Lipid nanoparticle formulations for optimal RNA-based topical delivery to murine airways.

INTRODUCTION: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19. METHODS: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging. RESULTS: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73-97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control. CONCLUSION: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.

Structural Properties of Inverted Hexagonal Phase: A Hybrid Computational and Experimental Approach.

Inverted/reverse hexagonal (HII) phases are of special interest in several fields of research, including nanomedicine. We used molecular dynamics (MD) simulation to study HII systems composed of dioleoylphosphatidylethanolamine (DOPE) and palmitoyloleoylphosphatidylethanolamine (POPE) at several hydration levels and temperatures. The effect of the hydration level on several HII structural parameters, including deuterium order parameters, was investigated. We further used MD simulations to estimate the maximum hydrations of DOPE and POPE HII lattices at several given temperatures. Finally, the effect of acyl chain unsaturation degree on the HII structure was studied via comparing the DOPE with POPE HII systems. In addition to MD simulations, we used deuterium nuclear magnetic resonance (2H NMR) and small-angle X-ray scattering (SAXS) experiments to measure the DOPE acyl chain order parameters, lattice plane distances, and the water core radius in HII phase DOPE samples at several temperatures in the presence of excess water. Structural parameters calculated from MD simulations are in excellent agreement with the experimental data. Dehydration decreases the radius of the water core. An increase in hydration level slightly increased the deuterium order parameter of lipids acyl chains, whereas an increase in temperature decreased it. Lipid cylinders undulated along the cylinder axis as a function of hydration level. The maximum hydration levels of PE HII phases at different temperatures were successfully predicted by MD simulations based on a single experimental measurement for the lattice plane distance in the presence of excess water. An increase in temperature decreases the maximum hydration and consequently the radius of the water core and lattice plane distances. Finally, DOPE formed HII structures with a higher curvature compared to POPE, as expected. We propose a general protocol for constructing computational HII systems that correspond to the experimental systems. This protocol could be used to study HII systems composed of molecules other than the PE systems used here and to improve and validate force field parameters by using the target data in the HII phase.

Coating of PLA-nanoparticles with cyclic, arginine-rich cell penetrating peptides enables oral delivery of liraglutide.

Until today, the oral delivery of peptide drugs is hampered due to their instability in the gastrointestinal tract and low mucosal penetration. To overcome these hurdles, PLA (polylactide acid)-nanoparticles were coated with a cyclic, polyarginine-rich, cell penetrating peptide (cyclic R9-CPP). These surface-modified nanoparticles showed a size and polydispersity index comparable to standard PLA-nanoparticles. The zeta potential showed a significant increase indicating successful CPP-coupling to the surface of the nanoparticles. Cryo-EM micrographs confirmed the appropriate size and morphology of the modified nanoparticles. A high encapsulation efficiency of liraglutide could be achieved. In vitro tests using Caco-2 cells showed high viability indicating the tolerability of this novel formulation. A strongly enhanced mucosal binding and penetration was demonstrated by a Caco-2 binding and uptake assay. In Wistar rats, the novel nanoparticles showed a substantial, 4.5-fold increase in the oral bioavailability of liraglutide revealing great potential for the oral delivery of peptide drugs.

Ionizable amino lipid interactions with POPC: implications for lipid nanoparticle function.

Lipid nanoparticles (LNPs) composed of ionizable cationic lipids are currently the leading systems for siRNA delivery in liver disease, with the major limitation of low siRNA release efficacy into the cytoplasm. Ionizable cationic lipids are known to be of critical importance in LNP structure and stability, siRNA entrapment, and endosomal disruption. However, their distribution inside the LNPs and their exact role in cytoplasmic delivery remain unclear. A recent study [Kulkarni et al., On the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and siRNA, ACS Nano, 2018, 12(5), 4787-4795] on LNP-siRNA systems containing the ionizable lipid DLin-KC2-DMA (also known as KC2 with an apparent pKa of ca. 6.7) suggested that neutral KC2 segregates from other components and forms an amorphous oil droplet in the core of LNPs. In this paper, we present evidence supporting the model proposed by Kulkarni et al. We studied KC2 segregation in the presence of POPC using molecular dynamics simulation, deuterium NMR, SAXS, and cryo-TEM experiments, and found that neutral KC2 has a high tendency to separate from POPC dispersions. KC2 confinement, upon raising the pH during the formulation process, could result in rearrangement of the internal structure of LNPs. As interactions between cationic KC2 and anionic endosomal lipids are thought to be a key factor in cargo release, KC2 confinement inside the LNP may be responsible for the observed low release efficacy.

