developed for encapsulating both conventional drugs and the new genetic drugs (plasmid DNA-containing therapeutic genes, antisense oligonucleotides, and small, interfering RNA [siRNA]) within LNs encompassing a very specific set of properties: a diameter centered on 100 nm, a high drug-to-lipid ratio, excellent retention of the encapsulated drug, and a long (>6 hours) circulation lifetime. Particles with these properties tend to accumulate at sites of disease, such as tumors, where the endothelial layer is "leaky" and allows extravasation of particles with small diameters. Thus, LNs protect the drug during circulation, prevent it from reaching healthy tissues, and permit its accumulation at sites of disease. We will discuss recent advances in this field involving conventional anticancer drugs as well as gene-delivery, immunostimulatory, and gene-silencing applications involving the new genetic drugs. LNMs have the potential to offer new treatments in such areas as cancer therapy, vaccine development, and cholesterol management.

Liposomal nanomedicines.

Liposomal nanoparticles (LNs) encapsulating therapeutic agents, or liposomal nanomedicines, represent an advanced class of drug delivery systems, with several formulations presently on the market and many more in clinical trials. Over the past 20 years, a variety of techniques have been developed for encapsulating both conventional drugs (such as anticancer drugs and antibiotics) and the new genetic drugs (plasmid DNA containing therapeutic genes, antisense oligonucleotides and small interfering RNA) within LNs. If the LNs possess certain properties, they tend to accumulate at sites of disease, such as tumours, where the endothelial layer is 'leaky' and allows extravasation of particles with small diameters. These properties include a diameter centred on 100 nm, a high drug-to-lipid ratio, excellent retention of the encapsulated drug, and a long (> 6 h) circulation lifetime. These properties permit the LNs to protect their contents during circulation, prevent contact with healthy tissues, and accumulate at sites of disease. The authors discuss recent advances in this field involving conventional anticancer drugs, as well as applications involving gene delivery, stimulation of the immune system and silencing of unwanted gene expression. Liposomal nanomedicines have the potential to offer new treatments in such areas as cancer therapy, vaccine development and cholesterol management.

Transfection properties of stabilized plasmid-lipid particles containing cationic PEG lipids.

Recent work has shown that plasmid DNA can be efficiently encapsulated in well-defined "stabilized plasmid-lipid particles" (SPLP) that have potential as systemic gene therapy vehicles [Gene Ther. 6 (1999) 271]. In this work, we examine the influence of ligands that enhance cellular uptake on the transfection potency of SPLP. The ligand employed is a cationic poly(ethylene glycol) (PEG) lipid (CPL) consisting of a lipid anchor and a PEG(3400) spacer chain with four positive charges at the end of the PEG (CPL(4)). It is shown that up to 4 mol% CPL(4) can be inserted into preformed SPLP, resulting in up to 50-fold enhancements in uptake into baby hamster kidney (BHK) cells. The addition of Ca(2+) to SPLP-CPL(4) (CPL(4)-incorporated SPLP) results in up to 10(6)-fold enhancements in transgene expression, as compared to SPLP in the absence of either CPL(4) or Ca(2+). These transfection levels are comparable to those observed for plasmid DNA-cationic lipid complexes (lipoplexes) but without the cytotoxic effects noted for lipoplex systems. It is concluded that in the presence of Ca(2+) and appropriate ligands to stimulate uptake, SPLP are highly potent transfection agents.

Entrapment of small molecules and nucleic acid-based drugs in liposomes.

