Pii: s0169-409x(97)00108-7

Advanced Drug Delivery Reviews 30 (1998) 73–83 Recent advances in liposome technologies and their applications for systemic aInex Pharmaceuticals Corporation, 1779 West 75th Street, Vancouver, BC, V6P 6P2, Canada bDepartment of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver V6T 1Z3, Canada Received 1 July 1997; accepted 11 July 1997 Abstract
The recent clinical successes experienced by liposomal drug delivery systems stem from the ability to produce well-defined liposomes that can be composed of a wide variety of lipids, have high drug-trapping efficiencies and have anarrow size distribution, averaging less than 100 nm in diameter. Agents that prolong the circulation lifetime of liposomes,enhance the delivery of liposomal drugs to specific target cells, or enhance the ability of liposomes to deliver drugsintracellularly can be incorporated to further increase the therapeutic activity. The physical and chemical requirements foroptimum liposome drug delivery systems will likely apply to lipid-based gene delivery systems. As a result, the developmentof liposomal delivery systems for systemic gene delivery should follow similar strategies.
Keywords: Liposomal gene delivery systems; Cationic lipids; DNA encapsulation; Prolonged circulation lifetimes;Targeting; Fusogenic liposomes; Intracellular delivery; Transfection Contents
2. Production of liposomes for conventional drug delivery .
3. Production of liposomes for gene delivery.
4. Liposomes with prolonged circulation lifetimes .
5. Targeted delivery to specific cells .
6. Fusogenic liposomes for intracellular delivery .
1. Introduction
al drugs with proven clinical utility [1,2]. Notableexamples are liposome formulations of doxorubicin Recent advances in liposome technologies for [3,4], all-trans retinoic acid [5], amphotericin B [6], conventional drug delivery have resulted in liposom- daunorubicin [7] and vincristine [8,9]. These ad-vances have led to the production of well-defined, relatively small liposomal systems that have the Corresponding author. Tel.: 1 1 604 2649954; fax: 1 1 604 ability to entrap drugs with high efficiencies, reside  1998 Elsevier Science B.V. All rights reserved.
P I I S 0 1 6 9 - 4 0 9 X ( 9 7 ) 0 0 1 0 8 - 7 A. Chonn, P.R. Cullis / Advanced Drug Delivery Reviews 30 (1998) 73 –83 in the circulation for extended periods, and accumu- specific target cells. This review will highlight late at regional sites of disease, such as inflammation several of the advances made in liposome tech- and tumor. The next generation of liposomal drug nologies and discuss how these advances may be delivery systems will include drug-loaded liposomes applied to resolve the challenges facing the develop- with surface-associated targeting information that ment of liposomes for the controllable and reproduc- will increase drug accumulation in specific cells, as well as fusogenic liposomes that will enable moreefficient intracellular drug delivery.
It is anticipated that these advances in liposome 2. Production of liposomes for conventional
technologies will be directly applicable to the design drug delivery
of liposomal systems for systemic gene delivery. Inmany ways, the challenges facing the development The major advances in liposome technology in the of liposomal gene delivery systems are not unlike past decade arise from the ability to produce well- those that have faced liposomal drug delivery sys- defined liposomes composed of a wide variety of tems. The therapeutic index of the conventional or lipids with different physical and chemical prop- gene-based (plasmid DNA or RNA transcripts) drug erties, having high drug-trapping efficiencies and will be enhanced by delivering more biologically having narrow size distributions, averaging less than active drug to target cells / tissues and less to non- 100 nm in diameter. These physical and chemical target cells / tissues, to avoid drug-related toxicities.
properties have been shown to significantly affect the With gene-based drugs, however, delivery into stability and pharmacokinetics of liposomes [17]. A appropriate cells represents only part of the problem; number of procedures have been established to a number of intracellular barriers exist that can produce well-defined liposomes (extensively re- inhibit the biologic activity of gene-based drugs viewed in [18,19]). These include extrusion, where [10,11]. It is not clear what role, if any, liposomes the liposomes are forced through filters with well- will play in overcoming these intracellular barriers.
