Issues Associated with Large-Scale Production of Liposomal Formulations

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Issues Associated with Large-Scale Production of Liposomal Formulations
By: Tony Nakhla, PhD; Mike Marek, MS, RPh; & Tom Kovalcik, PhD
Drug Delivery Technology
INTRODUCTION
It was reported more than 40 years ago that when phospholipids are subjected to aqueous environments, closed bilayer structures (liposomes) spontaneously form that can encapsulate part of the aqueous medium in their interior.1
Initially, liposomes were attractive to biophysicists as model systems for biological membranes. The lipid bilayer structures of liposomes mimic the barrier properties of biomembranes, and therefore, they offered the potential of examining the behavior of membranes of known composition. Thus, by altering the lipid composition of the bilayer or the material incorporated, it was possible to establish differences in membrane properties. Model membranes have been used extensively to study lipid-protein interactions, in addition to membrane function and structural properties.2-6
Liposomes can be formulated and processed to differ in size, composition, charge, and lamellarity and accordingly, a wide range of compounds may be incorporated into either the lipid or trapped aqueous space. Such flexibility has presented several potential applications to scientific investigators to which liposomes have since been adapted.7-10
The biodegradable and non-toxic nature of phospholipid vesicles proposes that these formulations are amenable to administration without serious side effects. As a result, liposomes are frequently used as drug delivery vehicles. Further, liposomes are regarded as suitable carriers because they can serve as a depot system for the sustained release of an associated compound.
One of the basic goals of chemical therapeutics is to deliver the drug efficiently and specifically to the site of disease. Some drugs may be delivered in their free form whereas others require a carrier in order to reach and enter their final destination because a) they are rapidly cleared from the area of introduction or the circulation or b) they are obstructed by biological barriers, which they cannot permeate. Liposomes can alter the biodistribution of entrapped substances and protect the enclosed materials from inactivation by the host defense mechanisms.11 Therefore, liposomes can be used as vehicles to achieve specific delivery of therapeutic drugs to target organs. In addition, liposomes can reduce toxicity of antimicrobial12, antiviral13, and chemotherapeutic7-9,14 agents, and they have demonstrated the ability to modulate or potentiate the immunogenicity of antigenic substances, that is, function as immunological adjuvants.15,16 Accordingly, there has been a myriad of drugs and antigens incorporated into liposomes to achieve those objectives. More recently, liposomes have been demonstrated to be efficient vehicles for gene therapy.10, 17, 18
SIZE REDUCTION
With the many potential uses presented by these model membranes, the therapeutic applications of liposomes are dependent on the physical integrity and stability of the lipid bilayer structure. Lipid-based formulations can be devised as site-specific drug delivery vehicles that a) are readily cleared by the Kupffer cells of the liver and the macrophages19,20 or b) evade detection of the active substance by the reticuloendothelial system (RES) and efficiently deliver liposome-incorporated material to target tissue, organ or tumor.21,22
There are numerous techniques for liposome preparation, and the resulting vesicles may be large, small, and of unilamellar or multilamellar nature.23 Multilamellar vesicles (MLV), composed of numerous concentric bilayers, are produced from mechanical agitation of a dispersion of dried lipid with an aqueous phase. Mechanical agitation is the simplest method for production of MLV that produces a suspension of large liposomes that are very heterogeneous in size and exhibit a relatively low level of aqueous encapsulation. In addition, these heterogenous liposome formulations manifest relatively short circulation half-lives in mammals, are cleared rapidly from sites of administration, and are distributed within the RES. However, homogenous liposome formulations that exhibit reduced vesicle diameters are advantageous with respect to extended circulation half-life, and consequently, enhanced uptake by tissues and organs. These parameters are critical to the in vivo behavior of liposomal drug delivery systems. Large unilamellar vesicles (LUV) can be prepared from MLV to exhibit the characteristics that are beneficial for enhanced delivery of the incorporated material. The most common method for LUV preparation is extrusion of MLV under pressure through membranes of known pore sizes.24 These LUVs are utilized to optimize the incorporation of a desired compound within liposomes, to limit the permeability of the membrane to the entrapped material, and to alter half-life in circulation in attempts to enhance the therapeutic efficiency of a liposomal formulation. Accordingly, size distribution is a critical parameter with respect to the pharmacokinetic and pharmacodynamic behavior of biologically active substances that are site-specifically targeted in vivo.
