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PHYSICOCHEMICAL PROPERTIES OF LIPOSOMESIN RELATION TO UPTAKE BY ALVEOLAR MACROPHAGES

ByJANICE L. CACACE

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOLOF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYUNIVERSITY OF FLORIDA

1991


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Copyright 1991by

Janice L. Cacace


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For My ParentsThomas and Diana Cacace

andMy Husband

John McDonnellFor Never QuestioningAnd Always Supporting


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TABLE OF CONTENTSpage

ACKNOWLEDGEMENTS HiLIST OF TABLES viiLIST OF FIGURES viiiGLOSSARY OF TERMS xiiiABSTRACT xivCHAPTERS1 INTRODUCTION 1

Background Alveolar Macrophages 5Reticuloendothelial System (RES 5Endocytosis 6Phagocytosis 9Intracellular Fate of Ingested Substances. 11Factors Affecting Phagocytosis 12Properties of Phagocytic Cells 24Alveolar Macrophages (AM) 25Macrophage Culture 27Lung Surfactant 2 9Background Liposomes 33Phospholipids 33Liposomes 4Physicochemical Parameters of Liposomes 45Thermotropic Phase Transitions 45Effects of Water on PhospholipidPhase Transitions 4 7Differential Scanning Calorimetry 51Factors Affecting ThermotropicTransitions of Phospholipids 55Thermal Transitions in PhospholipidMixtures and Phase Diagrams 58Use of DSC in Liposome Formulation 6 3Surface Charge of Liposomes 65Factors Affecting Electrophoresis 69Electrophoresis of Phospholipids 72Interactions Between Liposomes and Cells 79Selection of Marker 84

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Objective 902 MATERIALS AND METHODS 95

Chemicals and Reagents 95Liposome Preparation 95Cell Culture 95Experimental Procedures 97Phospholipid Purity by TLC 97Carboxyfluorescein Purification 98Fluorescence 99Liposome Preparation 99Liposome Separation from Unencapsulated CF. 100Particle Size Measurement 101Differential Scanning Calorimetry 101Zeta Potential 101Stability 103

Macrophage Culture 104Harvesting of Alveolar Macrophages 104AM Viability 105AM Phagocytic Activity 105Alveolar Lining Material (ALM) 106Lung Homogenate ( LH 10 7Qualitative Uptake 108Quantitative Uptake 108In vitro Stability 109Statistical Methods 109

3 RESULTS AND DISCUSSION 110Physicochemical Parameters 110Phospholipid Purity 110CF Fluorescence IllLiposome Size and Size Distributionby Laser Light Scattering 115Differential Scanning Calorimetry 117Tc of HEPC : DPPG Liposomes 117Tc of HEPC : DPPS Liposomes 121Tc of SPM 124Effect of ALM on Tc 130

Effect of Ca2 * on DSC Measurement 132Zeta Potential Measurements by RankBrothers Mark II 134Zeta Potential of HEPC:DPPG Liposomes 134Zeta Potential of HEPC: DPPSLiposomes 134Zeta Potential Measurements by MalvernZetasizer 134Zeta Potential of HEPC:DPPG Liposomes 134Zeta Potential of HEPC:DPPS Liposomes 138Discussion Zeta Potential of NegativelyCharged Phospholipids 14 3


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LIST OF TABLESTable page1. Composition of Pulmonary Surfactant 302. Phase Transitions of Anhydrous Phospholipids 463. Effect of Chain Length on T of DPPC 494

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Effect of Phospholipid Headgroup on Tc 495. Effect of pH on Tc of DPPG and DPPS 566

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Effect of Vesicular Structure on Tc 587 . Isomorphous Mixtures of Phospholipids 628. Effect of Temperature on Dielectric Constant andViscosity of Water 79

.

Effect of Sugars on Dielectric Constant andViscosity of Water at 25 C 7010. Dielectric Decrement Values of Salts 7111. Cation Effect on Zeta Potential ofPhosphatidylglycerol 7 712. Effect of Calcium and Temperature on ZetaPotential of Phosphatidylserine 7713. Effect of Ionic Strength on Zeta Potentialof Phosphatidylserine Liposomes 7814 . Composition of DMEM 9615. Thin Layer Chromatography of Phospholipids 11016. Particle Sizing of ,HEPC:DPPG Liposomes 11617. Particle Sizing of HEPC:DPPS Liposomes 116


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LIST OF FIGURESFigure Pa9e1. Structural representation of phospholipid (A)and sphingolipid (B). The circled area (top left)represents the hydrophilic headgroup withpossible X substituents (bottom box), and theshaded area (right) represents the hydrophobic,fatty acid portion of the molecule, with m and ndenoting the length of the hydrocarbon chain 342. Phospholipids with corresponding dynamic molecularshapes and polymorphic phases; (A) micellar,

(B) bilayer, (C) interdigitated, (D) hexagonal 373. Schematic diagram of a multilamellar liposome,an "onion skin" configuration with concentric lipidbilayers separated by aqueous spaces, surroundingan aqueous core 394. Preparation of multilamellar vesicles, and sizingby extrusion through a polycarbonate membrane 4 35. Representative endothermic phase transition forheating (A) and cooling (B) of hydrogenatedegg phosphatidylcholine 536. Idealized phase diagram for a binary mixture ofphospholipids whose components are completelymiscible in the liquid and solid state. Above thefluidus line, the phospholipid is in a liquidstate; below the solidus line, the phospholipid

is in a solid or "gel" state; in between the lines,both states exist in equilibrium 617. The electric double layer, represented by aspherical particle with a net negative surfacecharge suspended in aqueous medium and surroundedby a layer of opposite charge. Below the particle

is a diagram indicating the electrical potential asa function of distance from the surface of theparticle. The potential at the plane of shear isthe zeta potential 67

