LIPOSOME DRUG ENCAPSULATION TECHNIQUES

7. LIPOSOME DRUG ENCAPSULATION TECHNIQUES

The physicochemical characteristics of the drug as well as those of the lipids used determine drug loading into liposomal vesicles (6,7,92,163). Some of the commonly used techniques for loading of liposomal vesicles are passive and active encapsulation and complexation. The choice of the entrap- ment process is determined by the nature of the drug as well as that of the lipids (6,92,164). Water soluble drugs can be easily entrapped within the aqueous compartment of SUV or within the interlamellar spaces of MLV (6,7,92). Hydropho- bic drugs, primarily as a result of their affinity towards lipids, associate with the hydrocarbon chains. DNA-based therapeu- tics such as oligonucleotides and plasmids, due to their anio- nic charge, complex with cationic liposomes predominantly due to electrostatic interactions (111,112). Entrapment of DNA-based therapeutics into anionic liposomes is enhanced

using divalent cations (e.g., Ca 2 þ ) or polycations (27,28). Such cations can facilitate electrostatic interactions between

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negatively charged liposomes and DNA-based therapeutics (27,28,165).

On the basis of their interaction with the liposome bilayer, entrapment drug candidates can be classified into three major types (92,163):

1. Drugs with low oil =water and low octanol=water partition coefficients;

2. Drugs with low oil =water partition coefficients but high or variable octanol =water coefficients;

3. Drugs with high oil =water and high octanol=water partition coefficients.

Drugs in the first class are typically hydrophilic and freely water soluble, due to which they can be encapsulated into liposomes using passive encapsulation strategies (7,92). Class 2 drugs are usually amphiphilic whose membrane per- meability is dependent on the pH in the aqueous medium; thus they can easily be encapsulated into liposomes using active encapsulation techniques (7,92). Class 3 drugs are hydrophobic in nature and tend to strongly associate with lipid bilayers. Class 3 drugs are unsuitable for encapsulation into liposomes since they can phase separate easily, due to which such drug compounds are delivered using oil-in-water emulsions (7,92).

7.1. Passive Encapsulation Passive entrapment of drugs in liposomes involves preferential

partitioning of the drug either in the aqueous compartment or by association with the lipids (7). Passive entrapment of drug molecules in MLV typically takes place when these vesicles are formed in aqueous solutions of drugs (6,7,92). Passive entrapment in SUV is also facilitated during their extrusion and sequential passage through filters. Retention of drug inside liposomes is typically low and is determined by membrane per- meability, the stronger the membrane association the better the drug retention in the vesicles. For example, drugs such as meth- otrexate and hydroxyzine tend to remain in the liposomes for a long time due to their strong association with zwitterionic mem- bers, whereas, charged drugs such as adriamycin typically do not

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interact with the lipids and therefore are released rapidly (7). Membrane association and thus passive entrapment of drugs can be improved by synthesis of their lipophilic derivatives that have a higher oil =water partition coefficient. This deri- vatization approach has been used to improve the passive entrapment efficiency of 6-mercaptopurine from 1.92% for the parent drug to 91.8% for the lipophilic derivative (gly- ceryl monostearate drug-conjugate) (86); and that of triamci- nolone from 5% for the parent drug to 85% for triamcinolone acetonide 21-palmitate (166). The commercially available product AmBisome has been developed using passive loading technologies (163).

7.2. Active Encapsulation Active loading of liposomal vesicles was pioneered by Cullis

et al. (3,6,167). This method is based on the pH-dependent dif- ferential membrane permeability of ionization states of drugs (3). Active loading consists of initial suspension of empty lipo- somes with a pH gradient with respect to the external aqueous medium containing the drug of interest (168,169) and the

entrapped aqueous core. Depending on the pK a of the drug, the external pH is manipulated so the drug exists in a predomi- nantly non-ionized state. In response to the concentration gra- dient of the drug, which is developed across the bilayer membrane of the liposome, the non-ionized species is trans- ported to the internal aqueous space of the liposome. However, the internal pH of the liposomes is maintained at a value so that the drug is reverted to its ionized state and reverse flux of the drug into the medium is prevented. pH gradients across membranes can also be generated using ammonium sulfate to facilitate active loading (6,170). Active loading is suitable for amphiphilic weak acids or weak bases and can be used to have very high-loading efficiencies compared to passive encapsula- tion techniques. Active loading can also be used for liposomal entrapment of metal ions (6). This variation of the active load- ing strategy involves lipophilic carrier-mediated transmem- brane transport of metal ions into aqueous cores of vesicles that contain metal ion chelators. After being transported into

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the aqueous core, chelation prevents the reverse flux of the metal ions (6). The commercially available products Doxil, Myo- cet, and DaunoXome have been developed using active loading technologies (171). The reader is referred to the case study in this book on the development of AmBisome by Adler-Moore.

7.3. Drug Complexation Drug entrapment in liposomes by complexation with their

surface is based on the electrostatic interaction of the drug and the lipid component in the formulation. Due to the char- acteristic nature of this interaction and association of the drug with the lipids compared to classical drug entrapment in the aqueous core of liposomes; these products are termed drug-lipid complexes (26–28). Although many of the currently developed formulations for small molecular weight drug can- didates are based on drug loading into the liposomes, one of the original commercially licensed liposomal drug product Abelcet is a small molecular weight drug (amphotericin B)– lipid complex (15). The Abelcet formulation is amphotericin

B interdigitated and complexed with lipids in a 1:1 drug to lipid ratio. The lipid component in the formulation is com- posed of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in a 7:3 molar ratio. The formulation is based on the strong binding of this complex until drug release in the fungal cytoplasm.

Currently, drug complexation for loading drug into lipo- somes is most commonly used for cationic liposomes intended to deliver DNA-based therapeutics (111,112,165). Cationic liposomes interact with the anionic backbone of DNA-based therapeutics to form complexes as a result of electrostatic

attraction (for details, refer Sec. 5.3 ). Complexation of DNA-based therapeutics on liposome surfaces protects them from nuclease degradation and can also facilitate their entry into cells (172). Cationic lipids have been used to deliver a wide range of DNA-based therapeutics such as oligonucleotides and gene therapy plasmids in cell culture and animal models (118). Several cationic liposomal formula- tions of DNA-based therapeutics are currently in human clinical trials (120,173).

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