defined pore sizes under moderate pressures, re- The potential of liposomes to systemically deliver versed-phase evaporation, sonication and detergent- DNA was recognized as early as the late 1970s (extensively reviewed in [12]), however, gene-based Another significant advance has come from the drugs have presented interesting challenges for sys- ability to entrap drugs in liposomes with high temic delivery systems. First, gene-based drugs are efficiencies while maintaining the integrity of the highly susceptible to degradation by the nucleases liposome structure. Drug loading can be achieved present in plasma. Although liposomes have the either passively (i.e. the drug is encapsulated during potential to encapsulate gene-based drugs and pre- liposome formation) or actively (i.e. after liposome vent inactivation by nucleases, procedures to effi- formation). Hydrophobic drugs can be directly in- ciently encapsulate plasmid DNA in well defined, corporated into liposomes during vesicle formation, small liposomes or lipidic DNA particles have only and the extent of uptake and retention is governed by recently been realized. Second, the efficacy of gene- drug–lipid interactions. Trapping efficiencies of based drugs is completely dependent on gaining 100% are often achievable, but this is dependent on entry into the target cell cytosol in an intact form.
the solubility of the drug in the liposome membrane.
Therefore, for liposomes to be effective, they must Passive encapsulation of water-soluble drugs relies incorporate agents that promote intracellular deliv- on the ability of liposomes to trap aqueous buffer ery. With few exceptions (i.e. skeletal muscle [13,14] containing a dissolved drug during vesicle formation.
and hepatocytes [15,16]), naked plasmid DNA alone Trapping efficiencies (generally less than 30%) are is not taken up very efficiently by most cell types in limited by the trapped volume contained in the vivo. Third, for certain gene therapy approaches, liposomes and drug solubility. Another approach to such as those involving the delivery of suicide genes, enhance the passive encapsulation of water-soluble systemic gene delivery systems must have the po- drugs is to impart an amphipathic nature to the drugs tential to selectively deliver gene-based drugs to by conjugating or complexing the drugs to lipids A. Chonn, P.R. Cullis / Advanced Drug Delivery Reviews 30 (1998) 73 –83 [20,21]. Alternatively, water-soluble drugs that have cells in vitro [29] by (1) increasing the association of ionizable amine functions can be actively entrapped plasmid DNA with liposomes and (2) increasing the by employing pH gradients [22], which can result in binding of cationic liposome–plasmid DNA com- trapping efficiencies approaching 100%.
plexes to cells. This has prompted many researchersto synthesize different cationic lipids that exhibitimproved gene transfer and cell tolerability prop- 3. Production of liposomes for gene delivery
erties [30–32], as well as to develop novel pro-cedures to efficiently encapsulate plasmid DNA Based on our experience with liposomal drug within lipid-based carriers. The addition of plasmid delivery systems, it is envisioned that the ideal DNA to preformed cationic liposomes often results liposomes for systemic gene delivery will encapsu- in the formation of a heterogeneous mixture of late plasmid DNA with high efficiencies, will protect unstable complexes of cationic lipids and plasmid the DNA from degradation by plasma nucleases, will DNA [33–35]. This heterogeneity and instability are have a narrow size distribution, averaging 100 nm or undoubtedly responsible for the poor reproducibility less in diameter, in order that the liposomes can in the transfection activity observed in vivo when access extravascular regions, and will have the these complexes are administered intravenously [36– potential to incorporate a wide range of lipids, especially lipids that promote fusion with cellular In the past couple of years, there have been membranes and / or enhance liposome stability in the significant advances made in the formulation of circulation. The feasibility of passively encapsulating plasmid DNA into relatively small, stable plasmid DNA in liposomes was demonstrated in the late DNA-containing lipidic particles or liposomes that 1970s using a number of the methods indicated protect plasmid DNA from degradation by nucleases.
above. For example, high molecular weight DNA is For example, Gao and Huang [40] describe a pro- entrapped in egg phosphatidylcholine liposomes by cedure where the addition of polylysine or other hydrating the lipid film in the presence of DNA [23].