8. Structure of 5, 6-carboxyfluorescein 86


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9 . Concentration versus relative fluorescence for5,6-carboxyfluorescein at excitation X 492 ran andemission X. 524 run. Note the decrease influorescence due to self-quenching atconcentrations > 1 x lCT'M 891 . Phospholipid structures 9311. Calibration curve for 5,6-carboxyfluorescein inPBS and Triton X-100 plotted as molar concentrationCF versus relative fluorescence intensity atexcitation X 492 run and emission X.524 run 11212. Calibration curve for 5,6-carboxyfluorescein inPBS or PBS containing 1 X 10"8M HEPC or DPPS,plotted as molar concentration CF versusrelative fluorescence intensity at excitationX 492 run and emission X 524 nm 11313. Effect of the molar concentration of HEPC onthe relative fluorescence intensity of CF atexcitation X 492 nm and emission X 524 nm 11414. Representative DSC endotherm for DPPG (left, solidline), and HEPC (right, dashed line) 11815. Phase diagram of HEPC: DPPG liposomes based ononset temperature of heating (top) andcooling (bottom) 11916. Transition temperature versus mol% DPPG forHEPC: DPPG liposomes in PBS (time 0) or DMEM

(4 hours) 12017. Representative DSC endotherm for DPPS (50 mM)in 3 mM CF (left, solid line) and DPPS (25 mM)in PBS (right, dashed line) 12218. Phase diagram of onset temperature of heating

(top), and cooling (bottom) for HEPC:DPPSliposome series 12319. Transition temperatures of HEPC:DPPS liposomesin PBS (filled) or DMEM (diagonal) 12520. Tc of HEPC: DPPS liposomes encapsulatingPBS or CF (LIPCF) 12621. Representative thermogram for SPM (Left, solidline), and HEPC (right, dashed line) 127

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22. Phase diagram of HEPC:SPM liposomes representedby temperature of onset (bottom) andcompletion (top) 12923. Effect of incubation of liposomes in ALM for

4 hours on Tc of HEPC, HEPC:DPPG (50:50),and HEPC:DPPS (50:50) 131

24. Zeta potential of HEPC:DPPG liposomes by RankBrothers Mark II MicroelectrophoresisApparatus in PBS (bottom) and DMEM (top) 13525. Zeta potential of HEPC:DPPS liposomes by RankBrothers Mark II MicroelectrophoresisApparatus in PBS (bottom) and DMEM (top) 13626. Zeta potential of HEPC:DPPG liposomes by MalvernZetasizer 3 in PBS (bottom) and DMEM (top) 13727. Zeta potential of HEPC:DPPG liposomes by MalvernZetasizer 3 in DMEM (top) and DMEM withoutFBS (bottom) 13928. Zeta potential of HEPC:DPPG liposomes by MalvernZetasizer 3 in PBS (bottom), PBS + 5% FBS(middle), and PBS + Ca2+ (200mg/L) + Mg2+

(200 mg/L) (top) 14029. Zeta potential of HEPC:DPPS liposomes by Malvern

Zetasizer 3 in PBS (top) or 3mM CF (bottom) 14130. Zeta potential of HEPC:DPPS liposomes by MalvernZetasizer 3 in PBS (bottom) or DMEM (top) 14231. Zeta potential of HEPC:DPPS liposomes by MalvernZetasizer 3 in PBS (top) or 3 mM CF (bottom) 14432. Zeta potential of LIPCF versus mol% chargedphospholipid 14733. Zeta potential of HEPC:SPM liposomes in PBSand DMEM 14834. Stability of HEPC:DPPG liposomes in PBS, DMEM,ALM, and LH. A: HEPC; B: HEPC:DPPG (80:20);C: HEPC:DPPG (50:50); D: HEPC:DPPG (20:80);E: DPPG 15635. Stability of HEPC:DPPG liposomes plotted as % CFretained versus mol% DPPG in various media.

A: PBS; B: DMEM; C: ALM; D: LH 158


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36. Stability of HEPC:DPPG plotted as % CF remainingversus time in vitro in DMEM (open circle) or incell culture (closed circle) 15 937. Stability of HEPC:DPPS liposomes in PBS, DMEM,ALM, and LH. (A) HEPC:DPPS (50:50);

(B) HEPC:DPPS (80:20) 16038. Stability of HEPC:DPPS liposomes plotted as % CFretained versus mol% DPPS in various media.

A: PBS; B: DMEM; C: ALM; D: LH 16139. Stability of HEPC:DPPS liposomes in vitro inDMEM (open circle) or in cell culture (closedcircle). (A) HEPC:DPPS (80:20);

(B) HEPC:DPPS (50:50) 16340. Stability of CF in lung homogenate at 37Crepresented as % remaining versus time 16641. Representative SDS-Polyacrylamide gelelectrophoresis of alveolar lining material,with molecular weights of the protein standards(left side) and calculated molecular weightsfor the samples (right side) 16842. Log R t of SDS-PAGE protein standards versusmolecular weight of protein, used fordetermination of molecular weights of proteins

in ALM 16943. Qualitative uptake of liposomes by AM. Fluorescencemicrograph (left) with the corresponding phasemicrograph (right) (to locate cell position),after incubation for 15 minutes (top) or 1 hour(bottom). Note the distinct punctate vacuoleswhich are more prominent in the latter time point. 17144. Uptake of HEPC:DPPG liposomes by AM over 4 hours.

