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`Liposomal Formulations for Nucleic Acid Delivery
`
`CHAPTER 9
`
`Ian MacLachlan
`
`CONTENTS
`
`9.1 Liposomes for the Delivery of Nucleic Acid Drugs.............................................................237
`9.2 Liposome Constituents .........................................................................................................239
`9.2.1 Cationic Lipids .........................................................................................................239
`9.2.2 The Role of Helper Lipids in Promoting Intracellular Delivery..............................240
`9.2.3 PEG–Lipids...............................................................................................................241
`9.2.4 Active Targeting........................................................................................................242
`9.3 Methods of Encapsulating Nucleic Acids ............................................................................242
`9.3.1 Passive Nucleic Acid Encapsulation.........................................................................243
`9.3.2 The Ethanol Drop (SALP) Method of Nucleic Acid Encapsulation........................247
`9.3.3 Encapsulation of Nucleic Acid in Ethanol-Destabilized Liposomes .......................247
`9.3.4 The Reverse-Phase Evaporation Method of Nucleic Acid Encapsulation ...............248
`9.3.5 The Spontaneous Vesicle Formation by Ethanol Dilution (SNALP) Method of
`Nucleic Acid Encapsulation .....................................................................................249
`9.4 Analytical Methods...............................................................................................................251
`9.4.1 Measuring Particle Size ............................................................................................251
`9.4.2 Zeta Potential............................................................................................................253
`9.4.3 Encapsulation............................................................................................................253
`Pharmacology of Liposomal NA..........................................................................................254
`9.5.1 Pharmacokinetics and Biodistribution of Liposomal NA Following Systemic
`Administration ..........................................................................................................254
`9.5.2 Toxicity of Liposomal NA Formulations .................................................................256
`9.5.3 Immune Stimulation .................................................................................................258
`9.5.4 Immunogenicity........................................................................................................259
`9.5.5 The Efficacy of Liposomally Formulated NA Drugs...............................................260
`References ......................................................................................................................................262
`
`9.5
`
`9.1 LIPOSOMES FOR THE DELIVERY OF NUCLEIC ACID DRUGS
`
`Liposomes are artificial vesicles made up of one or more bilayers of amphipathic lipid encap-
`sulating an equal number of internal aqueous compartments. They are distinguished on the basis
`of their size and the number and arrangement of their constituent lipid bilayers (Figure 9.1).
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`Figure 9.1 Liposomes. Mulilamellar vesicles (MLVs) are large (hundreds of nm in diameter) complex structures
`containing a series of concentric bilayers separated by narrow aqueous compartments. Large
`unilamellar vesicles (LUVs) are between 50 and 500 nm in diameter, while the smallest liposomes
`namely small unilamellar vesicles (SUVs) are ⬍50 nm. LUVs are the preferred systems for delivery
`of NA drugs. Lipids are drawn roughly to scale.
`
`Multilamellar vesicles (MLVs) are formed by the aqueous hydration of dried lipid films. Typically
`hundreds of nanometers in diameter, they are large, complex structures containing a series of con-
`centric bilayers separated by narrow aqueous compartments. Simple unilamellar vesicles between
`50 and 500 nm in diameter are referred to as large unilamellar vesicles (LUVs) while the smallest
`liposomes, vesicles smaller than 50 nm in diameter, are small unilamellar vesicles (SUVs).
`Liposomes have received attention not only for their utility as model membrane systems, but
`also for use in drug delivery. Typically, liposomes are used as drug carriers, with the solubilized
`drug encapsulated in the internal aqueous space formed by the liposomal lamellae. Liposomal drug
`formulations can be used to overcome a drug’s nonideal properties, such as limited solubility,
`serum stability, circulation half-life, biodistribution, and target tissue selectivity. Experience with
`conventional small molecule drugs has shown that the drugs which benefit the most from liposo-
`mal delivery, are those that are chemically labile, subject to enzymatic degradation and have an
`intracellular site of action [1]. For this reason, there is considerable interest in exploiting lipo-
`somes as carriers of nucleic acids (NAs), either as plasmid vectors for gene therapy applications
`or to deliver smaller NA species such as antisense oligonucleotides, ribozymes and, more recently,
`siRNA for the purposes of downregulating target genes. Because of their ability to achieve favor-
`able drug/lipid ratios and their more predictable drug release kinetics LUV are the preferred lipo-
`some delivery system for NA drugs.
