Kim F. Wong

Rational design of cationic lipids for siRNA delivery

We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

Spontaneous Entrapment of Polynucleotides upon Electrostatic Interaction with Ethanol-Destabilized Cationic Liposomes

This study describes the effect of ethanol and the presence of poly(ethylene) glycol (PEG) lipids on the interaction of nucleotide-based polyelectrolytes with cationic liposomes. It is shown that preformed large unilamellar vesicles (LUVs) containing a cationic lipid and a PEG coating can be induced to entrap polynucleotides such as antisense oligonucleotides and plasmid DNA in the presence of ethanol. The interaction of the cationic liposomes with the polynucleotides leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. The degree of lamellarity as well as the size and polydispersity of the liposomes formed increases with increasing polynucleotide-to-lipid ratio. A direct correlation between the entrapment efficiency and the membrane-destabilizing effect of ethanol was observed. Although the morphology of the liposomes is still preserved at the ethanol concentrations used for entrapment (25-40%, v/v), entrapped low-molecular-weight solutes leak rapidly. In addition, lipids can flip-flop across the membrane and exchange rapidly between liposomes. Furthermore, there are indications that the interaction of the polynucleotides with the cationic liposomes in ethanol leads to formation of polynucleotide-cationic lipid domains, which act as adhesion points between liposomes. It is suggested that the spreading of this contact area leads to expulsion of PEG-ceramide and triggers processes that result in the formation of multilamellar systems with internalized polynucleotides. The high entrapment efficiencies achieved at high polyelectrolyte-to-lipid ratios and the small size and neutral character of these novel liposomal systems are of utility for liposomal delivery of macromolecular drugs.

Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients

The uptake of the anticancer agent doxorubicin into large unilamellar vesicles (LUVs) exhibiting a transmembrane pH gradient (inside acidic) has been investigated using both kinetic and equilibrium approaches. It is shown that doxorubicin uptake into the vesicles proceeds via permeation of the neutral form and that uptake of the drug into LUVs with an acidic interior is associated with high activation energies (Ea) which are markedly sensitive to lipid composition. Doxorubicin uptake into egg-yolk phosphatidylcholine (EPC) LUVs exhibited an activation energy of 28 kcal/mol, whereas for uptake into EPC/cholesterol (55:45, mol/mol) LUVs Ea = 38 kcal/mol. The equilibrium uptake results obtained are analyzed in terms of a model which includes the buffering capacity of the interior medium and the effects of drug partitioning into the interior monolayer. From the equilibrium uptake behaviour, a doxorubicin partition coefficient of 70 can be estimated for EPC/cholesterol bilayers. For a 100 nm diameter LUV, this indicates that more than 95% of encapsulated doxorubicin is partitioned into the inner monolayer, presumably located at the lipid/water interface. This is consistent with 13C-NMR behaviour as a large proportion of the drug appears membrane associated after accumulation as reflected by a broadening beyond detection of the 13C-NMR spectrum. The equilibrium accumulation behaviour of a variety of other lipophilic amines is also examined in terms of the partitioning model.

Anomalous solubility behavior of the antibiotic ciprofloxacin encapsulated in liposomes: a 1H-NMR study

Many drugs are weak bases and can be accumulated into liposomes in response to a pH gradient to achieve high internal drug concentrations. This study is aimed at gaining an understanding of the relationship between the retention of the fluoroquinolone antibiotic ciprofloxacin in liposomes and the intraliposomal form and location of this drug. 1H-NMR spectroscopy was used to probe the interactions experienced by ciprofloxacin following uptake into large unilamellar liposomes (LUV). It is shown that ciprofloxacin is located in the aqueous interior of the liposomes and is self-associated in the form of small stacks. It does not precipitate out of solution even though the intraliposomal ciprofloxacin concentration can exceed its solubility in aqueous solutions by almost two orders of magnitude. The results also indicate that little entrapped ciprofloxacin partitions into the inner monolayer of the LUV. As a result of the lack of precipitation and rapid exchange properties, ciprofloxacin can respond quickly to changes in electrochemical equilibria such as depletion of the pH gradient. This provides a rationale for the rapid leakage of this drug in response to serum destabilization or depletion of the pH gradient.

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.

Acyl chain orientational order in the hexagonal HII phase of phospholipid-water dispersions

The deuterium nuclear magnetic resonance (2H NMR) spectrum of perdeuterated tetradecanol in a mixture of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and water was used to compare the variation of the acyl chain orientational order parameter, S(n), with carbon position, n, in the liquid crystalline lamellar (L alpha) and hexagonal (HII) phases. The characteristics independence of S(n) with n (plateau) normally observed in the L alpha phase is replaced by a more rapid decrease of S(n) with n in the HII phase. It is suggested that as a consequence of the geometrical characteristics of the HII phase, there is an increase in conformational freedom available to different parts of the acyl chain.

Proton flux in large unilamellar vesicles in response to membrane potentials and pH gradients

The transport of protons across liposomes composed of phosphatidylcholine in response to electrical potentials or pH gradients has been investigated. The results support three major conclusions. The first of these concerns the need for reliable measurements of electrical potentials and pH gradients. It is shown that the potential probe tetraphenylphosphonium and the pH probe methylamine provide accurate and self consistent measures of electrical potentials and pH gradients respectively in these systems. Second, it is shown by two independent techniques that the pH gradients induced in response to valinomycin and potassium dependent electrical potentials are significantly smaller than would be expected for electrochemical equilibrium. The pH gradients observed are stable over an 8 h time course and are sensitive to the ionic composition of the buffers employed, where the presence of external sodium results in the smallest induced pH gradients. These results are discussed in terms of current models of proton conductance across membranes. In a final area of investigation, it is shown that valinomycin and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) can transport sodium ions in a synergistic manner.

Dopamine accumulation in large unilamellar vesicle systems induced by transmembrane ion gradients

Transmembrane movement of dopamine in response to K+ or H+ ion gradients has been investigated. It is shown that dopamine can accumulate rapidly into large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine exhibiting either a K+ diffusion potential (delta psi; negative inside) or a pH gradient (inside acidic). This can result in entrapped dopamine concentrations of 30-40 mM and inside-outside concentration gradients of nearly 300-fold. The transmembrane dopamine gradients formed in LUV systems exhibiting delta pH (inside acidic) indicate that the transport process can be dictated by movement of the neutral form of dopamine which redistributes according to a simple Henderson-Hasselbach equilibrium. The mechanism of dopamine transport in response to a valinomycin-induced K+ potential is more complex. Although generation of a K+ diffusion potential results in acidification of the vesicle interior, the magnitude of the induced delta pH (approx. 1 pH unit) is insufficient to account for the dopamine concentration gradient achieved (greater than 200-fold). Further, data presented here suggest that higher uptake levels of dopamine can be achieved when certain anions (ATP and citrate) are entrapped within the LUV system. These anions may complex with the protonated form of dopamine creating a non-equilibrium trapping phenomena resulting in interior concentrations of dopamine in excess of that predicted by a simple Henderson-Hasselbach equilibrium.

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.