(A) HEPC; (B) HEPC:DPPG (80:20); (C) HEPC:DPPG(63:35); (D) HEPC:DPPG (50:50); (E) HEPC:DPPG(20:80) 173

45. Uptake of HEPC:DPPG liposomes by alveolarmacrophages plotted against mol% DPPG, after 1 and4 hours incubation 175

46. Uptake of HEPC: DPPS liposomes by alveolarmacrophages over 4 hours. HEPC (solid);HEPC:DPPS (80:20) (diagonal); HEPC:DPPS(65:35) (cross hatch) 177

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47. Uptake of HEPC:DPPS (80:20) (diagonal) versusHEPC:DPPG (80:20) (solid) by AM over 4 hours 17848. In vitro uptake of HEPC:DPPS (63:35 (diagonal)versus HEPC:DPPG (65:35) (solid) by AM over

4 hours 17949. In vitro uptake of HEPC:DPPG (65:35) liposomesby AM in ALM (diagonal) versus DMEM (solid)over 4 hours 180

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GLOSSARY OF TERMSAbbreviation DefinitionCFDCPDLPGDMPCDMPEDOPCDPPCDPPEDPPGDPPSDSCDSPCDSPEDSPSHEPCLIPCFMLVPAPCPGPSPISPMSUVTcUNEXLIP

5 , 6-carboxyfluoresceinDicetylphosphateDilaurylphosphatidylglycerolDimyristoylphosphatidylcholineDimyristoylphosphatidylethanolamineDioleoylphosphatidylcholineDipalmitoylphosphatidylcholineDipalmitoylphosphatidylethanolamineDipalmitoylphosphatidylglycerolDipalmitoylphosphatidylserineDifferential scanning calorimetryDistearoylphosphatidylcholineDistearoylphosphatidylethanolamineDistearoylphosphatidylserineHydrogenated egg phosphatidylcholineExtruded liposomes encapsulating CFMultilamellar vesiclePhosphatidic acidPhsophatidylcholinePhosphatidylglycerolPhosphatidylserinePhosphatidylinos itolSphingomyelinSmall unilamellar vesicleTransition temperatureUnextruded liposomes encapsulating CF


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Abstract of Dissertation Presented to the Graduate Schoolof the University of Florida in Partial Fulfillment of theRequirements for the Degree of Doctor of PhilosophyPHYSICOCHEMICAL PROPERTIES OF LIPOSOMES

IN RELATION TO UPTAKE BY ALVEOLAR MACROPHAGESBY

Janice L. Cacace

December, 1991Chairman: Hans SchreierMajor Department: Pharmaceutical Sciences

Treatment of intracellular infections is limited by theinability of most therapeutic agents to cross the cellmembrane and interact with the infecting organism. Oneoption is to target the drug directly to the reservoir ofinfectionthe macrophageby utilizing the natural capacityof these cells to ingest particulate matter.

The studies presented here attempt to elucidate theinteractions between a colloidal dosage form, liposomes, andmacrophages of the lungs, i.e., alveolar macrophages. Theprimary variable was the percentage of negatively chargedphospholipid in the liposomal membrane. Characterization ofthe physicochemical properties of zeta potential, transitiontemperature, and stability, as well as uptake by alveolarmacrophages, was assessed in vitro . Stability and uptakestudies were performed by fluorescence using the self-


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quenching probe 5, 6-carboxyfluorescein. The majorphospholipids used were hydrogenated egg phosphatidylcholine(HEPC), dipalmitoylphosphatidylglycerol (DPPG) anddipalmitoylphosphatidylserine (DPPS)

.

These liposomes retained their contents under in vitroconditions simulating storage, cell culture, and the lunglumen. Their ability to release encapsulated materials uponenzyme contact was demonstrated by retention of < 20% ofencapsulated carboxyfluorescein after 4 hours incubationwith lung homogenate.

Several pitfalls were elucidated by the interaction ofliposomes with the culture mediumDulbecco's modified Eaglemedium (DMEM) . The presence of calcium ions in this mediumcaused considerable charge neutralization, in some casesliposome aggregation, and phospholipid phase separations.Even though charge neutralization occurred, the uptake ofliposomes varied with DPPG and DPPS content. Maximum uptakeoccurred with HEPC:DPPG (65:35 molar ratio). The uptake ofHEPC: DPPS liposomes was similar to HEPC alone; however,these results may have been complicated by the physicalinstability of DPPS containing liposomes.

When HEPC: DPPG (65:35) liposomes were incubated withalveolar macrophages in alveolar lining material (ALM) , theamount taken up was comparable to the same study in DMEM.However, the initial rate of uptake was faster in ALM.


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Thus, it appears that hydrogenated phospholipidvesicles provide a suitable carrier for substances targetedto alveolar macrophages. The uptake process is dependent onthe type and amount of phospholipid present, with anapparent preference for DPPG containing liposomes

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CHAPTER 1INTRODUCTION

Liposomes have been widely used for the study of modelmembranes and potential drug delivery systems for about 25years (Bangham 1986). Interest in their use as drug carriersis based on the potential to encapsulate a diverse number ofmaterials of biological interest (Gregoriadis 1976). Inaddition, it is possible to alter such parameters asliposome size, surface charge, bilayer fluidity andstability to adapt the carrier to a wide range of experimen-tal conditions.

Although considerable technological advances have beenmade, liposome use in drug delivery has achieved limitedsuccess. One of the major obstacles to attaining specificgoals was stated recently by Lopez-Berestein and Fidler(1989): "Selective targeting of therapeutic agents to appro-priate sites of action while avoiding the reticuloendo-thelial system is still a challenging and unresolved issue";therefore, the focus for drug delivery has switched to "newapproaches to selective targeting as well as elaboratemethods for prolonging drug availability and improvingtargeting to intracellular sites." Thus, although the use ofliposomes may improve drug therapy, by far the major


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rationale for their use involves targeting either to sitesof infection or away from sites of toxicity. This can be toa specific organ or cell population.

The rapid uptake of liposomes by cells of the reticulo-endothelial system (RES), mainly in the liver and spleen(Gregoriadis and Ryman 1972a, b, Segal et al. 1974, Kimelberget al. 1979, Abra and Hunt 1981), makes these cells theperfect target, since it enables the use of their naturalphysiologic function of phagocytosis to improve drugdelivery.

Since these cells can also be the host to a variety ofintracellular infections (e.g., mycobacteria, Brucella ,Listeria , and Salmonella sp. ) (DeDuve et al. 1974), therationale for cell-directed delivery is even moreattractive.

