Chapter one:Site-specific drug delivery using liposomes as carriers 7 packing of phospholipids in bilayers above the Tc.Cholesterol also reduces the of the bilayers ompounds Negatively charged lipids,such as stearylamine,are usually used in order to provide a surface charge to the liposomes.For drug molecules encapsulated in the aqueous space,the bilayer serves as a diffusion barrier permitting the liposomes to serve as a rate-controlling input device.Papah- tics to clinical applicationsThe introduction of this delivery system ery syst directly to the target site(such as the eye,lung,or bladder)is a well-estab- lished approach for treating local diseases,and liposomes have been shown to play a beneficial role when applied in this way. III.The liposome-drug concept Liposome size Liposomes have been used via a variety of administration routes,including intravenous,intramuscular,intraperitoneal,and oral.30-32 However,IV injec- tion is the most widely utilized route.The half-life of liposomes in the vascular system can range from a few minutes to many hours,depending (0.1 to 1.0 um)are taker up preferentially by cells of the reticuloendothelial system (RES),located principally in the liver and spleen,3 whereas liposomes larger than 3.0 um are deposited in the lungs.3 This preferential uptake of smaller-size lipo- somes by cells of the RES system has been utilized to deliver chemothera- peutic acrophages and tumors of the iver. e physical approaches based upon the ability to destabilize the liposome bilayer have led to the design of heat-sensitive,light-sensitive, and pH-sensitive liposomes.35- Targeting ligands chemical approach to achievingeiveryqures that ome hasa .variety of targeting ligand been proposed for this purpose,including antitumor monoclonal antibodies (MAb),carbohydrates,vitamins,and transport proteins.3 Only carbohydrate and MAb-modified liposomes have thus far shown promise in achieving targeting specificity. sful targeting of liposomes to cells other than those belo the RESis fairly re ricted,but appears to incude hepatocytes and n red blood cells.3 A high degree of specific liposome-cell association has been obtained in vitro by coating the vesicles with cell-specific ligands,such as MAbs or F(ab)fragments (see Figure 1.3).40-2
Chapter one: Site-specific drug delivery using liposomes as carriers 7 packing of phospholipids in bilayers above the Tc. Cholesterol also reduces the permeability of the bilayers to encapsulated compounds. Negatively charged lipids, such as stearylamine, are usually used in order to provide a surface charge to the liposomes. For drug molecules encapsulated in the aqueous space, the bilayer serves as a diffusion barrier, permitting the liposomes to serve as a rate-controlling input device. Papahadjopoulos and co-workers have done pioneering research in trying to establish and develop the liposomal delivery system from experimental therapeutics to clinical applications.25–29 The introduction of this delivery system directly to the target site (such as the eye, lung, or bladder) is a well-established approach for treating local diseases, and liposomes have been shown to play a beneficial role when applied in this way. III. The liposome-drug concept A. Liposome size Liposomes have been used via a variety of administration routes, including intravenous, intramuscular, intraperitoneal, and oral.30–32 However, IV injection is the most widely utilized route. The half-life of liposomes in the vascular system can range from a few minutes to many hours, depending on the size and lipid composition of the vesicles. Following IV administration, small liposomes (0.1 to 1.0 mm) are taken up preferentially by cells of the reticuloendothelial system (RES), located principally in the liver and spleen,33 whereas liposomes larger than 3.0 mm are deposited in the lungs.34 This preferential uptake of smaller-size liposomes by cells of the RES system has been utilized to deliver chemotherapeutic agents to macrophages and tumors of the liver.14 Alternative physical approaches based upon the ability to destabilize the liposome bilayer have led to the design of heat-sensitive, light-sensitive, and pH-sensitive liposomes.35–37 B. Targeting ligands The chemical approach to achieving site-specific delivery requires that the liposome has a targeting ligand bound to its surface, thereby enabling it to attach