David B. Fenske

Developments in liposomal drug delivery systems

Liposomes are the leading drug delivery systems for the systemic (iv.) administration of drugs. There are now liposomal formulations of conventional drugs that have received clinical approval and many others in clinical trials that bring benefits of reduced toxicity and enhanced efficacy for the treatment of cancer and other life-threatening diseases. The mechanisms giving rise to the therapeutic advantages of liposomes, such as the ability of long-circulating liposomes to preferentially accumulate at disease sites including tumours, sites of infection and sites of inflammation are increasingly well understood. Further, liposome-based formulations of genetic drugs such as antisense oligonucleotides and plasmids for gene therapy that have clear potential for systemic utility are increasingly available. This paper reviews the liposomal drug delivery field, summarises the success of liposomes for the delivery of small molecules and indicates how this success is being built on to design effective carriers for genetic drugs.

Influence of pH gradients on the transbilayer transport of drugs, lipids, peptides and metal ions into large unilamellar vesicles

Pieter R. Cullis , Michael J. Hope , Marcel B. Bally , Thomas D. Madden , Lawrence D. Mayer , David B. Fenske a a Department of Biochemistry and Molecular Biology, UniOersity of British Columbia, VancouOer, B.C., Canada V6T 1Z3 b Inex Pharmaceuticals Corporation, 1779 West 75th AOenue, VancouOer, B.C., Canada V6P 6P2 c British Columbia Cancer Agency, DiOision of Medical Oncology, VancouOer, B.C., Canada V5Z 4E6 d Department of Pharmacology and Therapeutics, UniOersity of British Columbia, VancouOer, B.C., Canada V6T 1Z3

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, anti-sense 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.

FACTORS INFLUENCING UPTAKE AND RETENTION OF AMINO-CONTAINING DRUGS IN LARGE UNILAMELLAR VESICLES EXHIBITING TRANSMEMBRANE PH GRADIENTS

The level of uptake and retention of amino-containing drugs in large unilamellar vesicles (LUVs) following uptake in response to a transmembrane pH gradient (DeltapH) can vary dramatically depending on the drug. For example, the anticancer drugs doxorubicin and epirubicin can be readily retained, whereas the anticancer drug vincristine and the antibiotic ciprofloxacin tend to leak out rapidly. In this investigation, we examine the influence of the hydrophobicity of the entrapped amines (that induce the DeltapH) and the anionic lipid content of the LUV on drug retention. It is shown that entrapment of increasingly hydrophobic monoamines (methylamine to amylamine) all lead to an induced DeltapH of 3 units and essentially complete drug uptake under the conditions employed, but do not lead to improved retention of vincristine and ciprofloxacin. However, significantly improved retention could be achieved by substitution of the anionic lipid distearoylphosphatidylglycerol (DSPG) for distearoylphosphatidylcholine (DSPC) in the LUV bilayer. Further, it is shown that if the induced DeltapH is reduced to 1.4 units (driven by entrapped diamine) nearly 100% accumulation of doxorubicin and epirubicin could be achieved, whereas only 25% loading for vincristine and ciprofloxacin was observed. Taken together these results provide methodology for improving (weak base) drug retention in liposomes and indicate that drugs that can partition into the lipid bilayer exhibit improved uptake and retention characteristics.

Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn2+ system exhibits superior drug retention over the nigericin/K+ system, and compares favorably with vesicles loaded by the standard DeltapH or amine methods. The unique features of this methodology and possible benefits are discussed.

Models of stratum corneum intercellular membranes: 2H NMR of macroscopically oriented multilayers

Deuterium NMR was used to characterize model membrane systems approximating the composition of the intercellular lipid lamellae of mammalian stratum corneum (SC). The SC models, equimolar mixtures of ceramide:cholesterol:palmitic acid (CER:CHOL:PA) at pH 5.2, were contrasted with the sphingomyelin:CHOL:PA (SPM:CHOL:PA) system, where the SPM differs from the CER only in the presence of a phosphocholine headgroup. The lipids were prepared both as oriented samples and as multilamellar dispersions, and contained either perdeuterated palmitic acid (PA-d31) or [2,2,3,4,6-2H5]CHOL (CHOL-d5). SPM:CHOL:PA-d31 formed liquid-ordered membranes over a wide range of temperatures, with a maximum order parameter of approximately 0.4 at 50 degrees C for positions C3-C10 (the plateau region). The quadrupolar splitting at C2 was significantly smaller, suggesting an orientational change at this position, possibly because of hydrogen bonding with water and/or other surface components. A comparison of the longitudinal relaxation times obtained at theta = 0 degrees and 90 degrees (where theta is the angle between the normal to the glass plates and the magnetic field) revealed a significant T1Z anisotropy for all positions. In contrast to the behavior observed with the SPM system, lipid mixtures containing CER exhibited a complex polymorphism. Between 20 and 50 degrees C, a significant portion of the entire membrane (as monitored by both PA-d31 and CHOL-d5) was found to exist as a solid phase, with the remainder either a gel or liquid-ordered phase. The proportion of solid decreased as the temperature was increased and disappeared entirely above 50 degrees C. Between 50 and 70 degrees C, the membrane underwent a liquid-ordered to isotropic phase transition. These transitions were reversible but displayed considerable hysteresis, especially the conversion from a fluid phase to solid. The order profiles, relaxation behavior, and angular dependence of these parameters suggest strongly that both the liquid-ordered CER- and SPM-membranes are bilayers. The unusual phase behavior observed for the CER-system, particularly the observation of solid-phase lipid at physiological temperatures, may provide insight into the functioning of the permeability barrier of stratum corneum.