polycationic polymers to plasmid DNA, prior to or In a similar manner, metaphase chromosomes are during the addition of cationic liposomes, results in passively entrapped in, or tightly associated with, particles with membranous structures of less than egg phosphatidylcholine–cholesterol (7:2, mol / mol) 100 nm in diameter. The plasmid DNA in the liposomes [24]. Alternatively, DNA can be encapsu- presence of polylysine alone or polylysine and lated in cochleate lipid cylinders that are formed cationic liposomes appears to be resistant to nuclease from the calcium-induced fusion of phosphatidylser- attack, remaining supercoiled when incubated with 5 ine liposomes [25]. Reversed-phase evaporation pro- ml of fetal bovine serum at 378C for 1 h. As cedures have also been employed to encapsulate characterized by negative stain electron microscopy, plasmid DNAs with good but variable encapsulation however, the plasmid DNA does not appear to be efficiencies [26,27]. More recently, freeze drying fully encapsulated by a lipid membrane. Moreover, methods have yielded DNA-containing multilamellar the particles formed are heterogeneous in nature, vesicles with encapsulation efficiencies of 50–60% [28]. For the most part, however, these procedures ratios that affect the transfection activity in Chinese yield relatively large multilamellar vesicles with low hamster ovary (CHO) cells in vitro. The transfection DNA encapsulating efficiencies and generally low active particle can be isolated by sucrose density gene transfer capabilities. Extrusion of the DNA- gradient ultracentrifugation and the purified particle containing multilamellar vesicles to reduce the par- is reported to be stable for up to three months at 48C, ticle size have resulted in poor recoveries of DNA- with no increase in particle size. Similar particles can be generated by the addition of DOPE, cholesteryl In the late 1980s, it was shown that cationic lipids, hemisuccinate and folate–poly(ethylene glycol)– phosphatidylethanolamine conjugates (6:4:0.01, mol / ethanolamine (DOPE)-containing liposomes, could mol / mol) to plasmid DNA–polylysine complexes enhance the efficiency of gene delivery to cultured [41]. These particles were shown to be highly active A. Chonn, P.R. Cullis / Advanced Drug Delivery Reviews 30 (1998) 73 –83 in transfecting KB cells in vitro, being 20–30 times mid DNA–cationic lipid complex formed by the more active than 3-b-[N-(N9,N9-dimethylethane)car- addition of cationic lipids, added in monomer or bamoyl]cholesterol–DOPE, (6:4, mol / mol) cationic micellar form, to plasmid DNA [45,46]. This com- liposome–plasmid DNA complexes. The stability plex can serve as a well-defined intermediate in the and pharmacokinetics of these particles upon in- preparation of plasmid DNA-containing liposomes travenous administration, or the ability of these with good properties for systemic gene delivery particles to transfect cells in vivo, have not been applications (unpublished results). For example, the Recently, Hofland et al. [42] described a detergen- dioleoylphosphatidylcholine or DOPE, to these inter- t-based procedure to form stable plasmid DNA– mediates results in the formation of plasmid DNA- lipidic particles by the addition of various amounts containing liposomes that have a narrow size dis- of cationic lipids [2,3-dioleoyloxy-N-(2(sperminecar- tribution, averaging 70–100 nm in diameter (Fig. 1).
boxamido)-ethyl)-N,N-dimethyl-1-propanaminium tr- Typically, plasmid DNA encapsulating efficiencies ifluoroacetate] and DOPE (1.5:1, mol / mol) solubil- of 70% are obtained using this procedure. A wide ized in buffered 1% octylglucoside to plasmid DNA, variety of lipids that alter the biodistribution of the followed by removal of the detergent by dialysis.
liposomes can be readily incorporated into these The particles can be stored frozen or as a suspension liposomes. For example, the incorporation of at least at 48C for 90 days with no loss in transfection 10 mol% poly(ethylene glycol) conjugated to phos- activity in NIH 3T3 cells in vitro. The physicalproperties of the active particles have not beendefined. However, the active particles can be pelletedby centrifugation at 3000 g for 15 min, indicatingthat they are relatively large particles. Moreover, invitro transfection efficiency is affected by the pres-ence of serum, with a 70% reduction in transfectionactivity in the presence of as little as 1% fetal bovineserum in the culture medium. Another detergent-based method that has yielded active particles hasrecently been described by Liu et al. [43,44]. In thisprocedure, stable emulsions of cationic lipids andplasmid DNA are produced by the addition of non-ionic surfactants. These particles are not well de-fined, but are relatively large in size. The averagediameter of lipid particles for emulsions containingvarious surfactants range from 170 to 250 nm. Uponmixing with plasmid DNA, the particle size increasesfive- to fourteen-fold in diameter, depending on thetype of non-ionic surfactant used for preparing theemulsions. The use of detergents containing branch-ed polyoxyethylene chains as the hydrophilic headgroup are more effective in preventing the formationof large DNA–emulsion complexes. The stabilityand biodistribution of these particles upon intraven-ous administration, or the ability of these particles totransfect cells in vivo have not yet been described inthe literature.