preferentially to the target site. A variety of targeting ligands have been proposed for this purpose, including antitumor monoclonal antibodies (MAb), carbohydrates, vitamins, and transport proteins.38 Only carbohydrate and MAb-modified liposomes have thus far shown promise in achieving targeting specificity. Successful targeting of liposomes to cells other than those belonging to the RES is fairly restricted, but appears to include hepatocytes and circulating red blood cells.39 A high degree of specific liposome-cell association has been obtained in vitro by coating the vesicles with cell-specific ligands, such as MAbs or F(ab1)2 fragments (see Figure 1.3).40–42
Drug delivery systems,second edition D-O&≥ F(ab -PE V Fab'-Vesicle re13 lustration of the chemical-coupng methodologyforniy The La atest Dev lopments C.Problems In,the obstacles to successful targeting that have to be overcome are substant ial.First,the liposomes have to escape nonspecific earance by the RES cells.Second,the vesicles have to cross the capillary endothelium and the basement membrane.Third,many cell types,including most tumor cells, display a low endocytotic capacity.Since it has been found that endocytosis is the dominant mechanism of liposome-cell interaction,this is a serious lim- itation to the delivery system. cells by virtue of their capacity to penetrate the liver's fenestrated endothe- lium.Once taken up by the cells,liposomes may be degraded in the lysos- omal compartment.Liposome-encapsulated drugs,when resistant to the intralysosomal environment,may slowly leak out of the lysosomes into the cytos and ma y be come available to exert their therapeutic c action.Drugs may also be released from liposomes phagocytized by macrophages. Another important aspect of the liposome-drug relationship involves reducing toxicity of the liposome-encapsulated agent.This is particularly important for antineoplastic agents with low therapeutic indices,such as adriamycin or antimicrobial drugs like amphotericin B.-45 D Manufacturing issues Liposomes are phospholipid vesicles composed of one or more phospholipid bilaver membranes and they carry aqueous or lipid lipids ard both hydrophobic and lipop hilic in aqueous d regions sequestrate into spherical bilayers.These layers are referred to as lamellae.Liposomes vary in charge and their size,depending on the method of preparation and the lipids used
8 Drug delivery systems, second edition C. Problems In vivo, the obstacles to successful targeting that have to be overcome are substantial. First, the liposomes have to escape nonspecific clearance by the RES cells. Second, the vesicles have to cross the capillary endothelium and the basement membrane. Third, many cell types, including most tumor cells, display a low endocytotic capacity. Since it has been found that endocytosis is the dominant mechanism of liposome-cell interaction, this is a serious limitation to the successful application of liposomes as a drug delivery system.14 Small-size liposomes may serve as drug carriers to liver parenchymal cells by virtue of their capacity to penetrate the liver’s fenestrated endothelium. Once taken up by the cells, liposomes may be degraded in the lysosomal compartment. Liposome-encapsulated drugs, when resistant to the intralysosomal environment, may slowly leak out of the lysosomes into the cytosol and may become available to exert their therapeutic action. Drugs may also be released from liposomes phagocytized by macrophages. Another important aspect of the liposome-drug relationship involves reducing toxicity of the liposome-encapsulated agent. This is particularly important for antineoplastic agents with low therapeutic indices, such as adriamycin or antimicrobial drugs like amphotericin B.43–45 D. Manufacturing issues Liposomes are phospholipid vesicles composed of one or more phospholipid bilayer membranes and they carry aqueous or lipid drugs. The lipids are both hydrophobic and lipophilic in aqueous media, and their hydrophobic regions sequestrate into spherical bilayers. These layers are referred to as lamellae. Liposomes vary in charge and their size, depending on the method of preparation and the lipids used. Figure 1.3 Illustration of the chemical-coupling methodology for antibody/liposomes. (From Pharmaceutical Technology, Conf. Proc., The Latest Developments in Drug Delivery Systems, Oct. 1985. With permission.)