`An advantage of liposomal drug delivery is that the pharmacokinetics, biodistribution, and intra-
`cellular delivery of the liposome payload are largely determined by the physicochemical properties
`of the carrier. For example, the biodistribution of a NA entrapped within a small, long circulating
`liposome is independent of the type of NA, which can be a relatively stable double-stranded plasmid
`DNA molecule or single-stranded antisense DNA, or one of the more labile ribonucleotide
`molecules such as ribozymes or a duplex siRNA. This is only true if the liposome is truly acting as
`a carrier, rather than a mere excipient. Liposomes function as excipients when used to formulate
`hydrophobic drugs that would otherwise be difficult to administer in aqueous dosage form.
`Hydrophobic drugs rapidly exchange into lipoproteins or other lipid-rich environments soon after
`injection, resulting in comparably uncontrolled pharmacology. In the context of NA drug delivery,
`liposomes are considered excipients if used to enable vialing and aqueous dosing of hydrophobic
`lipid–NA conjugates [2–5]. (These applications are not considered in this chapter, nor are those that
`use preformed, cationic lipid-containing vesicles to form “lipoplex” or “oligoplex” systems.)
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`
`An objective inherent in all pharmaceutical development is to minimize the risks associated with
`treatment while maximizing the benefit to patient health. The most important risk to patients is the
`toxicity associated with the administration of poorly tolerated compounds, often exacerbated by
`attempts to increase efficacy by escalating the administered dose. A well-designed liposomal delivery
`system will be capable of reducing the toxicity and increasing the potency of NA-based drugs by
`optimizing NA delivery to target tissues. Liposomal NA delivery will be determined by the physical
`and biochemical properties of the liposome including stability, size, charge, hydrophobicity, interac-
`tion with serum proteins, and interaction with nontarget cell surfaces. Ideally, liposomal carriers for
`NA delivery will have the following properties: (i) they will be safe and well tolerated; (ii) they will
`have appropriate pharmacokinetic attributes to ensure delivery to intended disease sites; (iii) they will
`mediate effective intracellular delivery of intact NA; (iv) they will be nonimmunogenic, enabling
`the use of multidosing treatment regimes; and (v) they will be stable upon manufacture so that large
`batches can be prepared with uniform, reproducible specifications. In this chapter we discuss the
`physical makeup, manufacturing methods, and pharmacological considerations specific to liposomal
`systems for the delivery of NA-based drugs, with emphasis on those that enable systemic delivery of
`synthetic polynucleotides such as antisense ODN, ribozymes, and siRNA.
`
`9.2 LIPOSOME CONSTITUENTS
`
`NA encapsulation was first described in the late 1970s, prior to the development of cationic lipid-
`containing lipoplex, using naturally occurring, neutral lipids to encapsulate high-molecular-weight
`DNA [6–8]. The first reports of low-molecular-weight oligo- or polynucleotide encapsulation
`similarly used passive techniques to entrap NA in neutral liposomes [9–11]. The advent of cationic
`lipid-mediated lipofection [12] saw a shift in emphasis away from encapsulated systems in favor of
`“lipoplex” or “oligoplex” systems. More recently, improvements in formulation technology have
`allowed for a return to encapsulated systems that contain cationic lipids as a means of facilitating
`both encapsulation and intracellular delivery. More advanced systems typically contain multiple lipid
`components, each of which play a role in determining the physical and pharmacological properties
`of the system as a whole.
`
`9.2.1 Cationic Lipids
`
`Cationic lipids play two roles in liposomal NA formulations. In the first case, they encourage
`interaction between the lipid bilayer and the negatively charged NA, allowing for the enrichment
`of NA concentrations over and above that which would be achieved using passive loading in charge neu-
`tral liposomes. Cationic lipids allow for encapsulation efficiencies greater than 40% when using
`coextrusion methods, and greater than 95% when using more sophisticated techniques [13–15].