One of the factors inhibiting the treatment of theseinfections in a traditional manner is the ability of thecausal agent to survive and proliferate intracellularly,most often within the confines of the vacuolar system intowhich it has been introduced through phagocytosis.Therefore, if one wishes to reach intracellular bacteriawith an appropriate agent (e.g., antibiotic delivery [Lopez-Berestein 1987, Swenson et al. 1988]), it must be capable ofentering the cell and of acting under the conditionsprevailing within the vacuoles occupied by the bacteria.


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Liposomes are potentially useful for this purpose.Their localization in cells harboring intracellular diseaseshas been discussed (Fidler et al. 1980, Yatvin and Lelkes1982), with the classic example being the treatment ofleishmaniasis, an intracellular parasitic disease localizedin Kupffer cells of the liver (Alving et al. 1978a, b, New etal. 1978, New 1990) .

Other potential sites for delivery to cells of the RESare the spleen and the lungs. The lungs, because of theirdaily interaction with airborn diseases, are prone toinfections and therefore a useful target organ for attemptsto localize drugs. It has been shown that intravenouslydelivered liposomes 1 um or greater in diameter exhibitimproved localization in the lungs compared to smaller-sizedparticles (Hunt et al. 1979, Fidler et al. 1980). Thiseffect has been attributed to trapping in the capillary bedsof the lungs (Hunt et al. 1979). It has also been shown thatpositively charged (stearylamine) and negatively charged(phosphatidylserine, phosphatidylglycerol and phosphatidicacid) liposomes accumulate in lungs to a greater extent thanneutral liposomes of similar size (Jonah et al. 1975,Kimelberg 1976, Kimelberg et al. 1979, Fidler et al . 1980,Abra et al. 1984). In each case, however, the majority ofthe liposome preparation still accumulated in the liverrather than in the lungs.


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Therefore, there has been increasing interest in thedelivery of liposomes directly into the pulmonary systemoffering a non-invasive route of administration by pulmonaryinstillation (Juliano and McCullough 1980) or aerosolization(Debs et al. 1987, Mihalko et al. 1988) as a way to achievesustained localized delivery while specifically targetingdrugs to the airways. Several anatomical and physiologicalproperties of the lungs and airways render them anattractive portal of entry for systemic drug delivery: alarge surface area (ca. 70 m2 ), a very thin (


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The cells of the MPS are produced from stem cells inthe bone marrow where they undergo proliferation and aredelivered to the blood as monocytes (Bellanti and Kadvec1985). After a period of maturation through a monoblastpromonocytemonocyte phase in the blood, the monocytesmigrate to their main site of action in various tissueswhere they differentiate further into macrophages. Thesecells include Kupffer cells of the liver and macrophages ofthe spleen, bone marrow, and lungs.

EndocytosisOne of the primary functions of macrophages is the

ingestion and destruction of foreign materials. They arehighly specialized to carry out this function by the processof endocytosis (Silverstein et al. 1977, Pratten and Lloyd1986), a general term which includes both phagocytosis(ingestion of particles) and pinocytosis (uptake ofnonparticulates, i.e., fluid droplets). Both represent theclearance of substances from the surrounding environmentthrough the formation of an intracellular vesicle andsubsequent delivery of entrapped materials to lysosomes

.

These substances can be endogenous or exogenous, includingbacteria, viruses, damaged or effete cells, neoplasticcells, macromolecules and colloidal materials includingparticles with synthetic (latex), denatured (boiled yeastcell walls, i.e., zymosan) and chemically modified surfaces


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(aldehyde-treated red blood cells) (DeDuve et al. 1974).However, macrophages are not attracted to viable undamagedanimal cells or encapsulated strains of pathogenic bacteria,unless these are coated with opsonins such as antibodies orcomplement (Poznansky and Juliano 1984).

The first encounter of host and foreign substance leadsto a stereotypical response causing mobilization ofphagocytic elements to the areas where the substance wasintroduced. This may occur as an isolated event or as partof the inflammatory response. Once mobilized, the phagocyticcells mount an attack on their target by a process calledphagocytosis, a multiphasic act requiring the followingsteps (Bellanti and Kadvec 1985):

(1) Recognition of material to be ingested. Circulatingmonocytes are attracted to an area of injury by a number offactors, some of which are derived from the complementsystem secreted by T-lymphocytes . Here they may furtherdifferentiate into macrophages and may be activated in avariety of ways. Once activated, the cells displayheightened metabolic activity and enhanced function.

(2) Movement towards the material (chemotaxis ) . Theinteraction of a particle with the plasma membrane of aphagocytic cell results in the generation of signals that,when relayed to the cell's interior, initiate the movementof cytoplasm, the formation of membrane pseudopods, and the


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remodeling and fusion of the membrane to form a phagocyticvacuole.

(3) Adhesion and ingestion. The process of adhesion isseparate from that of ingestion (internalization) and doesnot necessarily predestine a particle for ingestion.Ingestion of an attached particle requires the sequentialinteraction of membrane receptors on the phagocyte withligands distributed throughout the surface of the particle.This is referred to as the "zipper" model due to therequirement for a zipper-like interaction of cellularreceptors with particle-bound ligands. The zipper modelpredicts that a particle with ligands (antibodies)distributed over only one part of its surface will bind tothe macrophage plasma membrane but will not be ingested.This in part depends on the activity of the macrophage. Forexample, complement receptors lead to binding but notingestion for unstimulated (unprimed) macrophages, butbinding and ingestion occur for stimulated (inflammatory)macrophages (Silverstein et al. 1978). In addition,attachment has been shown to be independent of temperatureand energy expenditures, whereas ingestion is consideredhighly temperature dependent and requires active cellularmetabolism (Rabinovitch 1969, Silverstein et al. 1977,Pratten and Lloyd 1986).