Lipid nanoparticle delivery systems for siRNA-based therapeutics.

Therapeutics based on small interfering RNA (siRNA) have a huge potential for the treatment of disease but requires sophisticated delivery systems for in vivo applications. Lipid nanoparticles (LNP) are proven delivery systems for conventional small molecule drugs with over eight approved LNP drugs. Experience gained in the clinical development of LNP for the delivery of small molecules, combined with an understanding of the physical properties of lipids, can be applied to design LNP systems for in vivo delivery of siRNA. In particular, cationic lipids are required to achieve efficient encapsulation of oligonucleotides; however, the presence of a charge on LNP systems can result in toxic side effects and rapid clearance from the circulation. To address these problems, we have developed ionizable cationic lipids with pKa values below 7 that allow oligonucleotide encapsulation at low pH (e.g., pH 4) and a relatively neutral surface at physiological pH. Further optimization of cationic lipids to achieve maximized endosomal destabilization following uptake has resulted in LNP siRNA systems that can silence genes in hepatocytes at doses as low as 0.005 mg siRNA/kg body weight in mouse models. These systems have been shown to be highly effective clinically, with promising results for the treatment of hypercholesterolemia and transthyretin-induced amyloidosis among others. More LNP siRNA therapeutics, targeting different tissues and diseases, are expected to become available in the near future.

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.

On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids.

The mechanism whereby cationic lipids destabilize cell membranes to facilitate the intracellular delivery of macromolecules such as plasmid DNA or antisense oligonucleotides is not well understood. Here, we show that cationic lipids can destabilize lipid bilayers by promoting the formation of nonbilayer lipid structures. In particular, we show that mixtures of cationic lipids and anionic phospholipids preferentially adopt the inverted hexagonal (H(II)) phase. Further, the presence of 'helper' lipids such as dioleoylphosphatidylethanolamine or cholesterol, lipids that enhance cationic lipid-mediated transfection of cells also facilitate the formation of the H(II)phase. It is suggested that the ability of cationic lipids to promote nonbilayer structures in combination with anionic phospholipids leads to disruption of the endosomal membrane following uptake of nucleic acid-cationic lipid complexes into cells, thus facilitating cytoplasmic release of the plasmid or oligonucleotide.

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.

Long-circulating vectors for the systemic delivery of genes.

The development of vectors capable of treating systemic diseases is an important goal for gene therapy protocols. In order for a carrier system to preferentially accumulate at sites of systemic disease, such as tumors, sites of inflammation and sites of infection, the carrier must exhibit long circulation lifetimes following intravenous injection. Unfortunately, most gene delivery systems, including viral vectors as well as non-viral vectors, e.g., lipoplexes, polyplexes and lipopolyplexes, are rapidly cleared from the circulation and are preferentially taken up by the 'first-pass' organs such as liver, lung and spleen. Here we review recent literature concerning the ability of non-viral vectors to act as systemic gene therapy agents. The most promising systemic vectors are liposomal systems in which plasmid DNA is encapsulated within a lipid bilayer. The stabilized plasmid-lipid particle (SPLP) system, for example, exhibits circulation half-lives of the order of 6 h following intravenous injection, and preferentially accumulates in distal tumors with gene expression primarily localized to the tumor site.

Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures.

Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15--0.25 g ODN/g lipid) of polyanionic ODN in lipid vesicles. This process requires the presence of up to 40% ethanol (v/v) and initial formulation at acidic pH values where the DODAP is positively charged. In addition, the presence of a poly(ethylene glycol)-lipid was required during the formulation process to prevent aggregation. The 'stabilized antisense-lipid particles' (SALP) formed are stable on adjustment of the external pH to neutral pH values and the formulation process allows encapsulation efficiencies of up to 70%. ODN encapsulation was confirmed by nuclease protection assays and (31)P NMR measurements. Cryo-electron microscopy indicated that the final particles consisted of a mixed population of unilamellar and small multilamellar vesicles (80--140 nm diameter), the relative proportion of which was dependent on the initial ODN to lipid ratio. Finally, SALP exhibited significantly enhanced circulation lifetimes in mice relative to free antisense ODN, cationic lipid/ODN complexes and SALP prepared with quaternary aminolipids. Given the small particle sizes and improved encapsulation efficiency, ODN to lipid ratios, and circulation times of this formulation compared to others, we believe SALP represent a viable candidate for systemic applications involving nucleic acid therapeutics.

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.

Roles of lipid polymorphism in intracellular delivery.

Lipids, which adopt nonbilayer phases, have fascinated researchers as to the functional roles of these components in biomembranes. In particular, lipids capable of adopting the hexagonal H(II) phase have received considerable attention because of the observation that such lipids can promote membrane fusion. In the rational design of lipid-based delivery systems, H(II) phase lipids have been employed to endow systems with fusogenic, membrane-destabilizing properties. We will outline the molecular basis for the polymorphic phase behavior of lipids and highlight some of the uses of nonbilayer lipids in the preparation of lipid-based delivery systems. In addition, a distinction will be drawn between lipid-based systems which rely on the inclusion of nonbilayer lipids for activity, and systems which contain components which actively promote formation of nonbilayer structure within biological membranes.

Stabilized plasmid-lipid particles for systemic gene therapy.

The structure of 'stabilized plasmid-lipid particles' (SPLP) and their properties as systemic gene therapy vectors has been investigated. We show that SPLP can be visualized employing cryo-electron microscopy to be homogeneous particles of diameter 72 +/- 5 nm consisting of a lipid bilayer surrounding a core of plasmid DNA. It is also shown that SPLP exhibit long circulation lifetimes (circulation half-life >6 h) following intravenous (i.v.) injection in a murine tumor model resulting in accumulation of up to 3% of the total injected dose and concomitant reporter gene expression at a distal (hind flank) tumor site. In contrast, i v. injection of naked plasmid DNA or plasmid DNA-cationic liposome complexes did not result in significant plasmid delivery to the tumor site or gene expression at that site. Furthermore, it is shown that high doses of SPLP corresponding to 175 microg plasmid per mouse are nontoxic as assayed by monitoring serum enzyme levels, whereas i.v. injection of complexes give rise to significant toxicity at dose levels above 20 microg plasmid per mouse. It is concluded that SPLP exhibit properties consistent with potential utility as a nontoxic systemic gene therapy vector.

Doxorubicin entrapped within liposome-associated antigens results in a selective inhibition of the antibody response to the linked antigen.

The generation of an immune response can dramatically alter the circulation lifetime of a targeted liposome, particularly when the response is generated against the surface-coupled ligand. Following repeated administrations, rapid elimination of the carrier system is observed, thereby limiting potential applications for targeted liposomes in a therapeutic setting. In this study, we have investigated whether the encapsulation of a toxic drug within the carrier could prevent an immune response against a surface-bound protein. Liposome clearance and humoral immune response were monitored throughout multiple administrations of liposomes containing doxorubicin with surface-conjugated ovalbumin. The results show that low doses of encapsulated doxorubicin can prevent humoral immunity against repeated administration of liposomes conjugated with ovalbumin. The immunosuppressive effect was specific for the ovalbumin coupled to the liposome surface. This selective suppression of immunity against a surface conjugated protein could prove advantageous for safe repeated administration of protein containing liposomal systems.