Fig. 1. Cryo-electron micrographs of plasmid DNA encapsulated An alternative approach that has recently been in liposomes. Panel (A) represents vesicles formed in the presence developed takes advantage of the hydrophobic plas- of and (B) in the absence of plasmid DNA.
A. Chonn, P.R. Cullis / Advanced Drug Delivery Reviews 30 (1998) 73 –83 phatidylethanolamine results in plasmid DNA-con- the target cell of several gene therapies for blood taining liposomes that have a circulation half-life approaching 10–12 h in mice. Moreover, these The biodistribution of intravenously administered DNA-containing liposomes appear to be stable in the cationic liposome–plasmid DNA complexes is not circulation of mice, with the majority of the plasmid appropriate for such systemic applications. For in- DNA extracted from the circulating liposomes at 24 stance, it has recently been demonstrated that cat- ionic liposome–plasmid DNA complexes, exhibitingstrong positive zeta potentials, are cleared rapidlyfrom the circulation [57,58]. These intravenously 4. Liposomes with prolonged circulation
administered cationic liposome–plasmid DNA com- lifetimes
plexes [N-(2,3-bis(oleyloxy)propyl-N,N,N-trimethyl-ammonium The use of liposomes for systemic drug delivery monium bromide and DOPE-containing liposomes] requires that the liposomes have the ability to avoid are rapidly eliminated from the plasma, with 50– immediate uptake by phagocytic cells of the re- 60% of the dose taken up by the liver within 5 min, ticuloendothelial system (RES) and remain in circu- and 20–30% of the dose taken up by the lung within lation for extended periods of time in order to 1 min, falling to 10% after 5 min [57]. The cationic enhance the opportunity for the liposomal drugs to liposome–plasmid DNA complexes are predominant- reach non-RES target tissues. A significant advance ly taken up by the Kupffer cells in the liver.
in the development of liposomal drugs has come Moreover, a recent study has shown that cationic with the use of specialized lipids, such as mono- lipid–DNA complexes, harboring excess positive surface charge, are potent activators of the comple- )-modified phosphatidylethanolamine, that engender ment system, potentially a barrier to the efficient long circulation lifetimes when incorporated into delivery of genes when using high lipid doses [59].
liposomes [47–50]. It has been proposed that these Although there have been a few reports demon- PEG–lipid conjugates provide a ‘steric stabilization’ strating the feasibility of using these complexes to of the surface by virtue of the hydrophilic brush coat deliver genes to a number of different tissues (such provided by the PEG polymer [51]. This coat has as the liver, lung, spleen, heart, skeletal muscle, been shown to inhibit serum protein binding to the kidney, uterus, bone marrow cells, peripheral blood liposomal surface [52,53], which would otherwise and ovary) after intravenous administration [36– promote uptake by the RES, complement activation 39,57,58], the observed levels of gene delivery are and destabilization of the liposomal membranes. It low and often are not reproducible. This may be a has been demonstrated that increased circulation consequence of the rapid elimination of the majority lifetimes enhance the opportunity for liposomes, of the injected dose of cationic liposome–plasmid administered systemically, to leave the vascular compartment and enter certain extravascular regions Our recent findings show that DOPE-containing cationic liposomes can be stabilized in the circulation The ability to generate sustained circulating of mice by reducing the cationic lipid content of the liposomal gene delivery systems using the PEG– liposomes and incorporating at least 2 mol% PEG– lipid technology should prove useful for systemic phosphatidylethanolamine derivatives [60]. In vitro, gene delivery applications. For instance, the ability the addition of serum to cationic liposomes com- of long circulating liposomes to accumulate within tumors will be advantageous for cancer gene therapy DOPE (85:15, mol / mol) induces a rapid aggregation applications involving tumor suppressor genes or of the cationic liposomes, forming large fused aggre- suicide genes. Furthermore, the avoidance of RES gates ( . 1 mm in diameter) [60]. Amphipathic uptake, especially by Kupffer cells, the resident PEG–lipid conjugates can stabilize DOPE-containing macrophages of the liver, would enhance the oppor- liposomes by inhibiting the fusogenic activity of tunity for liposomes to deliver genes to hepatocytes, A. Chonn, P.R. Cullis / Advanced Drug Delivery Reviews 30 (1998) 73 –83 However, the fusogenic activity is essential for DNA-containing liposomes has recently been dem- efficient gene delivery [30,31,41,63,64] and, thus, an onstrated in vitro by a number of investigators. For essential property of the amphipathic PEG–lipid example, Lee and Huang [41] have shown that conjugates is that they have the ability to dissociate folate, conjugated to the distal end of PEG–phos- from the carrier at some later time, restoring the phatidylethanolamine, enhances the plasmid DNA fusogenic activity of the liposomes and allowing the uptake and transfection efficiency of KB cells in liposomes to fuse with target cells. The feasibility of vitro by employing plasmid DNA-containing pH- this approach has recently been demonstrated [61].
