LIPOSOME COMPOSITION: CHOICE OF LIPIDS

5. LIPOSOME COMPOSITION: CHOICE OF LIPIDS

The choice of lipids for drug encapsulation into liposomes is dependent on the drug characteristics and intended applica- tions. Liposomal composition determines the properties (including surface charge, rigidity, and steric interactions) and the in vitro and in vivo performance of liposomes. The specific properties of the liposomes are determined by the chemistry of the head and tail groups of the constituent lipids. Selection is often decided on a case-by-case basis, since pro- duct performance can be drastically affected by slight changes in the liposomal vesicle composition.

5.1. Conventional Liposomes Liposomes were traditionally prepared from a variety of

neutral and anionic lipids. Examples of some of these lipids include lecithins (85), sphingomyelins (86), phosphatidylcho- lines (87) and phosphatidylethanolamines (88) (neutral) and phosphatidylserines (89), phosphatidylglycerols (90) and phosphatidylinositols (91) (anionic). These liposomes have

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non-specific interactions with their environment and are often referred to as conventional or unmodified liposomes. These conventional liposomes are recognized by the mononuclear phagocytic cells and are removed from the circulation within

a few minutes to several hours (59). They are subsequently taken up by the liver and the spleen and therefore are very effective in targeting therapeutic agents to treat diseases of these organs (59). The addition of acidic phospholipids to mix- tures of zwitterionic phospholipids imparts a negative charge that helps to reduce liposomal aggregation and improve their stability (7).

Specific liposome characteristics can be altered by incor- poration of various liposomal components that have different properties (7). An example of an important liposomal property that is affected by lipid composition is the phase transition temperature (T m ) (7,92,93). This is the temperature of a lipo- some at which the membrane changes from ordered solid to disordered fluid and is dependent on the length and degree of saturation of the hydrocarbon chains (93,94). T m may be dependent on the acyl chain length (94), composition of the lipid bilayer (95), and the entrapped drug (96). For example, for lipids composed of phosphatidylcholine polar head group, the T m can vary from –15

C for dis- tearoyl chains (94). The fluid state of the membrane is rela- tively unstable, more elastic, can form transient hydrophilic channels and is permeable to the transport of materials. Consequently membranes above their T m tend to be ‘‘leaky’’ to entrapped drug substances (97). Conventional liposomes particularly those of high fluidity may disrupt on contact with the plasma, as a result of interactions with plasma components (7,97). The addition of cholesterol causes an ordering of the disordered fluid phase and therefore increas- ing amounts of cholesterol eventually lead to an elimination of the phase transition (95). Consequently, liposomes con- taining cholesterol are more cohesive and have high stability against proteins in vivo and against leakage of encapsulated materials (98). Addition of cholesterol to conventional lipo- some formulations increases their stability in the plasma (7,98).

C for dioleoyl chains to 55

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5.2. Sterically Stabilized Liposomes Sterically stabilized liposomes constitute an important class of

second generation liposomal vesicles that are engineered to have extended circulation times in vivo compared to conven- tional liposomes (99–101). Due to their ability to circumvent immune surveillance and recognition by the body as foreign and hence avoid opsonization and phagocytic uptake, steri- cally stabilized liposomes are also popularly known as stealth liposomes (99,101,102). Stealth liposomes are composed of lipids that have covalently linked polymers with hydrophilic head groups such as poly(ethylene glycol) (PEG) on their sur- face (100). The process of PEG conjugation to conventional lipids is known as pegylation and the lipids with covalently attached PEG can be included in the formulation at a desired ratio. Pegylation prevents the opsonization of proteins on the surface of stealth liposomal vesicles and prevents phagocytic uptake by the reticuloendothelial system, thus leading to their long circulating times in the systemic circulation (101,103). Commercially approved Doxil is a pegylated liposomal pro- duct, with surface grafted segments of the hydrophilic polymer methoxypolyethylene glycol (MPEG) (104). The MPEG seg- ments extend from the surface of the liposomes, reducing interactions between the lipid bilayer membrane and plasma components (103). The reader is referred to the case study on Doxil by Martin et al. in this book for more information. In addition to surface grafting with hydrophilic polymers, such other molecules as ganglioside and phosphatidylinositol have also been used to have a stealth effect (7,100). Addition of specific immunorecognition motifs such as integrin antibody segments on stealth liposomes can couple the advantages of cell targeting and improved circulation times, respectively (105).