Tunable pH-sensitive liposomes composed of mixtures of cationic and anionic lipids.

The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtures of anionic and cationic lipids have been investigated. It is shown that stable LUVs can be prepared from the ionizable anionic lipid cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl-N, N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH is reduced. The critical pH at which fusion was observed (pH(f)) was dependent on the cationic lipid-to-anionic lipid ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85 resulted in vesicles with pH(f) values that ranged from pH 4.0 to 6.7, respectively. This behavior is consistent with a model in which fusion occurs at pH values such that the DODAC/CHEMS LUV surface charge is zero. Related behavior was observed for LUVs composed of the ionizable cationic lipid 3alpha-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and (31)P NMR evidence is presented which indicates that pH-dependent fusion results from a preference of mixtures of cationic and anionic lipid for "inverted" nonbilayer lipid phases under conditions where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs composed of cationic and anionic lipids may be of utility for drug delivery applications. It is also suggested that the ability of cationic lipids to adopt inverted nonbilayer structures in combination with anionic lipids may be related to the ability of cationic lipids to facilitate the intracellular delivery of macromolecules.

Fluorescent-labeled poly(ethylene glycol) lipid conjugates with distal cationic headgroups.

The synthesis of a new class of fluorescent cationic poly(ethylene glycol) lipid conjugates (CPLs) is described. These lipids consist of a hydrophobic distearoyl-phosphatidylethanolamine (DSPE) anchor coupled to a highly fluorescent N(epsilon)-dansyl lysine moiety, which is attached to a hydrophilic poly(ethylene glycol) (PEG) spacer that is linked to a cationic headgroup made of lysine residues. Introduction of the dansyl moiety allows rapid and accurate quantification of CPLs within lipid bilayers using fluorescence techniques. The synthetic scheme is straightforward, using repeated amino-carboxyl coupling reaction steps, with purification by precipitation. A series of dansylated CPLs was synthesized with zero, one, three, and seven lysine residues located at the distal end of the PEG chain, giving rise to CPLs with one, two, four, and eight distal positive charges, respectively. The structures of the CPLs were confirmed by (1)H NMR spectroscopy and chemical analysis. CPLs provide a means of introducing positive charge to a bilayer that is localized some distance from the membrane surface, and are of particular interest for nonviral gene delivery applications. The usefulness of CPLs is demonstrated by the enhanced in vitro cellular binding and uptake of liposomes containing CPL(4).

Encapsulation of plasmid DNA in stabilized plasmid-lipid particles composed of different cationic lipid concentration for optimal transfection activity.

In previous work (Wheeler et al. (1999) Gene Therapy 6, 271-281) we have shown that plasmid DNA can be entrapped in "stabilized plasmid lipid particles" (SPLP) using low levels (5-10 mol%) of cationic lipid, the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). However, these SPLP exhibit low transfection potencies in vitro as compared to plasmid/cationic lipid complexes formed with liposomes composed of cationic and neutral lipid at a 1:1 lipid ratio. The objective of this study was to construct SPLPs with increased cationic lipid contents that result in maximum transfection levels. A phosphate buffer detergent dialysis technique is described resulting in formation of SPLP containing 7-42.5 mol% DODAC with reproducible encapsulation efficiency of up to 80%. An octanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permitting a quick exchange out of the SPLP to further optimize the in vitro and in vivo transfection. We have demonstrated that this technique can be used to encapsulate either linearized DNA or supercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution of approximately 75 +/- 6 nm as determined by cryo-electron microscopy. The high plasmid-to-lipid ratio observed corresponded to one plasmid per particle. The SPLP consist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electron microscopy. SPLP containing a range of DODAC concentrations were tested for in vitro and in vivo transfection. In vitro, in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the DODAC concentration in the SPLP was decreased from 42.5 to 24 mol% DODAC. Decreasing the cationic lipid concentration improved transfection in part due to decreased toxicity. In vivo studies using an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP showed maximum transfection activity for SPLP containing 24 mol% DODAC. Gene expression observed in tumor cells was increased by approximately one magnitude as compared to cationic lipid/DNA complexes. The SPLP were stable and upon storage at 4 degrees C no significant change in the transfection activity was observed over a one-year period. Thus this phosphate buffer detergent dialysis technique can be used to generate SPLP formulations containing a wide range of cationic lipid concentrations to determine optimal SPLP composition for high transfection activity and low toxicity.

