Sean C. Semple

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.

Interactions of liposomes and lipid-based carrier systems with blood proteins: Relation to clearance behaviour in vivo

Liposomes and lipid-based drug delivery systems have been used extensively over the last decade to improve the pharmacological and therapeutic activity of a wide variety of drugs. More recently, this class of carrier systems has been used for the delivery of relatively large DNA and RNA-based drugs, including plasmids, antisense oligonucleotides and ribozymes. Despite recent successes in prolonging the circulation times of liposomes, virtually all lipid compositions studied to date are removed from the plasma compartment within 24h after administration by the cells and tissues of the reticuloendothelial system (RES). Plasma proteins have long been thought to play a critical role in this process but only a few efforts were made to evaluate the relevant importance of plasma protein-liposome interactions in the clearance process. Strategies to increase the bioavailability of liposomal drugs have included altering lipid compositions and charge, increasing lipid doses, and incorporating surface coatings. All of these modifications can influence membrane-protein interactions. In this article, we will focus on our experiences with liposome-blood protein interactions and how alterations in the chemical and physical properties of the carrier system influence the interactions with blood proteins and circulation times.

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.

Beta 2 glycoprotein I is a major protein associated with very rapidly cleared liposomes in vivo, suggesting a significant role in the immune clearance of "non-self" particles.

Liposomes recovered from the blood of liposome-treated CD1 mice were previously reported to have a complex protein profile associated with their membranes (Chonn, A., Semple, S. C., and Cullis, P. R.(1992) J. Biol. Chem. 267, 18759-18765). In this study, we have further characterized and identified the major proteins associated with very rapidly cleared large unilamellar vesicles. These liposomes contained phosphatidylcholine, cholesterol, and anionic phospholipids (phosphatidylserine, phosphatidic acid, or cardiolipin) that dramatically enhance the clearance rate of liposomes from the circulation. These anionic phospholipids are normally found exclusively in the interior of cells but become expressed when cells undergo apoptosis or programmed cell death, and thus, they are believed to be markers of cell senescence. Analysis of the proteins associated with these liposomes by SDS-polyacrylamide gel electrophoresis revealed that two of the major proteins associated with the liposome membranes are proteins with electrophoretic mobilities corresponding to Mr of 66,000 and 50,000-55,000. The 66-kDa protein was identified to be serum albumin by immunoblot analysis. Using various biochemical and immunological methods, we have identified the 50-55-kDa protein as the murine equivalent of human β2-glycoprotein I. β2-glycoprotein I has a strong affinity for phosphatidylserine, phosphatidic acid, and cardiolipin inasmuch as the levels of β2-glycoprotein I associated with these anionic liposomes approach or even exceed those of serum albumin, which is present in serum at a concentration 200-fold greater than β2-glycoprotein I. Further, we demonstrate that the amount of β2-glycoprotein I associated with liposomes, as quantitated by an enzyme-linked immunosorbent assay, is correlated with their clearance rates; moreover, the circulation residency time of cardiolipin-containing liposomes is extended in mice pretreated with anti-β2-glycoprotein I antibodies. These findings strongly suggest that β2-glycoprotein I plays a primary role in mediating the clearance of liposomes and, by extension, senescent cells and foreign particles.

Influence of dose on liposome clearance: critical role of blood proteins

It is well established that the circulation half-life of liposomes increases with increasing dose. This effect is commonly attributed to saturation" of the fixed and free macrophages of the reticuloendothelial system resulting in reduced clearance rates. However, it is also known that the clearance rate of liposomes is dependent on the amount of associated blood protein, leading to the possibility that dose-dependent increases in circulation lifetimes could be due to decreases in the amount of blood protein associated per liposome. In order to test this hypothesis, the protein binding and clearance properties of large unilamellar liposomes composed of distearoylphosphatidylcholine/cholesterol and egg phosphatidylcholine/dioleoylphosphatidic acid/cholesterol were examined in mice. Liposomes were injected over a dose range of 10 to 1000 mg lipid/kg body weight, and the circulation lifetime and liver and spleen accumulation monitored. As expected, longer circulation half-lives were observed at higher doses for both liposome compositions. However, it was also found that at higher liposome doses, significantly less protein was bound per liposome. The results indicate that there is a limited pool of blood proteins that is able to interact with liposomes of a given composition. At higher lipid doses these blood proteins are distributed over more liposomes resulting in lower protein binding values and longer circulation lifetimes.

Encapsulation in liposomal nanoparticles enhances the immunostimulatory, adjuvant and anti-tumor activity of subcutaneously administered CpG ODN

Immunostimulatory oligodeoxynucleotides (ODN) containing cytosine-guanine (CpG) motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through triggering of the Toll-like receptor 9. We have previously shown that encapsulation in liposomal nanoparticles (LN) enhances the immunostimulatory activity of CpG ODN (LN-CpG ODN) (Mui et al. in J Pharmacol Exp Ther 298:1185, 2001). In this work we investigate the effect of encapsulation on the immunopotency of subcutaneously (s.c.) administered CpG ODN with regard to activation of innate immune cells as well as its ability to act as a vaccine adjuvant with tumor-associated antigens (TAAs) to induce antigen (Ag)-specific, adaptive responses and anti-tumor activity in murine models. It is shown that encapsulation specifically targets CpG ODN for uptake by immune cells. This may provide the basis, at least in part, for the significantly enhanced immunostimulatory activity of LN-CpG ODN, inducing potent innate (as judged by immune cell activation and plasma cytokine/chemokine levels) and adaptive, Ag-specific (as judged by MHC tetramer positive T lymphocytes, IFN-γ secretion and cytotoxicity) immune responses. Finally, in efficacy studies, it is shown that liposomal encapsulation enhances the ability of CpG ODN to adjuvanate adaptive immune responses against co-administered TAAs after s.c. immunization, inducing effective anti-tumor activity against both model and syngeneic tumor Ags in murine tumor models of thymoma and melanoma.