5.3. Liposomes for Gene Delivery Liposomes for gene delivery are typically composed of combina-

tions of cationic and zwitterionic lipids (42,106–108). Cationic lipids commonly used are 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)

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ethyl]-NN-dimethyl-1-propanaminium (DOSPA), 3,[N-(N 1 N- dimethylethylenediamine)- carbamoyl]cholesterol (DC-chol),

N-[1-[2,3-dioleyloxy]propyl]-NNN-trimethylammonium chlor- ide (DOTMA), and dioctadecyl amido glycil spermine (DOGS) (109). Commonly used zwitterionic lipids, also known as helper lipids, are 1,2-dioleoyl-sn-glycero-3-phosphoethanola- mine (DOPE) and cholesterol (109).

Cationic liposomes upon electrostatic attraction with the anionic DNA backbone form a cationic complex also known as

a lipoplex that is capable of transferring DNA molecules into cells by a process known as transfection (110). The cationic lipids in the formulation facilitate DNA complexation and condensation in the lipoplex (111,112). The zwitterionic lipids help in membrane perturbation and fusion. The overall posi- tive charge of the lipoplex facilitates cellular association and transfection (113). Excess cationic lipids also help to stabilize the liposomes in vivo and prevent release of DNA by anionic molecules in the serum.

Liposomal gene delivery vectors are believed to achieve transfection through the following sequence of events (114–116): (i) interaction with the cell membrane; (ii) receptor mediated endocytosis; (iii) release from the endosome into the cytoplasm, usually through destabilization and disruption of the endosome membrane; and (iv) uptake from the cytoplasm into the cell nucleus. X-ray diffraction studies have also indi- cated that cationic lipoplexes are successful in transfection

because of the formation of the H c II (hexagonal) phase instead of the La (lamellar) phase that is typically observed in liposo- mal bilayers (114). Formation of the hexagonal phase is attributed to due to the small less hydrated inverted-cone shape of the DOPE molecule (114,117).

The first use of cationic liposomes for gene delivery was demonstrated by Felgner et al. (110) when they successfully introduced a plasmid DNA encoding the chloramphenicol acet- yltransferase enzyme into mammalian cells using cationic lipo- somes composed of DOTMA. Since then numerous synthetic cationic lipids and their formulations have been successfully used for gene delivery in a wide range of cell culture and animal models (118–120). Some of the commercially available cationic

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liposomal formulations used for in vitro gene delivery include:

LipofectAmine Õ (Invitrogen, Carlsbad, CA, USA); Effectene (Qiagen, Valencia, CA, USA); and Tranfectam Õ (Promega, Madison, WI, USA) (119,121).

The human in vivo gene therapy trial for melanoma conducted by Nabel et al. (44,122). used cationic liposomes com- posed of cationic DC-chol and zwitterionic DOPE to transfer a gene encoding the foreign major histocompatibility complex protein, HLA-B7 into cancer nodules. The clinical trial demon- strated the feasibility of cationic liposomes for gene delivery in humans. Currently, cationic liposomes have progressed into clinical trials for several indications that include cystic fibrosis (123), metastatic head and neck carcinoma (124), breast cancer (124), and ovarian cancer (125). There is, however, no commer- cially approved cationic liposomal product on the market.

Despite this progress, cationic liposomes suffer from sev- eral undesirable issues that reduce their overall potential of DNA delivery. These include inactivation in the presence of serum, instability upon storage (109), and cytotoxic effects on cells, both in vitro (126,127) and in vivo (128–132). Cytotoxicity of cationic lipids has been demonstrated in a variety of cell types including phagocytic macrophages (127), pulmonary intratra- cheal tissue (128,129,132), and arterial cell walls (130). Toxicity is attributed to the production of reactive oxygen intermediates (128), induction of apoptosis (133), or stimulation of proinflam- matory cytokines (129) in response to the administration of cationic lipids. It is evident that there is a need for efficient and well-tolerated delivery systems to exploit the benefits of gene medicine. As a non-toxic alternative to cationic lipids, anionic liposomal formulations for the delivery of DNA-based therapeutics have been recently developed (27,28,134,135). The endogenous negative charge of these naturally occurring lipids is thought to be responsible for their low toxicity (27,28).