Stabilized plasmid-lipid particles: pharmacokinetics and plasmid delivery to distal tumors following intravenous injection.

A previous study has shown that plasmid DNA can be encapsulated in lipid particles (SPLP, "stabilized plasmid lipid particles") of approximately 70 nm diameter composed of 1,2-dioleoyl-3-phosphatidyl-ethanolamine (DOPE), the cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) and poly(ethylene glycol) conjugated to ceramide (PEG-Cer) using a detergent dialysis process (Wheeler et al. (1999) Gene Therapy 6, 271-281). In this work we evaluated the potential of these SPLPs as systemic gene therapy vectors, determining their pharmacokinetics and the biodistribution of the plasmid and lipid components. It is shown that the blood clearance and the biodistribution of the SPLPs can be modulated by varying the acyl chain length of the ceramide group used as lipid anchor for the PEG polymer. Circulation lifetimes observed for SPLPs with PEG-CerC14 and PEG-CerC20 were t(1/2) = approximately 1 and approximately 10 h, respectively. The SPLPs are stable while circulating in the blood and the encapsulated DNA is fully protected from degradation by serum nucleases. The accelerated clearance of SPLPs with PEG-CerC14 is accompanied by increased accumulation in liver and spleen as compared to PEG-CerC20 SPLPs. Delivery of intact plasmid to liver and spleen was detected. Significant accumulation (approximately 10% of injected dose) of the long circulating SPLPs with PEG-CerC20 in a distal tumor (Lewis lung tumor in the mouse flank) was observed following i.v. application and delivery of intact plasmid to tumor tissue at approximately 6% injected dose/g tissue is demonstrated.

Calcium enhances the transfection potency of plasmid DNA-cationic liposome complexes.

It is shown that calcium increases the in vitro transfection potency of plasmid DNA-cationic liposome complexes from 3- to 20-fold. The effect is Ca(2+) specific as other cations, such as Mg(2+) and Na(+), do not give rise to enhanced transfection and the effect can be inhibited by the presence of EGTA. It is shown that Ca(2+) increases cellular uptake of the DNA-lipid complexes, indicating that increased transfection potency arises from increased intracellular delivery of both cationic lipid and plasmid DNA in the presence of Ca(2+). In particular, it is shown that the levels of intact intracellular plasmid DNA are significantly enhanced when Ca(2+) is present. The generality of the Ca(2+) effect for enhancing complex-mediated transfection is demonstrated for a number of different cell lines and different cationic lipid formulations. It is concluded that addition of Ca(2+) represents a simple and useful protocol for enhancing in vitro transfection properties of plasmid DNA-cationic lipid complexes.

Cholesteryl hemisuccinate exhibits pH sensitive polymorphic phase behavior.

Cholesteryl hemisuccinate (CHEMS) is an acidic cholesterol ester that self-assembles into bilayers in alkaline and neutral aqueous media and is commonly employed in mixtures with dioleoylphosphatidylethanolamine (DOPE) to form 'pH sensitive' fusogenic vesicles. We show here that CHEMS itself exhibits pH sensitive polymorphism. This is evident from the fusogenic properties of large unilamellar vesicles (LUV) composed of CHEMS and direct visualization employing freeze-fracture electron microscopy. Below pH 4.3, LUV composed of CHEMS undergo fusion as monitored by lipid mixing assays and freeze-fracture electron micrographs reveal the characteristic striated signature of H( parallel) phase lipid. It is suggested that the pH dependent phase preferences of CHEMS contribute to the pH sensitivity of LUV composed of mixtures of CHEMS and DOPE.