`Cationic lipids also function by providing the liposome with a net positive charge, which in turn
`enables binding of the NA complex to anionic cell surface molecules. The most abundant anionic
`cell surface molecules, sulfated proteoglycans and sialic acids, interact with and are responsible for
`the uptake of cationic liposomes [16–18]. The role of cationic lipids in liposomal uptake presents
`a dilemma: highly charged systems are rapidly cleared from the blood, thereby limiting accumula-
`tion in target tissues. Particles with a neutral charge however, display good biodistribution profiles,
`but are poorly internalized by cells. This supports the concept of a modular delivery solution, that
`is, an engineered nanoparticle with individual components fulfilling different functions in the
`delivery process, and in particular, a system which responds to the microenvironment in a manner
`that facilitates transfection. Titratable, ionizable lipids are components that allow for the adjust-
`ment of the charge on the system by simply changing the pH after encapsulation [19]. At reduced
`pH when the system is strongly charged, NAs are efficiently encapsulated. When liposomes
`containing titratable, ionizable lipids are at a pH closer to the pKa of the cationic lipid, such as
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`physiological pH, they become more charge neutral and are able to avoid opsonization by blood
`components [19]. More recently, the use of novel, pH titratable cationic lipids with distinct
`physicochemical properties that regulate particle formation, cellular uptake, fusogenicity, and
`endosomal release of NA drugs have been described [20]. The chemical and biological properties
`of pH-titratable cationic lipids are influenced by their degree of lipid saturation. In particular, the
`phase transition properties, as measured using 31P-NMR, are affected. Above the phase transition
`temperature, Tc, lipids adopt the more highly fusogenic reverse hexagonal HII phase [20–22]. By
`noting the temperature at which this phase transition occurs, the relative ease with which lipids
`form the HII phase and become “fusogenic” can be determined. On this basis it has been shown
`that the fusogenicity of liposomal systems increases as the titratable cationic lipid becomes less
`saturated. The lipid pKa also correlates with the degree of saturation. pK measurements confirm
`that saturated lipids carry more residual charge at physiological pH. For this reason, liposomes
`containing the more highly saturated cationic lipids are taken up more readily by cells in vitro [20].
`However, liposomes containing the more fusogenic unsaturated cationic lipids DLinDMA and
`DLenDMA are more effective at mediating RNA interference in both in vitro cell culture systems
`and in vivo. The apparently conflicting results between cellular uptake and silencing potency are
`a reminder that cellular uptake per se is insufficient for effective delivery of NA. Cellular uptake,
`fusogenicity, and endosomal release are distinct processes, each of which need to be enabled by
`the delivery vehicle and each of which are profoundly affected by the physicochemical properties
`of the cationic lipids used.
`
`9.2.2 The Role of Helper Lipids in Promoting Intracellular Delivery
`
`Although we have just shown that cationic lipids may have inherent fusogenic properties of
`their own, cationic lipids were originally believed to require fusogenic “helper” lipids for effi-
`cient NA delivery [23–26]. Fusogenic liposomes facilitate the intracellular delivery of complexed
`plasmid DNA by fusing with the membranes of the target cell. Fusion may occur at a number of
`different stages in delivery, either at the plasma membrane, endosome or nuclear envelope.
`Fusion of first-generation, nonencapsulated lipoplex systems with the plasma membrane is
`expected to be a particularly inefficient method of introducing NA into the cytosol. Since
`lipoplex-NA is predominantly attached to the surface of the liposome, lipoplex fusion events
`resolve with NA, formerly attached to the liposome surface, deposited on the outside surface of
`the plasma membrane. Encapsulated systems are significantly different from lipoplex in this
`respect. Upon fusion with either the plasma or endosomal membrane(s), encapsulated carriers
`deliver their contents directly into the cytosol.
`Lipids that preferentially form nonbilayer phases, in particular the reverse hexagonal HII phase,
`such as the unsaturated phosphatidylethanolamine DOPE, promote destabilization of the lipid
`bilayer and fusion. Similar to fusogenic cationic lipids, decreasing the degree of lipid saturation
`increases the lipid’s affinity for the fusogenic HII phase [27–32]. However, some cationic lipids can
`function in the absence of these so-called helper lipids, either alone [24,25] or in the presence of
`the nonfusogenic lipid cholesterol [33]. This would suggest that either these lipids have properties
`which promote delivery through a mechanism which does not require membrane fusion, or that
`their own fusogenic properties are adequate to support delivery. As described above, cationic lipids
`are readily designed for optimal fusogenicity by controlling lipid saturation. This provides for
`multiple opportunities for modulating the fusogenicity of a liposomal lipid bilayer [20].
