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.

Antibody conjugation methods for active targeting of liposomes.

Liposomes are useful drug delivery vehicles since they may protect encapsulated drugs from enzymatic degradation and rapid clearance in vivo, or alter biodistribution, potentially leading to reduced toxicities (1,2). A major limitation to the development of many specialized applications is the problem of directing liposomes to tissues where they would not normally accumulate. Consequently, a great deal of effort has been made over the years to develop liposomes that have targeting vectors attached to the bilayer surface. These vectors have included ligands such as oligosaccharides (3,4), peptides (5,6), proteins (7,8) and vitamins (9). Most studies have focused on antibody conjugates since procedures for producing highly specific monoclonal antibodies (MAbs) are well established. In principle it should be possible to deliver liposomes to any cell type as long as the cells are accessible to the carrier. In practice it is usually not this simple since access to tissue, competition, and rapid clearance are formidable obstacles. It has also been shown that antibodies become immunogenic when coupled to liposomes (10,11), although in similar experiments with ovalbumin we have demonstrated that immunogenicity can be suppressed by formulating the liposomes with the cytotoxic drug doxorubi-cin (12). Such issues as these suggest that the development of antibody-targeted liposomes for in vivo applications will present difficult challenges.

Application of oligo-(14-amino-3,6,9,12-tetraoxatetradecanoic acid) lipid conjugates as steric barrier molecules in liposomal formulations.

Lipid conjugates of oligo-(14-amino-3,6,9,12-tetraoxatetradecanoic acid) (ATTAn) were synthesized as monodisperse analogues of poly(ethylene glycol) (PEG) derivatives used in liposomal drug delivery systems. The new lipids were shown to be at least equivalent to MePEGA-2000-DSPE in assays designed to evaluate the effectiveness of polymers as steric barrier molecules in liposomes. Liposomes containing 1-5% of ATTA8-DSPE (octamer) showed comparable long circulation behavior relative to PEG-2000-DSPE analogues. Surprisingly, the shorter ATTA4-DSPE (tetramer) appeared to be quite effective in reducing clearance. Liver enzyme levels and systemic single dose tolerability of ATTA8-DSPE liposomes were comparable to controls, suggesting that the new materials are nontoxic. Prolonged exposure of ATTA8-DSPE liposomes to splenocytes in vitro showed no evidence of mitogenicity relative to controls or MePEGA-2000-DSPE liposomes. ATTA8-DSPE was as effective as MePEGC-2000-DSPE in preventing complement activation by cationic liposome systems. Repeat dosage in vivo regimes in ICR mice using DSPC/cholesterol liposomes, with and without 5% ATTA8-DSPE and MePEGC-2000-DSPE, showed no evidence of enhanced clearance on successive doses. Splenocytes recovered after repeat doses showed no significant evidence of mitogenicity on restimulation with liposomes. Cellular differentiation and activation marker levels in splenocytes recovered after the fourth in vivo administration were at normal levels. These results suggest that ATTAn oligomers do not induce an immune response in isolation. It was demonstrated that ATTA8-DSPE could be used to replace PEG-lipids in the formulation of doxorubicin, plasmid DNA and oligonucleotides using a variety of formulation techniques. The study demonstrates that ATTAn oligomers can be safely and effectively used in place of poly(ethylene glycol) as well-defined biomaterials in liposomal applications where reproducible behavior is critical.

Preclinical pharmacology, toxicology and efficacy of sphingomyelin/cholesterol liposomal vincristine for therapeutic treatment of cancer.

PURPOSE: To establish the pharmacodynamic relationships between drug biodistribution and drug toxicity/efficacy, a comprehensive preclinical evaluation of sphingomyelin/cholesterol (SM/chol) liposomal vincristine and unencapsulated vincristine in mice was undertaken. METHODS: Pharmaceutically acceptable formulations of unencapsulated vincristine and liposomal vincristine at drug/lipid ratios of 0.05 or 0.10 (wt/wt) were evaluated for toxicity, antitumor activity and pharmacokinetics following intravenous administration. RESULTS: Mice given liposomal vincristine at 2 mg/kg vincristine had concentrations of vincristine in blood and plasma at least two orders of magnitude greater then those achieved after an identical dose of unencapsulated drug. One day after administration of the liposomal vincristine, there were at least tenfold greater drug quantities, relative to unencapsulated vincristine, in the axillary lymph nodes, heart, inguinal lymph nodes, kidney, liver, skin, small intestines and spleen. Increased plasma and tissue exposure to vincristine as a result of encapsulation in SM/chol liposomes was not associated with increased drug toxicities. Treatment of the murine P388 ascitic tumor with a single intravenous dose of unencapsulated drug at 2, 3 and 4 mg/kg, initiated 1 day after tumor cell inoculation, resulted in a 33 to 38% increase in lifespan. In contrast, long-term survival rates of 50% or more were achieved in all groups treated with the SM/chol liposomal vincristine formulations at doses of 2, 3 and 4 mg/kg. At the 4 mg/kg dose, eight of ten and nine of ten animals survived past day 60 when treated with SM/chol liposomal vincristine prepared at the 0.05 and 0.1 drug/lipid ratios, respectively. CONCLUSIONS: Overall, increased and prolonged plasma concentrations of vincristine achieved by liposomal encapsulation were correlated with dramatically increased antitumor activity in comparison with the unencapsulated drug, but no correlations could be established between pharmacokinetic parameters and toxicity.