(4) Intracellular digestion. Following ingestion, theparticle enters the cell in a vacuole composed of


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internalized plasma membrane (phagosome), which in turnfuses with a lysosome to form a phagolysosome where the cellattempts to destroy (digest) the foreign particle using awide array of enzymes (DeDuve et al. 1974).

PhagocytosisPhagocytosis has been described by Jacques (1970) as a

process during which a macrophage which appears to be movingwith a "flapping ruffle" at its leading edge, pushes outtowards the particle and quickly flows around it. Thisprocess can be very rapid, with complete ingestion takingplace within minutes. In addition, in the course of 10 to 20minutes, a phagocytic cell can internalize a quantity ofparticles whose combined surface area is equivalent to 30%to 50% of the area of the phagocyte's plasma membrane(Silverstein et al. 1977, Mahoney et al. 1977). This canproceed in either a specific or nonspecific manner (i.e.,with or without the involvement of immunologicallydetermined ligands). Nonspecific interactions occur as aresult of physicochemical conditions rather than as aconsequence of specific epitope-antibody binding.

While in some systems phagocytosis can proceed in theabsence of protein, in general it is stimulated by itspresence in the medium. This may be related to theadsorption of specific plasma proteins termed opsonins(Howard and Wardlaw 1958, Saba 1970, Silverstein et al.1978). These substances may be similar to natural


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antibodies, thus different from specific immunoglobulins.For instance, the presence of serum and divalent cations inculture medium was found necessary for the ingestion of redblood cells, but not for all particles (Wilkins and Bangham1964, Carr 1973), and in the case of bacteria, specificopsonic antibodies have been indicated. Aside fromincreasing the rate of phagocytosis, it has been suggestedthat opsonins in normal serum may be mediating in part therecognition mechanism of the nonimmune animal. This mayindicate that specific receptors on macrophage membranes arenecessary to initiate phagocytosis.

Indeed, the macrophage surface has been shown to bearmore than 30 receptors, which respond to particleopsonization by immunoglobulins (IgG), complement and otherproteins. There are two Fc portions of IgG involved,however, the Fab fragments are devoid of such activity(Ogmundsdottir and Weir 1980).

Complement is a series of sequentially reacting serumproteins (Esser 1982) that may possibly be an amplifier ofphagocytosis. Complement receptor activity is governed bythe physiological state of the macrophage (Silverstein etal. 1978). Complement receptors of unstimulated macrophagespromote binding but not ingestion of complement coatedparticles; complement receptors of inflammatory macrophagesmediate binding and ingestion of complement coated


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11particles. There is evidence to suggest the C3b component ofcomplement plays a part (Lambris and Ross 1982).

Of the other proteins, there are receptors formannosyl/fucosyl-terminated glycoproteins (Stahl and Gordon1982) as well as strong evidence for the involvement offibronectins in the opsonization process (Hsu and Juliano1982)

.

There is also evidence to suggest that the stimulus tophagocytize a particle is initiated by the particle. Themembrane response of the phagocyte to this stimulus is localand is confined to the segment of the plasma membraneadjacent to the particle initiating the stimulus. Membranecomponents that mediate this "non-specific" uptake ofparticles (e.g., polystyrene and latex particles) have beenproposed; however, they are purported to act independentlyof Fc receptors and complement receptors (Hsu and Juliano1982)

.

Intracellular Fate of Ingested SubstancesOnce ingested, the particle enters the cell in a

vacuole (phagosome) which in turn fuses with one or morelysosomes to form a phagolysosome where the cell attempts todestroy (digest) the foreign particle with a wide array oflysosomal enzymes (DeDuve et al. 1974). In order to survivein this vacuole, the ingested substance must be stable at pH4-5 (intracellular lysosomal pH) and withstand attack by 40or more digestive enzymes. The end product of digestion, the


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13These were comprehensively reviewed by Saba (1970) andinclude species, blood flow, metabolic activity, status ofthe cells, and particle dose. In general, species and bloodflow are not applicable to the in vitro study ofphagocytosis. However, the others, in conjunction withcertain physicochemical factors such as particle size,surface charge, surface affinity including opsonization, andmembrane fluidity, influence the phagocytic response in vivoand in vitro (Saba 1970, van Oss et al. 1975, Ilium andDavis 1982).

Metabolic activity . The energy sources for the cells ofthe MPS are dependent upon the degree of cellular maturity,the level of endocytic activity, and the environment.Following formation of a vacuole, the primary metabolicprocesses involved include the glycolytic pathway and thehexose monophosphate shunt. In addition, the main energysource for alveolar macrophages is the aerobic tricarboxylicacid (TCA) pathway. Collectively, stimulation of thesepathways is termed respiratory burst and consists of anincrease in glycolysis, hexose monophosphate shunt, oxygenconsumption, and hydrogen peroxide and lactic acidproduction. The increase in lactic acid is partlyresponsible for the decreased pH in the phagosome.Accompanying the respiratory burst is an increase in RNA andphospholipid turnover, which is important for proteinsynthesis and membrane formation. This is most prominent in


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neutrophils and to a lesser extent in mononuclearphagocytes. Both in vitro and in vivo phagocytosis areenergy dependent; however, particle adherence to thephagocytic cell membrane is not (Oren et al. 1963).

In addition, it has been shown that particle uptake invitro can be inhibited by low temperature (4C) or byinhibiting energy metabolism through the addition of NaF ornitrophenol (Steinman et al. 1974, Pratten and Lloyd 1986).Incubation at 4C causes an inhibition of uptake sincephagocytosis does not occur at these low temperatures, andany uptake here would likely be due to binding.