Lipid-based formulations of antisense oligonucleotides for systemic delivery applications.

Publisher Summary Increasing the specificity of therapeutic drugs and improving their delivery to sites of disease are primary goals of todays pharmaceutical industry. One of the most exciting advances in recent years has been the development of antisense technologies, which are capable of modulating protein expression with exquisite specificity. Unfortunately, this class of drugs is particularly sensitive to nuclease degradation, is eliminated rapidly from the circulation after intravenous administration, and is severely limited in its ability to penetrate through cellular membranes unaided. Attempts to address these problems through medicinal chemistry have produced several key advances. However, chemical alterations to improve one property of the molecule often affect other properties, potentially in a negative manner. The past decade has seen extensive use of liposomes and lipid-based delivery systems to improve the pharmacological properties of a variety of drugs. The principal benefits afforded therapeutic agents by liposomal encapsulation are enhanced plasma-circulation lifetimes, increased delivery to sites of disease, and changes in tissue distribution, which can result in reduced toxic side effects. Liposomal preparations of doxorubicin (DOXIL) and daunorubicin (DaunoXome) have been approved for the treatment of HIV-associated Kaposis sarcoma, whereas lipid-based formulations of amphotericin B (AmBisome, ABELCET, AMPHOTEC) are successful clinical products employed in the treatment of fungal infections. It is not surprising, therefore, that considerable interest has been on developing lipid-based delivery systems to overcome the problems associated with the systemic administration of DNA- and RNA-based therapeutics. The intent of this chapter is to introduce the reader to the various lipid-based formulations applied to polynucleic acid drugs, with emphasis on the generation and characterization of delivery vehicles for intravenous applications. The majority of examples apply to antisense oligodeoxynucleotides (ASODN) as they represent the most widely available class of DNA-based drugs.

Contact hypersensitivity: a simple model for the characterization of disease-site targeting by liposomes.

A murine model of delayed-type hypersensitivity (DTH) is characterized with respect to liposome accumulation at a site of inflammation. Mice were sensitized by painting the abdominal region with a solution of 2,4-dinitrofluorobenzene (DNFB) and inflammation was induced 5 days later by challenging the ear with a dilute solution of DNFB. The inflammatory response was readily monitored by measuring ear thickness (edema) and radiolabeled leukocyte infiltration. Maximum ear swelling and cellular infiltration occurred 24 h after the epicutaneous challenge with the ear returning to normal size after approximately 72 h. We demonstrate that large unilamellar vesicles (LUV) accumulate at the site of inflammation to a level more than 20-fold higher than that measured in the untreated ear. Vesicle delivery to the ear correlated with increased vascular leakage resulting from endothelium remodeling in response to DNFB challenge, and was not a consequence of increased local tissue blood volume. Extravasation occurred only during the first 24 h after ear challenge; after this time the permeability of the endothelium to vesicles returned to normal. We further showed that LUV with a diameter of 120 nm exhibit maximum levels of accumulation, that a polyethylene glycol surface coating does not increase delivery, and that the process can be inhibited by the application of topical corticosteroids at the time of induction. These data and the inflammation model are discussed with respect to developing lipid-based drug delivery vehicles designed to accumulate at inflammatory disease sites.

Liposome-Blood Protein Interactions in Relation to Liposome Clearance

AbstractOur recent in vivo studies have investigated the surface adsorption property of various circulating liposomes to blood proteins, and have related this property to liposome clearance behavior. In particular, we have investigated liposomes composed of different charged or neutral lipids, fatty acyl chain length and saturation, and cholesterol content. From these studies an apparent inverse relationship between the amount of blood protein that associates with large unilamellar vesicles and the circulation half-lives of the liposomes is observed, indicating that protein-mediated liposome clearance mechanisms are dominant. Furthermore, by comparing the protein profiles of rapidly cleared liposomes with liposomes exhibiting enhanced circulation times, key blood proteins have been identified and implicated in the clearance process.

Protein-Membrane Interactions in the Complex Biological Milieu

Alterations in the lipid composition of biological membranes can have dramatic effects on their ability to interact with soluble proteins. In a series of studies employing large unilamellar vesicles (LUVs) produced by an extrusion technique, we have characterized the influence of membrane components on protein-membrane interactions. Much of our understanding of how and why proteins interact with seemingly inert membrane surfaces stems mainly from studies involving one or two protein component systems. These studies, however, do not accurately reflect the interactions that occur in the complex biological milieu (reviewed by Horbett and Brash, 1987). There have been very few studies reported on the interactions of proteins with liposomal systems incubated with whole blood. There are two main reasons for this. First, the large majority of studies on the association of plasma proteins with liposomes in vitro have been performed employing multilamellar systems. Due to the variable lamellarity of liposomes of different lipid compositions, quantification of the amount of various proteins associated per liposome has not been possible. Second, convenient techniques have not been available for the isolation of liposomes, particularly LUVs, from blood components. We have recently described a rapid method for the isolation of well-defined large unilamellar liposomes from the blood of liposome-treated mice (Chonn et al., 1991). With such a procedure now available, we have started to biochemically and immunologically characterize the amount and type of proteins associated with liposomes exposed to the complex biological milieu.