The role of tumor-associated macrophages in the delivery of liposomal doxorubicin to solid murine fibrosarcoma tumors.

Murine fibrosarcoma tumors arising from subcutaneous inoculation of FSa-N cells exhibit 4-fold higher tumor-associated macrophage (TAM) levels than those from the FSa-R line. These solid tumors were used to assess the role of TAMs in the accumulation of liposomal anticancer drugs. Two liposomal formulations of doxorubicin were investigated: a conventional formulation composed of distearoylphosphatidylcholine (DSPC) and cholesterol and a sterically stabilized liposomal formulation composed of DSPC/cholesterol/poly (ethylene glycol)-modified distearoylphosphatidyethanolamine (PEG-PE). Circulating concentrations of PEG-PE containing liposomes 24 h after i.v. administration were 3-fold greater than those observed after administration of conventional liposomes. No differences were observed in drug retention or tumor (FSa-R or FSa-N) drug and liposomal lipid delivery when comparisons were made between different liposomal formulations. However, tumor doxorubicin concentrations were increased as much as 4-fold for liposomal formulations relative to free drug. Further, there was a 1.5- to 2-fold increase in doxorubicin delivery to TAM-enriched FSa-N tumors compared with FSa-R tumors. Fluorescence microscopy studies revealed a poor correlation between CD11b (Mac-1) positive cells (TAMs) and the appearance of doxorubicin fluorescence. These results suggest that uptake of liposomal drugs by TAMs does not account for the enhanced accumulation of liposomal drugs in solid tumors. Rather, the increased tumor drug delivery may be related to alternative TAM-mediated processes that increase tumor vascular permeability. Therapeutic studies demonstrated that increased tumor drug uptake observed for the liposomal doxorubicin formulations led to marginal improvements in antitumor activity, and it is suggested that much of the drug delivered in liposomal form is not biologically available.

Intratumor distribution of doxorubicin following i.v. administration of drug encapsulated in egg phosphatidylcholine/cholesterol liposomes.

PURPOSE: A pharmacological evaluation of an egg phosphatidylcholine/cholesterol (55:45 mole ratio, EPC/Chol) liposome doxorubicin formulation was carried out. The objective was to define liposomal lipid and drug distribution within sites of tumor growth following intravenous (i.v.) administration to female BDF1 mice bearing either Lewis lung carcinoma, B16/BL6 melanoma, or L1210 ascitic tumors. METHODS: Mice were injected i.v. with EPC/Chol liposomal doxorubicin, and plasma and tumor levels of lipid and drug were determined 1, 4 and 24 h late with radiolabeled lipid and fluorimetry or fluorescence microscopy, respectively. In addition, single-cell suspensions of the Lewis lung and B16/BL6 tumors were prepared and the presence of macrophages was determined with an FITC-labeled rat antimouse CD11b (MAC-1) antibody. RESULTS: For mice bearing the Lewis lung solid tumors, there was a time-dependent accumulation of liposomal lipid, with a plateau of approximately 500 micrograms lipid/g tumor at 48 h. In contrast, the apparent plateau (microgram doxorubicin/g tumor) for doxorubicin was achieved at 1 h and remained constant over a 72-h time course. In comparison with free drug administered at the maximum tolerated dose (MTD, 20 mg/kg) doxorubicin levels in tumors were two- to threefold greater when the drug was administered in liposomal form. The increase in drug delivery was comparable for both solid tumors. With animals bearing the L1210 ascitic tumor, drug exposure was as much as ten times greater (in comparison with free drug) when doxorubicin was administered in liposomes. An evaluation of single-cell suspensions prepared from the two solid tumors suggested that more than 98% of the tumor-associated drug and liposomal lipid was not tumor cell-associated. Histological studies with the Lewis lung carcinoma, however, revealed that a proportion of the drug did colocalize with tumor-associated macrophages. Analysis of cells obtained from mice bearing ascitic tumors showed that more than 80% of the cell-associated drug could be removed by procedures designed to remove adherent cells. CONCLUSION: The results summarized here suggest drug concentrations within a solid tumor, such as the Lewis lung carcinoma, are constant over time when the drug is given in a "leaky" EPC/Chol formulation. The results also suggest that liposomal lipid within sites of tumor growth is primarily localized within the interstitial spaces or tumor-associated macrophages.

An immune response to ovalbumin covalently coupled to liposomes is prevented when the liposomes used contain doxorubicin.