Status of the reticuloendothelial cells (activated ornon-activated) . The RES does not function solely as apassive scavenger. It can be activated or depressed by avariety of endogenous and exogenous factors . Activation canoccur naturally during bacterial infection, neoplasticdiseases, and diseases of autoimmunity, while reticuloendo-thelial depression associated with circulatory failure maybe a crucial factor in the development and progression of adisease process. Reticuloendothelial depression can also beinduced by the administration of a bacterial cell wallcomponent, such as muramyl dipeptide (Fidler 1986) or lipid-containing emulsions (e.g., Intralipid*) (Fischer et al.1980). Ilium and Davis (1984) have used this approach toproduce reticuloendothelial blockade resulting in aredirection of colloids from the liver to other organs by


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15administering a placebo colloid or a macromolecular material(e.g., dextran sulfate).

Particle dose . Small colloid doses removed primarily bythe liver cannot be used as an index of reticuloendothelialactivity. With increments in dose administered, there is anassociated decrease in clearance rate and a progressiveincrement in relative extrahepatic colloid localization.This may be due to progressive saturation of reticulo-endothelial cell capacity or to a progressive saturation ofthe available opsonin pool. With a large enough dose,reticuloendothelial blockade can be induced. This conditioncan persist for several days, in which no further uptake canproceed (Saba and DiLuzio 1966, Gregoriadis and Ryman 1972a,Gregoriadis and Neerunjum 1974, Kavet and Brain 1980). Thisappears to be related to the total number and surface areaof particles administered rather than particle composition(Abra and Hunt 1981)

.

Particle size . In vivo , large particles (>7 urn) will beremoved from the blood rapidly and efficiently by thefiltering propensity of the lung capillaries (Fidler et al.1980, Davis and Ilium 1986). Smaller particles that are notremoved by the lung will normally be removed by macrophages.

In relation to liposomes, the longest half life valueshave been obtained with the smallest (


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16phospholipid, small unilamellar vesicles have a much largertotal surface area and greater particle numbers comparedwith those of larger multilamellar vesicles (Pidgeon andHunt 1981) .

In vitro , the rate of particle uptake by peritonealmacrophages has been shown to be linear over time and toincrease with particle size for polyvinylpyrrolidoneparticles (Pratten and Lloyd 1986). Polyvinlypyrrolidoneparticles of 100 or 1,100 run diameter were phagocytized in arate-dependent manner over 2 . 5 hours , with the largerparticles being captured more rapidly. This effect was alsoshown by Hsu and Juliano (1982) using large multilamellarliposomes which were able to more efficiently deliver theircontents to mouse peritoneal macrophages than smallerunilamellar vesicles, although a greater number of smallerparticles were captured.

Surface charge . Cell membranes contain large amounts ofsurface carbohydrates, in particular surface sialic acidresidues which impart a negative charge to the surface(Allen and Chonn 1987). When these sialic groups areenzymatically removed from the cell surface, rapid uptake ofcells occurs by Kupffer cells of the liver. Likewise, whensulfatides and gangliosides are used to impart surfacehydrophilicity and negative charge (by expression of sialicacid groups), blood circulation times have been shown toincrease. However, the expression of a negative charge (by


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incorporation of phosphatidylserine) may lead to enhancedtrapping in the lung vasculature (Fidler et al. 1980, Fidler1986)

.

Juliano and Stamp (1978) found that liposomes ofnegative charge due to the presence of PS were cleared morerapidly from the circulation after intravenousadministration than positive and neutral liposomes,regardless of size. However, positively charged and neutralliposomes were cleared more rapidly if they were of largersize. In addition, large positively and negatively chargedliposomes were cleared at a similar rate, which was morerapid than for neutral particles.

In vitro , it has been consistently shown thatmacrophages possess a negatively charged surface and thatinteractions with positively charged particles lead toenhanced endocytosis (Schwendener et al. 1984, Mutsaers andPapadimitriou 1988). However, there is conflicting evidenceon the effects of the interactions of negatively chargedparticles with macrophages. There were measurabledifferences in the uptake rate in vitro for negativelycharged colloids, which also showed differences with respectto rate of vascular clearance and relative hepaticdistribution in vivo . Although these particles had differentelectrophoretic mobilities initially, after plasmaincubation, there was no measurable difference, presumablydue to the adsorption of plasma proteins (Wilkins and Meyers


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18

1966, Wilkins 1967). A similar reversal of charge was seenby Black and Gregoriadis (1976) where neutral and positiveliposomes acquired a negative charge on incubation with ratplasma; however, the surface charge on negatively chargedliposomes was essentially unchanged. Thus, while it isrecognized that the surface of the entity (bacteria,particles, etc.) being phagocytized will have significanteffects on its rate of removal, the relationship between thecharge in vitro and that acquired instantaneously in vivoremains to be ascertained.

Opsonins . In addition to the uptake of solubleproteins, macrophage surface receptors can also mediateparticle uptake. Thus coating a particle with proteins

capable of interacting with a macrophage (opsonization) canenormously enhance the uptake of the particle. The surfacecharacteristics of a particle determine whether or not itwill be opsonized and by which component. As a consequence,the mechanism of adhesion will be different depending on thenature of the opsonic component and the particular receptormediated process. The effect of opsonization of liposomeswas demonstrated by Hsu and Juliano (1982), by coatingliposomes with IgG, thus promoting interaction with themacrophage Fc receptors, and causing a 1000-fold increase inthe rate of phagocytosis. It was also demonstrated bySunamoto et al. (1984) that coating liposomes withpalmitoylamylopectin increased their uptake in alveolar


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19macrophages in vitro and their accumulation in lungs invivo . Similar enhancement would be expected with other typesof carriers.

The acquisition of an opsonic coat is many times anunavoidable process, as seen with gelatin microspheres thatinevitably acquire a coating of fibronectin upon injectioninto the circulation and are rapidly cleared by macrophages(Saba et al. 1978). In addition, studies by Saba and DiLuzio(1966) showed that in some cases diminished opsonin levelscould cause decreased particle uptake by macrophages

.

Surface affinity . In some instances, phagocytosis willoccur regardless of opsonization. This may be due to theaffinity of the particle for the macrophage surface as inthe case of the lipophilic surface of triglyceride emulsionsand certain latex particles (Saba and DiLuzio 1966).