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.

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.

Cationic poly(ethyleneglycol) lipids incorporated into pre-formed vesicles enhance binding and uptake to BHK cells

This paper describes a new method for enhancing the interaction of liposomes with cells. A novel class of cationic poly(ethyleneglycol) (PEG)-lipid (CPL) conjugates have been characterized for their ability to insert into pre-formed vesicles and enhance in vitro cellular binding and uptake of neutral and sterically-stabilized liposomes. The CPLs, which consist of a distearoylphosphatidylethanolamine (DSPE) anchor, a fluorescent dansyl moiety, a heterobifunctional PEG polymer (M(r) 3400), and a cationic headgroup composed of lysine derivatives, have been described previously [Bioconjug. Chem. 11 (2000) 433]. Five separate CPL, possessing 1-4 positive charges in the headgroup (referred to as CPL(1)-CPL(4), respectively), were incubated (as micellar solutions) in the presence of neutral or sterically-stabilized cationic large unilamellar vesicles (LUVs), and were found to insert into the external leaflet of the LUVs in a manner dependent on temperature, time, CPL/lipid ratio, and LUV composition. For CPL/lipid molar ratios < or =0.1, optimal insertion levels of approximately 70% of initial CPL were obtained following 3 h at 60 degrees C. The insertion of CPL resulted in aggregation of the LUVs, as assessed by fluorescence microscopy, which could be prevented by the presence of 40 mM Ca(2+). The effect of CPL-insertion on the binding of LUVs to cells was examined by fluorescence microscopy and quantified by measuring the ratio of rhodamine fluorescence to protein concentration. Neither control LUVs or LUVs containing CPL(2) displayed significant uptake by BHK cells. However, a 3-fold increase in binding was observed for LUVs possessing CPL(3), while for CPL(4)-LUVs values as high as 10-fold were achieved. Interestingly, the increase in lipid uptake did not correlate with total surface charge, but rather with increased positive charge density localized at the CPL distal headgroups. These results suggest that incorporation of CPLs into existing liposomal drug delivery systems may lead to significant improvements in intracellular delivery of therapeutic agents.

Structural and motional properties of vesicles as revealed by nuclear magnetic resonance

Correspondence to: David B. Fenske, Department of Biochemistry, Faculty of Medicine, University of British Columbia, 2146 Health Sciences Mall, Vancouver, B.C., Canada V6T 1Z3. Abbreviations: CHOL, cholesterol; CL, cardiolipin; D, lateral diffusion coefficient; DMPC, L-t~-dimyristoyl phosphatidylcholine; DOPA, L-ct-dioleoyl phosphatidic acid; DOPC, L-c~dioleoyl phosphatidylcholine; DOPE, L-ct-dioleoyl phosphatidylethanolamine; DOPS, L-~-dioleoyl phosphatidylserine; I)PPC, L-t~-dipalmitoyl phosphatidylcholine; DSC, differential scanning calorimetry; DSPC, t-a-distearoyl phosphatidylcholine; DSPG, L-c~-distearoyl phosphatidylglycerol; EPC, egg phosphatidylcholine; ESR, electron spin resonance; FRAP; fluorescence recovery after photobleaching; FT-IR, Fourier transform infrared spectroscopy; Gd-DTPA, gadoliniumdiethylenetriamine-pentaacetic acid; HDL, high density lipoprotein; LDL, low density lipoprotein; LPC, lysophosphatidylcholine; LUV, large unilamellar vesicle; MLV, multilamellar vesicle; MRI, magnetic resonance imaging; NOESY, nuclear Overhauser enhancement and exchange spectroscopy; NMR, nuclear magnetic resonance; NMRD, nuclear magnetic relaxation dispersion; NOE, nuclear Overhauser enhancement; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; P1, phosphatidylinositol; PIP, PI-4-phosphate; PIP 2, PI-4,5-diphosphate; PS, phosphatidylserine; POPC, lpalmitoyl-2-oleoyl phosphatidylcholine; POPC-d31 , l-[2H31]Palmitoyl-2-oleoyl phosphatidylcholine; QELS, quasi-elastic light scattering; ROESY, rotating frame nuclear Overhauser effect spectroscopy; SCD , carbon-deuterium bond order parameter; SPM, sphingomyelin; SUV, small unilamellar vesicle; TI, spin-lattice or longitudinal relaxation time; T2, spin-spin or transverse relaxation time; Tin, gel-to-liquid crystalline phase transition temperature; VLDL, very low density lipoprotein. One of the most powerful techniques that has been applied to the study of biological and model membranes is nuclear magnetic resonance (NMR). Questions regarding both static and dynamic aspects of membrane structure can be probed via an almost limitless number of pulse sequences, thereby providing a wealth of information not easily obtained from other techniques. One reason for the usefulness of NMR in the elucidation of membrane structure stems from the anisotropic nature of molecular motions within liquidcrystalline lipid bilayers. This is true for intramolecular reorientations of lipid functional groups (acyl chains, headgroups) as well as motions of the whole molecule (rotational and lateral diffusion, transbilayer transport). NMR allows characterization of these motions because many magnetic interactions are themselves anisotropic (e.g., the chemical shift anisotropy, dipolar and quadrupolar interactions). Information on molecular ordering and orientation, rate and type of motion(s) and polymorphism have been obtained from such techniques as solid-state 2H[1-6] 31p_ [2,7-9] and 13C-NMR [2]. Unlike other methods such as fluorescence and ESR which rely on bulky reporter groups, NMR is entirely non-perturbing. A wide range of nuclei are available for study,