`Attempts to address the role of fusogenic lipids in vivo have yielded confounding results. In this
`regard it is important to distinguish the effect of fusogenic lipids on NA delivery to target tissue
`from their effect on intracellular delivery. Fusogenic formulations are more likely to interact
`with the vascular endothelium, blood cells, lipoproteins, and other nontarget systems while in the
`blood compartment. For this reason there may be an advantage to transiently shield the fusogenic
`potential of systemic carriers using shielding agents such as polyethylene glycol (PEG).
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`9.2.3 PEG–Lipids
`
`An ideal delivery system would be one that is transiently shielded upon administration, facili-
`tating delivery to the target site, yet becomes increasingly charged and fusogenic as it reaches the
`target cell. PEG lipids partially address this challenge. PEG–lipid conjugates are readily incorpo-
`rated in liposomal NA formulations. They provide a benefit during the formulation process, stabi-
`lizing the nascent particle and contribute to formulation stability by preventing aggregation in the
`vial [13]. PEG conjugates sterically stabilize liposomes by forming a protective hydrophilic layer
`that shields the hydrophobic lipid layer. By shielding the liposome’s surface charge they prevent the
`association of serum proteins and resulting uptake by the reticuloendothelial system when liposomes
`are administered in vivo [34,35]. In this way, cationic liposome NA formulations are stabilized in a
`manner analogous to PEGylated liposomal drug formulations that exhibit extended circulation life-
`times [36–41]. Although this approach has been investigated with a view towards improving the sta-
`bility and pharmacokinetics of lipoplex containing either plasmid DNA [42] or antisense
`oligonucleotides [43], PEG–lipid-containing lipoplex systems suffer from the heterogeneity and
`suboptimal pharmacology common to most nonencapsulated NA–cationic lipid complexes.
`Although PEG–lipid-containing systems are promising with respect to their ability to deliver NA
`to disease sites, improvements are required to increase their potency. Early PEGylated liposomes for
`the delivery of small molecule chemotherapeutic drugs utilized stably integrated PEG lipids such as
`PEG-DSPE [39]. These systems are designed to function as carriers that facilitate the accumulation of
`active drug compound at disseminated disease sites. The drug is released at the cell surface at a “leak-
`age rate” determined by the liposomal bilayer composition. NA-based drugs differ in this respect in
`that they require effective intracellular delivery, hence the use of the cationic and fusogenic lipids
`described earlier. PEGylated systems typically exhibit relatively low-transfection efficiencies. This is
`mainly due to the ability of the PEG coating to inhibit cell association and uptake [23,44,45]. Ideally,
`PEG–lipid conjugates would have the ability to dissociate from the carrier and transform it from a sta-
`ble, stealthy particle to a transfection-competent entity at the target site. Various strategies have been
`applied to this problem. A number of investigators have explored the use of chemically labile
`PEG–lipid conjugates [46–52], in particular those that are “pH sensitive.” Typically, these systems
`invoke a chemically labile linkage between the lipid and PEG moieties that reacts via acid-catalyzed
`hydrolysis to destabilize the liposomes by removal of the sterically stabilizing PEG layer. Although
`this approach results in improved performance both in vitro and in vivo, it may be regarded as sub-
`optimal for two reasons. First, pH-sensitive PEG lipids are designed to be rapidly hydrolyzed in the
`reduced pH environment encountered within the endosome, but since PEG lipids are known to inhibit
`cellular uptake, a prerequisite to endosomal localization and hydrolysis, their use actually limits the
`amount of material delivered to the endosome [53]. Second, the incorporation of pH-sensitive or oth-
`erwise chemically labile lipids results in a truncation of formulation shelf life relative to systems that
`use more stable PEG–lipids. An alternative to the use of acid-labile PEG–lipids involves the use of
`chemically stable, yet diffusible PEG lipids.