Ilium and Davis (1984) reasoned that the opsonizationof particles followed by adhesion to macrophages would begreatly modified if particles were given a hydrophilicsurface that provided a steric repulsive barrier since thiswould create a high potential energy barrier and negateshort-range attractive forces, thus diminishing proteinadsorption and/or cell adhesion events. This could alsoaccount for the increased blood circulation times seen byAllen and Chonn (1987) when gangliosides and sulfatides wereused to impart surface hydrophilicity and negative charge(by expression of sialic acid groups) to liposomal surfaces.


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20Several studies using this technique (Ilium and Davis

1984, Ilium et al. 1986a, b, 1987, Davis and Hansrani 1985)showed colloid phagocytosis by mouse peritoneal macrophageswas altered in relation to surface hydrophobicity. Largehydrophobic groups induced rapid and efficient (ca. 90%)particle clearance, whereas large hydrophilic groupsresulted in much slower clearances. This is thought to bedue to the hydrophilic group inhibiting uptake of plasmacomponents (opsonization) and adhesion between the particlesand the macrophages in vivo , i.e., a steric repulsioneffect. This also led to a redistribution of particles intoother organs. For example, uncoated particles were taken upby the liver, particles coated with Poloxamer 407 wentalmost exclusively to the bone marrow, and particles coatedwith Poloxamine 908 were retained in the vascularcompartment. In each case, the adsorbed layer thickness wassimilar, therefore, chemical and physical differences musthave been contributing to the recognition of particlesurfaces.

Other polymers such as polyethylene glycols (PEG) werefound to decrease the uptake of liposomes by the RES. Blumeand Cevc (1990) were able to demonstrate approximately tentimes less uptake into cultured cells for PEG-coatedvesicles with the corresponding particle removal from blooddecreasing to only 15% of the rate characteristic foruncoated liposomes. Whereas, Woodle et al. (1990) were able


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23speculated that this effect was most likely due to thedecreased interaction with high density lipoproteins . Thismay be due to increased membrane rigidity, thus increasedstability of the liposomes with higher cholesterol content.This is consistent with later studies by Moghimi and Patel(1987 and 1989) where liver and splenic cells showed lessaffinity for liposomes composed of sphingomyelin andsaturated phospholipids, all of which would be in a solidphase at the temperature of study compared tophosphatidylcholine. However, in the presence of serum, theuptake was decreased for the liver cells, but increased forthe splenic cells.

Thus, any of these factors could be used to alter theuptake of particles by macrophages. In addition,optimization of several factors, such as surface sialic acidand bilayer viscosity properties, has been shown to have asynergistic effect in increasing circulation times ofliposomes (Allen and Chonn 1987).

Most research efforts in this area have centered on themechanisms by which this process could be inhibited orcircumvented for the delivery of therapeutic agents in theform of colloidal particles. However, as mentioned above, insome cases it may be desirable to deliver the particle tothe cells or tissues where the cells are localized.


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24Properties of Phagocytic Cells

Although phagocytic cells are functionally similar,there is no reason to assume they are identical. Laskin etal. (1988) found Kupffer cells and peritoneal macrophages tobe functionally and biochemically distinct, although bothdisplayed the morphologic and histochemical characteristicsof mononuclear phagocytes . While both phagocytize in a timedependent manner, Kupffer cells were 2 to 3 times morephagocytic. Upon stimulation, peritoneal macrophages releasegreater amounts of superoxide anion and hydrogen peroxide.In addition, differences in protein production by thesecells were observed.

Some other differences seen between macrophages includethe response to chemotactic (movement initiation) factors.Both rat (Laskin et al. 1988) and mouse (Hashimoto et al.1984) peritoneal macrophages responded to the chemotacticfactors complement C5 and phorbol ester tumor promoter.Whereas, it had previously been seen by Ward (1968) thatalveolar macrophages are much more responsive to chemotacticfactors than peritoneal macrophages. In many cases, thesedifferences arise due to the tissue of origin andspecialization of the macrophage cell type. This isparticularly important when discussing the energyrequirements for phagocytosis.

The energy sources for the cells of the mononuclearphagocyte system are dependent upon the degree of cellular


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25maturity, the level of endocytic activity, and theenvironment. Following formation of a vacuole, the primarymetabolic processes involved are the glycolytic pathway andthe hexose monophosphate shunt. However, the main source ofenergy for alveolar macrophages is the aerobic TCA pathway.This is accompanied by a burst of respiration, which is muchgreater in alveolar macrophages than in polymorphs orperitoneal macrophages (Carr 1973). It has been shown byOren et al. (1963) that alveolar macrophages differ fromboth polymorphonucleocytes and monocytes in that the restingrespiration of alveolar macrophages is much higher andphagocytosis causes only a small increase in oxygen uptakeand glucose metabolism. Monocytes and polymorphonucleocytesphagocytize efficiently without aerobic metabolism, butrequire glycolysis for the process. Inhibition of glycolysisin this case can block particle uptake. With alveolarmacrophages, interference with aerobic metabolism oroxidative phosphorylation can depress particle uptake, whichis depressed even further if glycolysis is inhibited. Inaddition, alveolar macrophage respiration can be stimulatedin culture by the presence of serum and glucose. This iscontrary to the depression of respiration seen inpolymorphonucleocytes in the presence of glucose.

Alveolar Macrophages (AM)Of particular interest here are cells found in the

pulmonary tract. In pulmonary tissue, cells which resemble


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26macrophages elsewhere may be found in interstitialconnective tissue of the alveolar wall, forming part of thelining epithelium of the alveolus, the great alveolar cellsof Sorokin, and free in the lumen of the alveolus (Carr1973). These alveolar macrophages belong to the group offree macrophages which are scattered diffusely throughoutthe mammalian body that also includes macrophages ofconnective tissue or histiocytes, macrophages of serosalsacs, and macrophages of inflammatory exudates. Pulmonary AMare derived from circulating monocytes originating from bonemarrow (van oud Alblas and van Furth 1979), as areperitoneal macrophages and Kupffer cells. This influx ofmonocytes is a steady state process, responsible for cell

renewal in the alveoli. They are considered end cells, asthey do not divide appreciably upon arrival (Myrvik andKohlweiss 1980). Most AM leave the alveoli by way of theairways and are expelled via the mucociliary pathway, with amean turnover time around 27 days.