Chapter one:Site-specific drug delivery using liposomes as carriers 9 Drug entrapped in carrie Lysosome D Fusion and the carrier Endocytosis Endocytic vesicles Macrophage Figure 1.4 Schematic of phagocytosis of particulate carriers by macrophages.Macro by the pro sis.Drugs are elease 940.202 Two major methods are usually used to make liposomal systems for drug incorporation.The first method deals with hydration ofalipid followe by high-intensity agitation using sonication or a high-shear propeller.Lipo- somes are subsequently sized by filtration or extrusion.In the second method,a phospholipid is first dissolved in an organic solvent and then added to an aqueous medium by vigorous agitation.The organic solvent is ved unde vacuum,and the resulting mal dis or emulsior lamellae (see Figure 1.4). Liposomes produced by the high-encapsulation injection process are found to exhibit a broad size distribution in the range of 0.2 to 1.5 um; downsizing such liposomes results in a loss of encapsulated materials.An alternative method involves the extrusion of a hete fairly large liposomes through polycarbonate membranes un er moderate pressures.This technique can reduce a heterogeneous population to a sus- pension of vesicles that exhibit a mean particle size near that of the pores through which they are extruded(see Figure 1.5). Incorporation of drugs into liposomes is achieved by using one of the Encapsu ation is useful for water-soluble drugs,and es hydration of a lipid with an aqueous solution of a drug.The dissolved drug remains in the intralamellar spaces.In the process of partitioning,the drug is dis- solved along with the phospholipids in a suitable organic solvent.It is then eous phase.The residual solvent is dred frst added directly to the in a vacuum The reverse s is used for weak acidic atexist in both chared and ucharred forms dependinson the
Chapter one: Site-specific drug delivery using liposomes as carriers 9 Two major methods are usually used to make liposomal systems for drug incorporation. The first method deals with hydration of a lipid followed by high-intensity agitation using sonication or a high-shear propeller. Liposomes are subsequently sized by filtration or extrusion. In the second method, a phospholipid is first dissolved in an organic solvent and then added to an aqueous medium by vigorous agitation. The organic solvent is removed under vacuum, and the resulting liposomal dispersion or emulsion is sized by filtration or extrusion. Generally, the first method yields multiple lamellae (see Figure 1.4). Liposomes produced by the high-encapsulation injection process are found to exhibit a broad size distribution in the range of 0.2 to 1.5 mm; downsizing such liposomes results in a loss of encapsulated materials. An alternative method involves the extrusion of a heterogeneous population of fairly large liposomes through polycarbonate membranes under moderate pressures. This technique can reduce a heterogeneous population to a suspension of vesicles that exhibit a mean particle size near that of the pores through which they are extruded (see Figure 1.5). Incorporation of drugs into liposomes is achieved by using one of the three primary mechanisms: encapsulation, partitioning, and reverse loading. Encapsulation is useful for water-soluble drugs, and it involves hydration of a lipid with an aqueous solution of a drug. The dissolved drug remains in the intralamellar spaces. In the process of partitioning, the drug is dissolved along with the phospholipids in a suitable organic solvent. It is then dried first or added directly to the aqueous phase. The residual solvent is removed in a vacuum. The reverse-loading process is used for weak acidic drugs that exist in both charged and uncharged forms, depending on the Figure 1.4 Schematic of phagocytosis of particulate carriers by macrophages. Macrophages take up the carriers by the process of endocytosis. Drugs are released from the carriers following intralysosomal degradation of the carriers. (With permission, Elsevier, J. Control. Rel., 79, 29–40, 2002.) Drug entrapped in carrier Lysosome Fusion and release of drug from the carrier Endocytic vesicles Drug release Macrophage Endocytosis
Drug delivery systems,second edition volume Covalently coupled peptides 80-100nm figutre 15 molecular schematic of a surface-modified liposomal drug delivery vehi cle for intravascular targeting.(A)The liposome surface consists of a glycocalyx-like oligosaccharide layer to minimize nonspecific interactions and peptide ligands to mediate selective receptive targeting.(B)Composite molecular model showing gly colipids hydrating the surface of the phospholipid bilayer (a),an RGD peptide couplet thelime thouhpoly(ethylenoxidepb)a hypothatical coagulation factor VII peptide for targeting endothelial TF (c).(With pe Elsevier.I.Control.Rel.78.235-247.2002.) pH of the environment.Such drug molecules are added to an aqueous phase to create a ch d drug molecule is no lipophilic enough to pass through the lipid bilayer and return to the external medium (see Figure 1.6). Concentrations of the drug and lipids in the vesicles,measurements of captured volume,size distribution,and lamellarity characterize lipid vesi- cle The size of lingsomes is an important aspect in mein me-c omplement interactions.The compl ement system discriminate according to the liposomal size.Mean vesicle size and size distribution are important parameters for the physical properties and bio- logical fate of liposomes and their entrapped substances in vivo.One of the most commonly used methods to determine size and size distribution is ight-scattering analysis.New methods use la ing.If the sadspouou heteroge neous liposomes,accurate estimate of their size-frequency distribution is necessary.Other systems,such as dispersion,emulsions,and suspensions, are used frequently (see Figure 1.7). By utilizing a dehydration-rehydration process,a number of molecules lipos esicles are mi mes ith a used fo entrapment.The mixture is dehydrated by freeze drying,and the powder thus obtained is rehydrated under controlled conditions.Microfluidization of the drug containing dehydration-rehydration vesicles in the presence of