In the past two decades there have been major advances in the development of liposomal drug delivery systems suitable for applications ranging from cancer chemotherapy to gene therapy. In general, an optimized system consists of liposomes with a diameter of approximately 100 nm that possess a long circulation lifetime (half-life >5 h). Such liposomes will circulate sufficiently long to take advantage of a phenomenon known as disease site targeting, wherein liposomes accumulate at sites of disease, such as tumors, as a result of the leaky vasculature and reduced blood flow exhibited by the diseased tissue. The extended circulation lifetime is achieved by the use of saturated lipids and cholesterol or by the presence of PEG-containing lipids. This chapter will focus on the methodology required for the generation of two very different classes of liposomal carrier systems: those containing conventional small molecular weight (usually anticancer) drugs and those containing larger genetic (oligonucleotide and plasmid DNA) drugs. Initially, we will examine the encapsulation of small, weakly basic drugs within liposomes in response to transmembrane pH and ion gradients. Procedures will be described for the formation of large unilamellar vesicles (LUVs) by extrusion methods and for loading anticancer drugs into LUVs in response to transmembrane pH gradients. Three methods for generating transmembrane pH gradients will be discussed: (1) the use of intravesicular citrate buffer, (2) the use of transmembrane ammonia gradients, and (3) ionophore-mediated generation of pH gradients via transmembrane ion gradients. We will also discuss the loading of doxorubicin into LUVs by formation of drug-metal ion complexes. Different approaches are required for encapsulating macromolecules within LUVs. Plasmid DNA can be encapsulated by a detergent-dialysis approach, giving rise to stabilized plasmid-lipid particles, vectors with potential for systemic gene delivery. Antisense oligonucleotides can be spontaneously entrapped upon electrostatic interaction with ethanol-destabilized cationic liposomes, giving rise to small multilamellar systems known as stabilized antisense-lipid particles (SALP). These vectors have the potential to regulate gene expression.

Stabilized plasmid-lipid particles: a systemic gene therapy vector.

The ability of a systemically administered gene therapy vector to exhibit extended circulation lifetimes, accumulate at a distal tumor site, and enable transgene expression is unique to SPLP. The flexibility and low toxicity of SPLP as a platform technology for systemic gene therapy allows for further optimization of tumor transfection properties following systemic administration. For example, the PEG coating of SPLP is necessary to engender the long circulation lifetimes required to achieve tumor delivery. However, PEG coatings have also been shown to inhibit cell association and uptake required for transfection. The dissociation rate of the PEG coating from SPLP can be modulated by varying the acyl chain length of the ceramide anchor, suggesting the possibility of developing PEG-Cer molecules that remain associated with SPLP long enough to promote tumor delivery, but which dissociate quickly enough to allow transfection. Alternatively, improvements may be expected from inclusion of cell-specific targeting ligands in SPLP to promote cell association and uptake. Finally, the nontoxic properties of SPLP allow the possibility of higher doses. A dose of 100 micrograms plasmid DNA per mouse corresponds to a dose of approximately 5 mg plasmid DNA per kg body weight. This compares well to small molecules used for cancer therapy, which typically are used at dose levels of 10 to 50 mg per kg body weight. In summary, SPLP consist of plasmid encapsulated in a lipid vesicle that, in contrast to naked plasmid or complexes, exhibit extended circulation lifetimes following intravenous injection, resulting in accumulation and transgene expression at a distal tumor site in a murine model. The pharmacokinetics, biodistribution, and tumor transfection properties of SPLP are highly sensitive to the nature of the ceramide anchor employed to attach the PEG to the SPLP surface. The SPLP-CerC20 system in which the PEG-Cer does not readily dissociate exhibits good serum stability, long circulation lifetimes, and high levels of tumor accumulation and mediates marker gene expression at the tumor site. The flexibility of the SPLP system offers the potential of further optimization to achieve therapeutically effective levels of gene transfer and clearly has considerable potential as a nontoxic systemic gene therapy vehicle with general applicability. These features of SPLP contrast favorably with previous plasmid encapsulation procedures. Plasmid DNA has been encapsulated by a variety of methods, including reverse phase evaporation, ether injection, detergent dialysis in the absence of PEG stabilization, lipid hydration and dehydration-rehydration techniques, and sonication, among others. The characteristics of these protocols are summarized in Table I. None of these procedures yields small, serum-stable particles at high plasmid concentrations and plasmid-to-lipid ratios in combination with high plasmid-encapsulation efficiencies. Trapping efficiencies comparable with the SPLP procedure can be achieved employing methods relying on sonication. However, sonication is a harsh technique that can shear nucleic acids. Size ranges of 100 mm diameter or less can be achieved by reverse-phase techniques; however, this requires an extrusion step through filters with 100 nm or smaller pore size which can often lead to significant loss of plasmid. Finally, it may be noted that the plasmid DNA-to-lipid ratios that can be achieved for SPLP are significantly higher than those achievable by any other encapsulation procedure.