`The concept of diffusible PEG lipids arose from the observation that the length of the PEG lipid
`anchor has an influence on PEG lipid retention and the stability and circulation lifetime of empty
`lipid vesicles [54]. It has been found that by modulating the alkyl chain length of the PEG lipid
`anchor [55–59], the pharmacology of encapsulated NA can be controlled or “programmed” in a
`predictable manner. Upon formulation, the liposome contains a full complement of PEG in steady-state
`equilibrium with the contents of the vial. In the blood compartment, this equilibrium shifts and the
`PEG–lipid conjugate is free to dissociate from the particle over time, revealing a positively charged
`and increasingly fusogenic lipid bilayer that transforms the particle into a transfection-competent
`entity. Diffusible PEG lipids differing in the length of the their lipid anchors have been incorporated
`into liposomal systems containing plasmid DNA (SPLP) [13,55], antisense oligonucleotides (PFV,
`SALP) [19,56,60], and siRNA (SNALP) [14,15,61]. This approach may help to resolve the two
`conflicting demands imposed upon NA carriers. First, the carrier must be stable and circulate long
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`enough to facilitate accumulation at disease sites. Second, the carrier must be capable of interact-
`ing with target cells to facilitate intracellular delivery.
`
`9.2.4 Active Targeting
`
`Active targeting refers to processes that aim to increase the accumulation, retention or internal-
`ization of a drug through the use of cell-specific ligands. This is to be distinguished from the passive
`“disease site targeting” or the “enhanced permeability and retention” (EPR) effect, which results
`in the accumulation of appropriately designed carriers in target sites such as tumor tissue. Active
`targeting has been successfully applied to liposomal small molecule drug formulations and generally
`has the effect of improving the therapeutic index of the liposomal drug when measured in preclinical
`studies. NA delivery systems stand to benefit from targeting in two ways, first through improving the
`accumulation and binding of formulations to target cells and second by facilitating intracellular
`delivery through endocytosis. The perceived benefits of active targeting have encouraged numerous
`investigators in this area and targeting of NA formulations has been achieved through the use of
`molecules as diverse as antibodies directed against cell surface proteins [62–65], protein ligands of
`cell surface receptors [66–69], vitamins [70–72], and glycolipids [73,74].
`The earliest reports of targeted liposomal formulations of encapsulated NA were attempts to
`improve the intracellular delivery characteristics of charge neutral liposomes encapsulating either
`synthetic antisense DNA [63,65] or in vitro transcribed antisense RNA [64]. The results of these
`studies were encouraging, suggesting a significant benefit associated with the use of targeted
`systems. Although these in vitro studies effectively demonstrated the potential advantage of targe-
`ting at the level of intracellular delivery, they were unable to address important pharmacological
`considerations such as those that influence accumulation at disease sites. It is unlikely that addition
`of targeting ligands to delivery systems that are rapidly removed from the circulation will result
`in delivery exceeding that achieved by systems that display passive disease site targeting. For this
`reason many investigators have pursued approaches involving the addition of targeting ligands to
`sterically stabilized and charge shielded systems, such as those containing PEG lipids [71,72,75–77].
`This approach has been advanced, in part, by the development of the so-called postinsertion technique
`[78]. Postinsertion allows for the insertion of ligand–PEG–lipid conjugates into preformed liposomes
`containing encapsulated NA. This represents a significant improvement on earlier approaches in
`which ligands were chemically coupled to preformed liposomes, an approach limited by suboptimal
`coupling efficiencies, or where ligand–lipid conjugates were incorporated in the first stages of the
`formulation process, an approach limited by the resulting negative impact on NA encapsulation
`efficiency and subsequent suboptimal presentation of the targeting ligand.
`A number of reports suggest that it is possible to design encapsulated systems containing
`targeting ligands that retain extended circulation lifetimes and passive disease site targeting the
`following systemic administration. It remains to be seen if the benefits of active targeting outweigh
`the increased cost, manufacturing complexity and immunogenicity that often accompanies the use
`of such technology.
`
`9.3 METHODS OF ENCAPSULATING NUCLEIC ACIDS
`
`To capitalize on the pharmacology of liposomal drug carriers it is necessary to completely entrap
`NA within the contents of a liposome. In this regard it is important to distinguish first-generation
`“lipoplex” or “oligoplex” systems from those that truly encapsulate their NA payload. Lipoplex are elec-
`trostatic complexes formed by mixing preformed cationic lipid-containing vesicles with NA [12,79,80].