5.4. Lipid Specifications Lipids in liposomal formulations can be synthetic, semi-

synthetic, or derived from natural sources such as egg yolk or soybeans (136). For FDA approval of liposomal

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formulations, strict control of lipid excipients is mandated and specific information is required to be submitted prior to product approval (7,136). Natural lipids contain a mixture of lipid chains with different head groups, whereas synthetic lipids can be pure. For completely synthetic products, the source and process specifications should be supplied (7). For- mulations comprised of mixtures of natural lipids or natural starting products for semi-synthetic lipids are required to spe- cify individual lipid composition, degree of saturation, and relative percentages of fatty acids. Lipids if obtained from genetically modified plant and animal sources have also to

be indicated. In addition, lipids in human formulations are also mandated to be free of contamination from animal pro- teins and viruses. Typically lipids and their impurities (syn- thetic by-products if applicable and =or degradants) can be identified using spectroscopic techniques, which can be used to determine the acceptance criteria for starting materials (2,136). Some of the quality-determining specifications are adapted from the egg yolk phospholipid monograph (7).

6. MANUFACTURE OF LIPOSOMES As their clinical potential for diverse drug delivery applica-

tions has begun to be realized, the last few decades have witnessed the development of a large number of techniques for the manufacture of liposomal formulations (3,137–139). Early protocols were suitable for small laboratory scale lipo- some production; however, newer protocols are more sophisti- cated and amenable to expedited large-scale industrial manufacture and processing under cGMP conditions (137). The selection of a particular protocol is primarily dictated by the nature of the therapeutic in the liposomal formulation and should ensure preservation of its stability and biological activity during processing. Protocols that necessitate prolonged exposure to organic solvents or high temperature are unsuitable for protein therapeutics. In addition, the pro- duction method should maximize drug entrapment in liposo- mal vesicles. The following are some of the commonly used methods for the preparation of liposomes:

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6.1. Liposomes Preparation from Lipid Films Preparation of liposomes by hydration and agitation of lipid

films is one of the oldest and most widely used laboratory scale methods. This method exploits the natural self-assembly process of bilayer membranes and involves the formation of MLV from dried lipid films upon their exposure to an aqueous medium (6,92). This is usually achieved by the dissolution of lipids (in the desired ratio) in an organic solvent such as chloroform followed by its complete evaporation which leads to the deposition of a thin lipid film (7,140). Evaporation of the organic solvent can be assisted using a steady stream of nitrogen gas over the lipid surface (7). Use of inert nitrogen prevents oxidation of lipids and prevents chemical instability (for details refer Sec. 8.3 ). The resulting dried film of lipids is then dispersed in a solution of the material to be encapsu- lated. As the lipids hydrate, they assemble and form a suspen- sion of MLV. Mechanical agitation and sonication during hydration can assist the formation of MLV from lipid films (7). However, sonication produces unstable SUV that are sus- ceptible to physical degradation-related fusion (3). Drug entrapment in liposomal MLV is dependent on the volume of their enclosed aqueous compartments. The trapped aqueous volume of MLV is very small ( <1 mL=mmol lipid), thereby redu- cing the entrapping efficiency (140). Liposome preparation from lipid films yields large polydisperse vesicles and typically MLV are further treated to achieve vesicles with desired and consistent properties (6,92).