Calcium enhances the transfection potency of stabilized plasmid-lipid particles.

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

Distal cationic poly(ethylene glycol) lipid conjugates in large unilamellar vesicles prepared by extrusion enhance liposomal cellular uptake.

Cationic poly(ethylene glycol)-lipid conjugates (CPLs), a class of lipid designed to enhance the interaction of liposomes with cells, possess the following architectural features: 1) a hydrophobic lipid anchor of distearoylphosphatidylethanolamine (DSPE); 2) a hydrophilic spacer of poly(ethylene glycol); and 3) a cationic head group prepared with 0, 1, 3, or 7 lysine residues located at the distal end of the PEG chain, giving rise to CPL possessing 1, 2, 4, or 8 positive charges, respectively (CPL1 to CPL8). Previously we have described the synthesis of CPL, have characterized the postinsertion of CPL into PEG-containing LUVs and SPLP (stabilized plasmid-lipid particles), have shown significant increases in the binding of CPL-LUV to cells, and have observed dramatically enhanced transfection (up to a million-fold) of cells with CPL-SPLP in the presence of calcium [Chen et al. (2000) Bioconjugate Chem. 11, 433-437; Fenske et al. (2001) Biochim. Biophys. Acta 1512, 259-272; Palmer et al. (2003) Biochim. Biophys. Acta 1611, 204-216]. In the present study, we examine a variety of CPL properties (such as polarity and CMC) and characterize CPL-vesicular systems formed by extrusion and examine their interaction with cells. While CPL polarity was observed to increase dramatically with increasing charge number, CMC values were all found to be low, in the range of other PEGylated lipids, and exhibited only a small increase, going from CPL1 (1.3 microM) to CPL8 (2 microM). The CPLs were almost quantitatively incorporated into large unilamellar vesicles (LUVs) prepared by the extrusion method and were evenly distributed across the lipid bilayer. Lower levels of incorporation were obtained when CPLs were incubated with preformed liposomes (DSPC/Chol, 55:45) at 60 degrees C. The binding of CPL-LUVs to BHK cells in vitro was found to be dependent on the distal charge density of the CPL rather than total surface charge. Liposomes possessing CPL4 or CPL8 were observed to bind efficiently to cell surfaces and enhance cellular uptake in BHK cells (as observed with both lipid and aqueous content markers), whereas those possessing CPL1 or CPL2 exhibited little or no binding. These results suggest new directions for the design of liposomal systems capable of in vivo delivery of both conventional and genetic (plasmid and antisense) drugs.

Alkylated derivatives of poly(ethylacrylic acid) can be inserted into preformed liposomes and trigger pH-dependent intracellular delivery of liposomal contents.

Poly(ethylacrylic acid) (PEAA) is a pH-sensitive polymer that undergoes a transition from a hydrophilic to a hydrophobic form as the pH is lowered from neutral to acidic values. In this work we show that pH sensitive liposomes capable of intracellular delivery can be constructed by inserting a lipid derivative of PEAA into preformed large unilamellar vesicles (LUV) using a simple one step incubation procedure. The lipid derivatives of PEAA were synthesized by reacting a small proportion (3%) of the carboxylic groups of PEAA with C10 alkylamines to produce C10-PEAA. Incubation of C10-PEAA with preformed LUV resulted in the association of up to 8% by weight of derivatized polymer with the LUV without inducing aggregation. The resulting C10-PEAA-LUV exhibited pH-dependent fusion and leakage of LUV contents on reduction of the external pH below pH 6.0 as demonstrated by lipid mixing and release of calcein encapsulated in the LUV. In addition, C10-PEAA-LUV exhibited pH dependent intracellular delivery properties following uptake into COS-7 cells with appreciable delivery to the cell cytoplasm as evidenced by the appearance of diffuse intracellular calcein fluorescence. It is demonstrated that the cytoplasmic delivery of calcein by C10-PEAA-LUV could be inhibited by agents (bafilomycin or chloroquine) that inhibit acidification of endosomal compartments, indicating that this intracellular delivery resulted from the pH-dependent destabilization of LUV and endosomal membranes by the PEAA component of the C10-PEAA-LUV. It is concluded that C10-PEAA-LUV represents a promising intracellular delivery system for in vitro and in vivo applications.