`The result is a heterogenous, metastable aggregate that is effective when used to transfect cells in cul-
`ture but has relatively poor performance in vivo. Upon systemic administration, lipoplex systems are
`rapidly cleared from the blood, accumulating in the capillary bed of first-pass organs such as the lung.
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`Lipoplex are effectively taken up by the cells of the innate immune system, contributing to their pro-
`found toxicities and off-target effects. These side effects may manifest as “efficacy” in antitumor or anti-
`infective applications, confounding data interpretation and encouraging the acceptance of false-positive
`results. For these reasons, an abundance of caution is encouraged when initiating in vivo studies that use
`liposomes to deliver NA. Of particular importance is the use of appropriate analytical methodology,
`described in Section 9.4, to properly characterize lipid-based systems prior to and during use.
`
`9.3.1 Passive Nucleic Acid Encapsulation
`
`Liposomal encapsulation of small molecule drugs may be achieved by either “passive” or “active”
`loading. Unlike small molecule drugs, NAs are not readily packaged in preformed liposomes using
`pH gradients or other similar active loading techniques. This is predominantly due to the large size
`and hydrophilic nature of NA, which conspire to prevent them from crossing intact lipid bilayers. For
`this reason, much of the work on NA encapsulation has utilized passive loading technology.
`Passive encapsulation typically involves the preparation of a “lipid film,” the lipidic residue that
`remains after evaporation of the organic phase of a lipid solution (Figure 9.2). Rehydration of the
`
`Lipid solution in solvent
`
`Nucleic acid solution
`in buffer
`
`Dried lipid film
`
`Lipid hydration with
`nucleic acid solution
`
`MLV formation
`by freeze/thaw (5−10×)
`
`MLV extrusion (10x)
`
`LUV collection
`
`Free nucleic acid removal
`
`Sample concentration
`
`Sterile filtration
`
`Figure 9.2 Passive method of NA encapsulation. Passive encapsulation utilizes a dried lipid film prepared by
`evaporating the organic phase of a lipid solution. The resulting lipid film is rehydrated in an aqueous
`solution of NA in buffer, forming MLV. Multiple freeze-thaw cycles increase the extent of NA encap-
`sulation within the MLV bilayers. The vesicles are then extruded through polycarbonate filters
`producing LUV.
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`lipid film in aqueous media, typically buffer containing NA, followed by vigorous mixing, results
`in the formation of MLV. This is followed by multiple cycles of freezing and thawing to increase
`the extent to which the NA solute is entrapped by the MLV bilayers. The MLV preparation is then
`subjected to multiple rounds of extrusion through polycarbonate filters to produce LUV (Figure 9.2
`and Figure 9.3) [81]. The size of the LUV is determined by the size of the filter pores. This process
`suffers from a number of limitations. When used to encapsulate NA, the efficiency of passive encap-
`sulation is generally quite low, ranging from 3 to 45%, depending on the composition of the lipid
`bilayer and other factors (Table 9.1). The low encapsulation efficiency, consequently, necessitates
`the incorporation of a postencapsulation separation step such as dialysis, size exclusion chro-
`matography or ultrafiltration to remove nonencapsulated NA. In an effort to improve the efficiency
`of encapsulation, excess lipid is often incorporated in the formulation process, resulting in low
`NA/lipid ratios which ultimately impact toxicity and cost of goods. Finally, the extrusion process is
`inherently difficult to scale. Preparation of large batches requires the use of custom-built extruders
`to accommodate large filters. The probability of filter tears, resulting in batch failure, increases
`as the size and cost of the batch increases. In spite of these process limitations, extrusion-based
`methods for liposome preparation have been successfully adopted by many laboratories, presum-
`ably because the technology is readily accessible to the casual investigator. Furthermore, significant
`progress has been made adapting or enhancing extrusion-based processes for the liposomal formu-
`lation of NA-based drugs. These include the use of cationic and anionic lipids [82,83], ionizable
`cationic lipids [19,84], PEG lipids [85], and detergent or organic solvents such as ethanol [19,60]
`to control bilayer assembly.