6.2. Liposome Preparation by Freeze–Thaw Cycling of MLV

To improve the drug entrapment efficiencies, frozen and thawed multilamellar vesicles (FATMLV) were developed by Mayer et al. (141,142). FATMLV are generated from MLV by repeated freeze–thaw cycling of MLV. This procedure involves rapid freez- ing of MLV suspensions using liquid nitrogen followed by thaw- ing at 40

C. Microscopic investigations have revealed that subjecting MLV to freeze–thaw cycling leads to breakdown of the characteristic concentric lamellae of MLV. Although the

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exact mechanism still remains unknown, formation of ice crys- tals is speculated to be a contributing factor to MLV disruption. It is interesting to note that the average size ( >1 mm) and over all size distribution (high polydispersity index) of FATMLV remain similar to those of MLV (140). The choice of lipids and their concentration along with the nature of the drug to be encap- sulated in the formulation determine the entrapment efficiencies of FATMLV (141,142). Typical drug entrapment efficiencies of FATMLV are higher than those of MLV and can range from 2 to 17 mL =mmol lipid (140).

6.3. Liposome Preparation by Extrusion Techniques

Though FATMLV improve the entrapment efficiency of drugs in liposomes, such liposomes are large in size and generate non-homogenous suspensions (7,143). Extrusion techniques have been used to produce SUV from MLV (143). These tech- niques typically involve passage of an MLV suspension through polycarbonate membranes or filters of definite size. Typically, smaller size vesicles are obtained by the sequential passage of the MLV suspension through a series of progres- sively smaller pore size filters. In addition to yielding lipo- somes with homogenous populations, extrusion techniques can also handle higher lipid concentrations, as high as 400 mg =mL lipids. The entrapped volumes of liposomes generated by the extrusion technique range from 1 to 3 =mmol lipid and are higher than those of conventional MLV (7,138,140). Extrusion techniques can easily be adapted to industrial production and can be compliant to cGMPs and other regulatory requirements (7).

6.4. Liposome Preparation by Dehydration/ Rehydration

In this technique, SUVs and the solute to be entrapped are dispersed in buffer and the solution is frozen and dehydrated by the passage of liquid nitrogen till complete evaporation of the aqueous medium takes place (74). The dried film of lipids and solutes is then reconstituted by rehydration with the

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necessary buffer. Upon rehydration of this solid mixture MLV are produced. These MLV can be further subjected to size reduction using microfluidization or sequential passage through membranes. Liposomes prepared by the dehydra- tion =rehydration technique have been used in the production of recombinant protein vaccines due to high entrapment effi- ciencies (74). Antigens have also been entrapped using such techniques; however, instead of SUVs, giant vesicles that can incorporate larger particulates have been used in the original freeze-drying step (74).

6.5. Liposome Preparation by Reverse Phase Evaporation

Preparation of liposomes using reverse phase evaporation technique involves the introduction of an aqueous medium (buffer) containing the solute to be entrapped into a solution of lipids in an organic solvent (7,138,144). The two-phase sys- tem is sonicated to form a temporary unstable emulsion. The organic solvent is eventually removed by evaporation under reduced pressure. The resultant suspension of lipids can also

be processed further as discussed in Sec. 6.3 till the vesicles are in the desired size range and with similar lamellar char- acteristics. The volume of the aqueous component of the initial emulsion and the concentration of the lipids are some of the factors that affect the characteristics of vesicles pre- pared by reverse phase evaporation (145). In general, since these vesicles have substantially larger entrapped internal aqueous volumes they have significantly higher entrapment efficiencies compared to MLV (7,138,144).

Liposomes generated by reverse phase evaporation can

be subclassified into two types based on modifications to the evaporation process made in the general method: stable plurilamellar vesicles (SPLV) (146) and multilayered vesicles prepared by the reverse-phase evaporation method (MLV- REV) (145,147). SPLV are prepared by simultaneous sonica- tion and concurrent evaporation of the initial emulsion, whereas MLV-REV does not involve the sonication process. MLV-REV have uniformly dispersed homogenous lamellae

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and higher entrapment efficiencies compared to SPLV (145,147).