The rate at which fusogenic activity is recovered is mol / mol) liposomes, particularly when the lipo- shown to be controlled to a large extent by the same somes carry an overall negative surface charge. This parameters that regulate spontaneous transfer of study clearly demonstrates that components which enhance the binding of liposomes to cells, mediatedeither by the use of targeting ligands or by a strongpositive surface charge, are essential for efficient 5. Targeted delivery to specific cells
liposomal gene delivery systems. The addition oftransferrin to cationic liposome–plasmid DNA com- In general, liposomes are effective delivery sys- plexes increases the amount of DNA taken up by tems because they alter the pharmacokinetics of the human hepatoma HepG2 cells in vitro twofold, free drug, leading to enhanced drug bioavailability to accompanied by a significant increase in the number specific target cells that reside in the circulation or, of b-galactosidase-positive cells (98–100% in the more importantly, to extravascular disease regions.
presence of transferrin compared to 3–4% in the The ability to selectively deliver drugs to specific absence of transferrin) [83]. Transferrin presumably cells, such as tumor cells, within these regions will acts to further facilitate the uptake of cationic further enhance the therapeutic index of liposomal liposome–DNA complexes via a receptor-mediated drugs. Targeted delivery and improved therapeutic process. Similarly, asialofetuin [79,80] and galac- activity of liposomal drugs in vivo has been achieved tose-containing lipids [84] have been shown to by coupling site-directive targeting ligands, such as increase the transfection efficiency of HepG2 cells in monoclonal antibodies [65–68], to the surface of vitro. Kikuchi et al. [85] have shown that the liposomes by either covalent or non-covalent meth- addition of epidermal growth factor to cationic ods [68,69]. A significant advance in this area has liposomes enhances the in vitro luciferase gene been the advent of novel PEG–phosphatidylethanol- expression in epidermal growth factor receptor-over- amine lipids that allow targeting ligands to be expressing HEC-A cells and not in epidermal growth conjugated at the distal ends of the PEG spacer factor receptor-deficient HRA cells. The coupling of [70–73]. These conjugates increase target cell bind- antibodies to pH-sensitive liposomes [86] or to ing in vitro, as well as prolong circulation times.
cationic liposomes [87] has been shown to also Furthermore, in addition to antibodies, glycolipids enhance transfection activity in vitro compared to [74–77], proteins [78–80] and vitamins [41,71] have that found in non-targeted DNA-containing lipo- been used to selectively target specific cells via cell For liposomal gene delivery systems, targeting ligands need to function not only to increase the 6. Fusogenic liposomes for intracellular delivery
binding of the liposomes to specific target cells, suchas hepatocytes, but also to promote the cellular Fusogenic liposomes can potentially facilitate the uptake of the liposomes via an endocytic pathway.
intracellular delivery of encapsulated drugs by fusing Endocytosis is believed to play a major role in with the target cell. A variety of approaches can be plasmid DNA delivery to cultured cells in vitro envisioned for constructing fusogenic liposomes.