It is now well established that liposomes with surface associated proteins are immunogenic. Repeated administration of protein coated liposomes elicits the generation of antibodies and the elimination of proteoliposome increases markedly in animals 'immunized' with such liposomes. This immune response compromises the therapeutic potential of liposomal formulations that rely on the use of protein- or peptide-based targeting ligands to enhance cell specificity. Strategies to suppress or inhibit such immune responses must be developed if this technology is going to prove therapeutically viable. This study evaluates whether an immune response to a protein, covalently attached to liposomes by a thioether bond between N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP)-modified-protein and N-(4-(P-maleimidophenyl)butyryl) (MPB)-activated lipids, can be suppressed when the liposomes used contain the anti-cancer drug doxorubicin. To assess this, the highly immunogenic protein ovalbumin was conjugated onto liposomes composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) with sufficient poly(ethylene glycol)-modified distearoyl phosphatidylethanolamine (PEG-DSPE) (2 mol%) to prevent liposome aggregation during protein coupling and to engender increased circulation lifetimes. The immune response to these liposomes with and without encapsulated doxorubicin was measured by: (1) monitoring liposome elimination after 3 weekly i.v. injections in C3H/HeJ mice and (2) measuring the anti-ovalbumin antibody levels by an ELISA assay. One week after a single dose of ovalbumin-coated PEG liposomes (50 microg protein/mouse) the immune response resulted in rapid elimination of a second dose of ovalbumin-coated PEG liposomes. Rapid liposome elimination was correlated to generation of high levels (> 9 microg/ml plasma) of circulating anti-ovalbumin IgG. In contrast, anti-ovalbumin antibodies were not detected when the liposomes used contained doxorubicin. Plasma elimination of these drug loaded protein coated liposomes decreased following repeated weekly i.v. doses, an effect that is consistent with liposomal doxorubicin mediated suppression of phagocytic cells in the liver.

Sphingomyelin-cholesterol liposomes significantly enhance the pharmacokinetic and therapeutic properties of vincristine in murine and human tumour models.

This study reports on the development of a liposomal formulation of vincristine with significantly enhanced stability and biological properties. The in vitro and in vivo pharmacokinetic, tumour delivery and efficacy properties of liposomal vincristine formulations based on sphingomyelin (SM) and cholesterol were compared with liposomes composed of distearoylphosphatidylcholine (DSPC) and cholesterol. SM/cholesterol liposomes had significantly greater in vitro stability than did similar DSPC/cholesterol liposomes. SM/cholesterol liposomes also had significantly improved biological properties compared with DSPC/cholesterol. Specifically, SM/cholesterol liposomes administered intravenously retained 25% of the entrapped vincristine after 72 h in the circulation, compared with 5% retention in DSPC/cholesterol liposomes. The improved retention properties of SM/cholesterol liposomes resulted in plasma vincristine levels 7-fold higher than in DSPC/cholesterol liposomes. The improved circulation lifetime of vincristine in SM/cholesterol liposomes correlated with increased vincristine accumulation in peritoneal ascitic murine P388 tumours and in subcutaneous solid A431 human xenograft tumours. Increased vincristine delivery to tumours was also accompanied by increased anti-tumour efficacy. Treatment with SM/cholesterol liposomal formulations of vincristine resulted in greater than 50% cures in mice bearing ascitic P388 tumours, an activity that could not be achieved with the DSPC/cholesterol formulation. Similarly, treatment of mice with severe combined immunodeficiency (SCID) bearing solid human A431 xenograft tumours with SM/cholesterol vincristine formulations delayed the time required for 100% increase in tumour mass to > 40 days, compared with 5 days, 7 days and 14 days for mice receiving no treatment or treatment with free vincristine or DSPC/cholesterol formulations of vincristine respectively.

Poly(ethylene glycol)-modified phospholipids prevent aggregation during covalent conjugation of proteins to liposomes.

Liposome aggregation is a major problem associated with the covalent attachment of proteins to liposomes. This report describes a procedure for coupling proteins to liposomes that results in little or no change in liposome size. This is achieved by incorporating appropriate levels of poly(ethylene glycol)-modified lipids into the liposomes. The studies employed thiolated avidin-D coupled to liposomes containing the thio-reactive lipid N-(4-(p-maleimidophenyl)butyryl)dipalmitoyl phosphatidylethanolamine (1 mol % of total lipid) and various amounts of MePEG-S-POPE (monomethoxypoly(ethylene glycol) linked to phosphatidylethanolamine via a succinate linkage). The influence of PEG chain length and density was also assessed. The presence of PEG on the surface of liposomes is shown to provide an effective method of inhibiting aggregation and the corresponding increase in liposome size during the covalent coupling of avidin-D. A balance between the size of the PEG used and the amount of PEG-lipid incorporated into the liposome had to be achieved in order to maintain efficient coupling. Optimal coupling efficiencies in combination with minimal aggregation effects were achieved using 2 mol % MePEG2000-S-POPE (PEG of 2000 MW) or 0.8 mol % MePEG5000-S-POPE (PEG of 5000 MW). At these levels, the presence of PEG did not affect the biotin binding activity of the covalently attached avidin. The ability of the resulting liposomes to specifically target to biotinylated cells is demonstrated.