10 Drug delivery systems, second edition pH of the environment. Such drug molecules are added to an aqueous phase in the uncharged state to permeate into liposomes. The pH is then adjusted to create a charge on the drug molecule. The charged drug molecule is not lipophilic enough to pass through the lipid bilayer and return to the external medium (see Figure 1.6). Concentrations of the drug and lipids in the vesicles, measurements of captured volume, size distribution, and lamellarity characterize lipid vesicles. The size of liposomes is an important aspect in measuring liposome-complement interactions. The complement system is not known to discriminate according to the liposomal size. Mean vesicle size and size distribution are important parameters for the physical properties and biological fate of liposomes and their entrapped substances in vivo. One of the most commonly used methods to determine size and size distribution is light-scattering analysis. Newer methods use laser light scattering. If the liposomes are monodisperse, light-scattering analysis is used. For heterogeneous liposomes, accurate estimate of their size–frequency distribution is necessary. Other systems, such as dispersion, emulsions, and suspensions, are used frequently (see Figure 1.7). By utilizing a dehydration–rehydration process, a number of molecules can be quantitatively entrapped into the aqueous phase of liposomes. Small, unilamellar vesicles are mixed with a solution of the drug and used for entrapment. The mixture is dehydrated by freeze drying, and the powder thus obtained is rehydrated under controlled conditions. Microfluidization of the drug containing dehydration–rehydration vesicles in the presence of Figure 1.5 Molecular schematic of a surface-modified liposomal drug delivery vehicle for intravascular targeting. (A) The liposome surface consists of a glycocalyx-like oligosaccharide layer to minimize nonspecific interactions and peptide ligands to mediate selective receptive targeting. (B) Composite molecular model showing glycolipids hydrating the surface of the phospholipid bilayer (a), an RGD peptide coupled to the liposome through a poly(ethylene oxide) spacer (b), and a hypothetical coagulation factor VII peptide for targeting endothelial TF (c). (With permission, Elsevier, J. Control. Rel., 78, 235–247, 2002.) A Hydrated oligosaccharide interface Covalently coupled peptides 80-100 nm Encapsulated volume B a b c
Chapter one:Site-specific drug delivery using liposomes as carriers 11 Vesicular Membrane CP(2) Complex 66 >O DPPC 8CL2- CP(2)Positive Units Figure 1.6 Adsorption of CP(2)on the membrane of liquid mixed-negative vesicles (schematic presentation).(With permission,Elsevier,J.Control.Rel.,78,267-271,2002.) DSPE-PEG(2000)-Biotin NeutrAvidinTM PEG-Biotin ine 777777777777777777777 Figure 1.7 Schematic drawing(not to scale)of the multilayer construct used for immobilizing PEG-biotinylated liposomes onto solid polymeric carrier materials via NeutrAvidinbinding.(With permission,Elsevier,J.Control.Rel.,80,179-195,2002.) nonentrapped solute generates smaller vesicles.In gene delivery,cationi liposomes that interact with negatively charged nucleic acid polymers are used.Relatively homogenous and physically stable suspensions can be obtained by carefully controlling the complex conditions. Liposome stability is determined by using controlled systems,which are stabilized electrostatically rically.Besides self-assembling can undergo fusion or phase normal col. change afte aggregation.Liposome dispersions exhibit both physical and chemical sta- bility.Physically stable formulations preserve both liposomal size distribu- tion and the quantity of the material entrapped.Stability depends on the
Chapter one: Site-specific drug delivery using liposomes as carriers 11 nonentrapped solute generates smaller vesicles. In gene delivery, cationic liposomes that interact with negatively charged nucleic acid polymers are used. Relatively homogenous and physically stable suspensions can be obtained by carefully controlling the complex conditions. Liposome stability is determined by using controlled systems, which are stabilized electrostatically, sterically, or electrosterically. Besides normal colloids, self-assembling colloids can undergo fusion or phase change after aggregation. Liposome dispersions exhibit both physical and chemical stability. Physically stable formulations preserve both liposomal size distribution and the quantity of the material entrapped. Stability depends on the Figure 1.6 Adsorption of CP(2) on the membrane of liquid mixed-negative vesicles (schematic presentation). (With permission, Elsevier, J. Control. Rel., 78, 267–271, 2002.) Figure 1.7 Schematic drawing (not to scale) of the multilayer construct used for immobilizing PEG-biotinylated liposomes onto solid polymeric carrier materials via NeutrAvidin™ binding. (With permission, Elsevier, J. Control. Rel., 80, 179–195, 2002.) Vesicular Membrane CP(2) CL2− CP(2) Positive Units Interfacial Complex + DPPC DSPE-PEG(2000)-Biotin Lipid vesicle Lipid vesicle NeutrAvidinTM PEG-Biotin Poly(ethylenimine) Acetaldehyde plasma polymer