[10,63,81,82]. The feasibility of using targeting Examples include the inclusion of lipids that are able ligands to increase the cellular uptake of plasmid to form non-bilayer phases, such as DOPE, which A. Chonn, P.R. Cullis / Advanced Drug Delivery Reviews 30 (1998) 73 –83 can promote destabilization of the bilayer, inducing propane) can function in the absence of helper lipids fusion events [88,89]. Furthermore, alterations in the [30,31] or in the presence of cholesterol (such as lipid composition can render liposomes pH sensitive, dioctadecylammonium bromide) [38] suggests that leading to enhanced fusogenic tendencies in low pH these cationic lipids may, by themselves, possess compartments such as endosomes [41,86,90]. Non- properties that promote endosomal release of plas- phospholipid fusogenic liposomes composed primari- mids via a mechanism other than a membrane fusion ly of dioxyethylene acyl ethers and cholesterol have event. As previously mentioned, plasmid DNA-con- been shown to fuse with plasma membranes of taining pH-sensitive liposomes are efficient gene erythrocytes and fibroblasts [91]. Alternatively, effi- delivery systems in vitro, provided that they have cient fusogenic liposomes can be achieved by incor- targeting ligands coupled to their surface [41,86].
porating fusogenic proteins into the liposome mem-brane [92–94] or entrapped within liposomes [95].
The feasibility of this approach has been demon- 7. Conclusions
strated for the delivery of the diphtheria toxin Asubunit using liposomes produced from influenza The development of controllable and reproducible virus envelopes [94]. Fusogenic peptides can be liposomal systems for systemic gene delivery neces- conjugated to the liposomes [96–98] and may also sitates the establishment of methods to efficiently promote intracellular delivery. The encapsulation of encapsulate gene-based drugs in well-defined, rela- a 30-amino acid fusogenic peptide has recently been tively small liposomes. Traditional methods for shown to promote relatively efficient endosomal encapsulating drugs in liposomes have proven to be release of propidium iodide, with 20–25% of the ineffective for gene-based drugs. However, recently encapsulated propidium iodide gaining access to KB developed detergent-based procedures to produce cell chromosomal DNA after 48 h [99].
stable plasmid DNA lipidic particles or plasmid The effectiveness of liposomal gene-based drugs is DNA-containing liposomes appear promising. In dependent on their ability to access the cytosol of vitro studies have shown that these systems are target cells. For optimum efficiency, therefore, lipid- active in delivering plasmid DNA to a number of based gene delivery systems should exhibit fusogenic cultured established cell lines. The in vivo studies activity. A number of studies illustrate that the above are certainly forthcoming. Although these are early approaches to enhance the fusogenic activity of stages for liposomal gene delivery systems, several liposomes can be applied to enhance the efficiency of of the advances made in liposomal drug delivery lipid-based gene delivery systems. For example, the technologies can be directly applied to these systems.
addition of replication-deficient adenovirus, which Noteworthy is the use of exchangeable PEG–lipid enhance endosomal escape, to cationic liposome– conjugates to stabilize the plasmid DNA-containing plasmid DNA complexes results in an approximately lipid-based carriers in the circulation. This should fivefold increase in chloramphenicol acyl transferase expedite the development of systemic liposomal gene activity detected in FAO hepatoma and 3T3-F442A delivery systems that exhibit targeted and enhanced adipocyte cells in vitro [100], and up to a 1000-fold increase in luciferase expression in human smoothmuscle cells in vitro [101]. Similarly, the incorpora-tion of the fusogenic protein from Sendai virus, by References
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Design of a short membrane-destabilizing peptide covalently

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Combination Therapy for Uncomplicated Falciparum Malaria in Ugandan Children A Randomized Trial Context Combination therapy is now widely advocated as first-line treatment for uncomplicated malaria in Africa. However, it is not clear which treatment regimensare optimal or how to best assess comparative efficacies in highly endemic areas. Objective To compare the efficacy and safety of 3


About myself I’m not a professional mathematician, I work as a pro-grammer. I have been studying in a university in Russia but have notfinished my study. So, I know little beyond my specialization. Nevertheless in my free time I discovered a new theory whichwould completely overturn general topology. About this lesson In this lesson I present my discovery, the theory of funcoidsand reloid

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