STABILIZATION OF LIPOSOMES
The stability of liposomes should meet the same standards as conventional pharmaceuticals. It is imperative that the chemical and physical stability of the vesicles in question are maintained. Chemical stability involves prevention of both the hydrolysis of ester bonds in the phospholipid bilayer and the oxidation of unsaturated sites in the lipid chain. Chemical instability can lead to physical instability or leakage of encapsulated drug from the bilayer and fusion and aggregation of vesicles. Approaches that can be taken to increase liposome stability involve efficient formulation and lyophilization. Formulation involves the selection of the appropriate lipid composition and concentration of the bilayer, in addition to the aqueous phase ingredients, such as buffers, antioxidants, metal chelators, and cryoprotectants. Charge-inducing lipids, such as phosphatidylglycerol can be incorporated into the liposome bilayer to decrease fusion, while cholesterol and sphingomyelin can be included in formulations in order to decrease permeability and leakage of encapsulated drugs. Buffers at neutral pH can decrease hydrolysis. If a low pH buffer is necessary during processing, for example, in active loading of drug, the low pH buffer should be readjusted to neutral pH or exchanged by ultrafiltration. Addition of an antioxidant, such as sodium ascorbate can decrease oxidation. Oxygen potential can be kept to a minimum during processing by nitrogen purging solutions whereas ethylendiamine tetraacetic acid (EDTA) can be added as a metal chelator to decrease free radical damage. High temperature and excessive shear that may be encountered during processing can be avoided by sizing lipids with an extrusion device rather than high-pressure homogenization. Freeze-dried liposome formulations should include a lyoprotectant, preferably a non-reducing disaccharide, such as trehalose and sucrose, which have been shown to stabilize liposomes during freezing and dehydration/rehydration. In general, successful formulation of stable liposome drug products includes the following25:
1. Processing with fresh, purified lipids and solvents.
2. Avoidance of high temperatures and excessive shear force.
3. Maintenance of low oxygen potential (nitrogen purging).
4. Use of antioxidants and/or metal chelators.
5. Formulating at neutral pH.
6. Use of a lyoprotectant when freeze-drying.
FREEZE-DRYING OF LIPOSOMES
Freeze-drying involves freezing of the product then removal of water at low temperature and high vacuum via sublimation of ice. This part of the freeze-drying cycle is referred to as primary drying. Adsorbed water is further removed at above freezing temperatures and low vacuum during secondary drying, resulting in the freeze-dried product. In general, moisture levels of freeze-dried products are designed to be less than 3%. A typical freeze-drying cycle is shown in Figure 1.26 Liposome bilayer membranes may be damaged during the freeze-drying cycle both by mechanical stress caused by high pressures vesicle membranes are exposed to during ice crystal formation and chemically from increased concentrations of solute during freezing and dehydration. Cryoprotectants have been shown to decrease vesicle fusion and leakage caused by both freeze-thaw and the freeze-drying process. Cryoprotectants are noneutectic in nature, that is they do not crystallize, forming an amorphous frozen matrix upon cooling. The freeze process generally occurs very quickly in the presence of cryoprotectants upon cooling to the freezing point depression. This effect is known as supercooling. Supercooling decreases the vesicle exposure to high concentrations of solute caused by slow ice crystallization and results in a more uniform frozen matrix. Solutes that act as cryoprotectants typically undergo a transition upon freezing from a viscous gel to a hard glass with less molecular mobility. This is referred to as the glass transition temperature (Tg?). Glass transition temperatures of various cryoprotectants are listed in Table 1.27 Critical to successful freeze-drying is that the product temperature remain below Tg? during primary drying to avoid ?shrinkage? or product collapse. Disaccharides have collapse temperatures between ?30?C and -35?C. The use of cryoprotectants with high collapse temperatures allows for faster rates of freeze-drying at higher product temperatures. The use of the non-reducing disaccharide sugars sucrose and trehaloses have been shown effective in decreasing physical damage of liposomes during freezing and freeze-drying. This protective effect appears to be sugar specific as monosaccharides glucose and fructose have less effect.28,29 Disaccharides appear to interact directly with