`
`Figure 9.3 The Lipex™ thermobarrel extruder for the preparation of uniformly sized liposomes by extrusion. An
`MLV or other vesicle preparation is introduced to the top of the extruder and the extruder is pres-
`surized with nitrogen, forcing the MLV through a polycarbonate filter of defined pore size. The resulting
`LUVs are collected via the outlet port at the bottom of the device. Extrusion is repeated, typically for
`a total of 10 passes. The unit permits thermostatic operation by virtue of the thermobarrel, which
`can be coupled to a circulating water bath. Photo courtesy Northern Lipids Inc., Vancouver, Canada,
`http://www.northernlipids.com.
`
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`
`(Continued)
`
`[183]
`[182]
`[181]
`[180]
`[72]
`[179]
`[178]
`[177]
`[176]
`
`[175]
`[174]
`[173]
`[172]
`[93]
`[171]
`[170]
`[169]
`[168]
`[167]
`[92]
`[166]
`[165]
`[164]
`[163]
`[162]
`[161]
`[160]
`[159]
`[158]
`[157]
`[85]
`[156]
`#1626
`
`[155]
`[154]
`[63]
`[153]
`
`[65]
`
`Reference
`
`Payload
`
`Encapsulation (%)
`
`Size (nm)
`
`Lipid Composition
`
`Formulation Method
`
`Table 9.1Liposomal Formulations of Oligo- and Polynucleotide Drugs
`
`ODN
`ODN
`ODN
`ODN
`Various
`ODN
`ODN
`ODN
`
`ODN
`ODN
`ODN
`ODN
`ODN
`ODN
`TFD, ODN
`ODN
`ODN
`pDNA
`ODN
`ODN
`RNA Aptamer
`Plasmid/ODN
`ODN
`ODN
`Ribozyme
`ODN
`ODN
`ODN
`ODN
`ODN
`ODN
`
`ODN
`ODN
`ODN
`ODN
`ODN
`
`Up to 20
`
`10–30
`
`ND
`2–5
`ND
`
`ND
`7–15
`ND
`⬎85
`
`43.5⫾4
`
`ND
`
`ND
`
`10–15
`80–100
`⬃16
`ND
`10
`ND
`ND
`
`ND
`88
`60
`⬍10
`⬎90
`⬍10
`⬍10
`30–40
`⬍10
`
`10–75
`
`ND
`
`24–32
`15–20
`
`2–3
`10
`⬍2
`
`3
`
`⬍10
`
`467.2⫾72.0
`
`250–300
`
`ND
`
`90–100
`90–110
`240–370
`
`130
`ND
`ND
`
`⬍2000
`220⫾55
`110⫾40
`100–140
`
`ND
`
`ND
`ND
`
`100–150
`
`ND
`ND
`
`50–65
`
`200–300
`
`400–500
`316–562
`
`ND
`
`110⫹30
`⬍200
`
`ND
`ND
`ND
`ND
`
`50–70
`
`ND
`
`100–140
`
`170
`
`460⫾200
`
`⬃200
`220⫾55
`
`DSPC:Chol:CPL
`HVJ liposome:PS:PC:Chol
`Thiocationic lipid:oleic acid:Vitamin D
`PE:CHEMS:LLO
`Folate liposomes:EPC:Chol:DSPE-PEG-Pteroate
`PE:CHEMS:LLO
`HVJ liposome:PS:PC:Chol
`DDAB:EPC:Chol
`CHEMS:DOPE or conventional SPC liposomes
`immunoliposomes
`HVJ liposome:PE-DTP:PS:PC:Chol
`DPPC:DMPG
`HVJ liposome:PS:PC:Chol
`EPC:Chol
`PC40:Chol:PEG-DSPE:DOTAP
`DOPE:CHEMS or SPC
`HVJ liposome
`DPPE:Cetyltrimethyl ammonium bromide
`HVJ liposome:PC:DOPE:Sph:PS:Chol
`HVJ liposome:PS:PC:Chol
`DPPC:Chol:DPPS or DPPA
`EPC:Chol:Folate-PEG-DSPE
`DSPC:Chol
`HVJ liposome:Chol:PC:PS
`HVJ liposome
`DOGS:DOPE
`HVJ liposome:PC:Chol:DC-Chol
`PC:Chol:PS
`DDAB:PC:Chol
`DOPE:Chol:Oleic Acid:Palmitoyl-CD4
`PC:Ch