6.6. Preparation of Liposomes Based on Lipid–Alcohol–Water Injection Technology

This technique of liposome preparation involves the injection of alcoholic solutions of lipids into aqueous media (6,92,148). The lipids undergo precipitation in the form of polydisperse unila- mellar vesicles. The size and polydispersity is affected by lipid concentration, relative percentage of alcohol to the aqueous phase as well as the dilution effect (148). Newer methods utiliz- ing the same general principles of alcohol injection technology with minor modifications have been recently developed to yield SUVs with greater homogeneity (6,92,149). One such method involves introduction of the aqueous phase into the lipid etha- nol solution followed by complete evaporation of ethanol using evaporation (149). First described by Batzri and Korn (150), the ethanol injection technique has been developed for entrap- ment of pharmaceutical proteins (151) and used for the com- mercial manufacture of Pevaryl Lipogel Õ , the first approved dermatological liposomal formulation (51,152).

6.7. Liposome Preparation Using Detergent Dialysis

Developed by Weder et al. (153) for topical liposomal formula- tions, detergent dialysis technique is similar to the alcohol injection technology in terms of general principles of facili- tated lipid solubilization. Instead of using alcohol, this techni- que accomplishes lipid solubilization in the form of mixed micelles in aqueous media using detergents (80,153). The detergents are then removed by dialysis, which leads to the disruption of the mixed micelles and the solubility of the phospholipids is lowered in the aqueous medium. Conse- quently, the mixed micelles are converted into liposomes. Commonly used detergents in this method of liposome manu- facture include bile salts such as sodium cholate, sodium taurocholate, and sodium deoxycholate, and other ionic and non-ionic tensides such as sodium dodecyl sulfate and dodecyl

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maltoside (154). This technique has been used in the prepara- tion of protein liposomes (155) and stabilized plasmid-lipid particles (156). However, detergent dialysis techniques are protracted and incomplete removal of the residual detergent may compromise liposome stability (3).

6.8. Freeze-Drying of Liposomes Though lyophilization or freeze-drying itself cannot generate

liposomes, it is included as a manufacturing process since liposomes prepared by any method described above can be converted into dry solid formulations using this technique. It can be appended as a continuation to any manufacturing process after liposomes with desired characteristics have been produced. Lyophilization of liposomes is one of the best ways to circumvent many of the stability problems associated with liquid liposome suspensions (for details refer Sec. 8.3 ) (157,158). Lyophilization involves three major processes: (i) freezing; (ii) primary drying; and (iii) secondary drying (157,158). The freezing process involves cooling of the lipo- some suspension at very low temperatures such that the water component of the liposomal suspension is frozen into solid ice and the viscosity of the suspension is significantly reduced by the formation of an amorphous glass. The frozen matrix is then subjected to the second phase of primary dry- ing. Primary drying involves removal of ice by sublimation under high vacuum and low temperature. At the end of the primary drying process, the frozen matrix is converted into

a freeze-dried porous cake. This resulting porous cake is then brought to shelf temperature (usually 25

C) and subjected to secondary drying to facilitate complete removal of water in the formulation. The headspace in the vials is replaced by nitrogen to minimize phospholipid oxidation. The process thus yields an elegant dry formulation that is recon- stituted with a recommended buffer prior to admin- istration (157,158).

It should be noted that during the lyophilization process, the liposomal bilayer structure may be disrupted or punctured due to the temperature stresses generated or due to the ice

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crystals formed during the initial freezing phase. This damage may lead to leakage of entrapped components, liposome fusion, and aggregation. Freeze-drying may also affect the sta- bility of some of the sensitive entrapped molecules such as pro- teins. To minimize this structural damage, lyophilization is conducted in the presence of cryoprotectants such as sorbitol, mannitol, trehalose, lactose, and sucrose (158–160). Cryopro- tectants decrease vesicle fusion and aggregation and improve liposome stability by forming a low mobility amorphous glass surrounding the vesicles during the freezing phase as well as due to interactions between them and the phospholipid head groups during the freezing cycle (161,162). The temperature of formation of this amorphous glass is characteristic of each cryoprotectant and is known as the glass transition tempera-

ture (T g ). From a regulatory perspective, commonly used sac- charide-based cryoprotectants currently qualify as generally recognized as safe (GRAS) food ingredients and thus can easily

be incorporated into parenteral formulations.