Particulates less than 10 (im can penetrate the airwaysin varying degrees and those in the range of 1 to 2 urn canenter the respiratory space without difficulty. It isprobable that up to 50% of airborne particles in this sizerange reach the alveoli. Particles that reach the terminalairways and alveoli are destined to be phagocytosed byalveolar macrophages (Green and Kass 1964, Green 1970).


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27Once phagocytized, normal macrophages are quite

efficient in killing many avirulent organisms as well assome microorganisms of relatively low virulence. Incontrast, normal alveolar macrophages are totallyincompetent to handle highly virulent intracellularparasites like Mycobacterium tuberculosis and Francisellatularensis (Myrvik and Kohlweiss 1980). Only immunologicallyactivated macrophages which are mobilized and activated bythe mechanisms of cell-mediated immunity can successfullycope with these virulent organisms.

Macrophage CulturePerhaps the best way to obtain a pure culture of

macrophages is by washing out a serosal cavity, usually theperitoneum (Carr 1973). These macrophages will be eitherresident or elicited, if they normally populate a givenanatomic site in an untreated animal, or have theirproduction stimulated (Jacques 1970), respectively. Thisprocess may affect the surface of the macrophages. It hasbeen shown by Silva Filho et al. (1987) that resident,elicited, and activated mouse peritoneal macrophages allpossess a negative charge, however, the charge is greatestwhen the macrophages are activated.

Since cells of the monocyte-macrophage lineage arecapable of rapid firm adherence to solid surfaces, whichenables the enrichment of the macrophage population toproduce macrophage rich monolayers by adherence to plastic


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28or glass as first described by Mosier (1967) and laterreviewed by Pennline (1981). This procedure allows for theseparation of mixed-cell suspensions into adherent(macrophage-rich) and non-adherent (lymphocyte rich)populations. Other cells present either degenerate rapidly,as do the polymorphonuclear cells, or fail to stick to theglass and are washed off with the first change of medium, asare the lymphocytes . The adherent fraction is heterogeneousand has been shown to contain populations of cells thatdiffer in their ability to bind antigen, form rosettes withantibody-coated erythrocytes, and kill tumor cells.Functional and morphological differences have been describedbetween adherent populations isolated from sourcescontaining activated, elicited, or resident macrophages aswell as within a given population between poorly adherentand strongly adherent cells.

The procurement of macrophages from the lung isespecially useful when a highly purified population ofmacrophages is required to study morphological, metabolicand functional parameters . When macrophages were recoveredfrom rabbit lungs, 90% to 95% of the total cells recoveredwere macrophages with 85% to 95% cell viability (McGee andMyrvik 1981). The same was found for AM of mice, rats andguinea pigs (Oren et al. 1963, Kirkawa and Roneda 1974, vanoud Alblas and van Furth 1979). The lower respiratory tractin a healthy individual is normally sterile. It has been


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29reported that >98% of the cells obtained via tracheal lavageare pulmonary alveolar macrophages with 1% to 2% lymphocytesand granulocytes as contaminants (Coggle and Tarling 1984).

Almost all macrophages isolated by lavage are viable;morphological, cytochemical and functional studies can beperformed as usual for comparison to other mononuclearphagocytes. In vitro , AM exhibit rapid spreading and highmitotic rate, and their cytoplasm is notably pyrominiphilic(Carr 1973). Pulmonary macrophages are positive for esteraseand negative for peroxidase, carry Fc receptors and showavid phagocytosis and pinocytosis. However, mature AM inlavage fluid rarely carry C3 receptors (van oud Alblas andvan Furth 1979) .

Lung SurfactantMammalian lung stability requires the presence of a

tensioactive material at the air/water interface, able tosupport very high surface pressures on dynamic compression,to prevent alveolar collapse at the end of expiration(Clements et al. 1961, Schwick et al. 1982, Keough 1985,Dobbs 1989). Not only do these structures not collapse dueto surface tension, they also can undergo appreciableexpansion and contraction. The surface forces regulating themechanics and stability of lung alveoli are modulated by thepresence of amphipathic substances at the air-waterinterface. This material is known as pulmonary surfactant


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30and has been well characterized, particularly in connectionwith infant respiratory distress syndrome (Ivey et al. 1977,Fujiwara et al. 1980, Haagsman and van Golde 1985, Jobe andIkegami 1987, Robertson and Lachmann 1988).

This surface-active material is a complex of lipids andprotein, as can be seen in Table 1. Dipalmitoylphosphatidyl-choline (DPPC) accounts for -60% of the phospholipidfraction, the other 40% being unsaturated phosphatidyl-choline (PC), and about 5% neutral lipid which is mostlycholesterol (Keough 1985).

TABLE 1Composition of Pulmonary Surfactant

Composition by Weight (%)

Phospholipids 85Saturated phosphatidylcholine 60Unsaturated phosphatidylcholine 20Phosphatidylglycerol 8Phosphatidylinositol 2Phosphatidylethanolamine 5Sphingomyelin 2Others 3

Neutral lipids and cholesterol 5Proteins 10

Contaminating serum proteins 8Sp-A: 32 to 36,000 kDa -1Lipophilic proteins: 6 to 18,000 kDa -1from Jobe and Ikegami (1987)

When Chapman (1975) analyzed the lung lipids of 7animal species, all contained considerable quantities of a


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31fully saturated palmitate lipid. Around 50% of these weredisaturated like DPPC and rigid at 37C, which he postulatedto be due to the aerobic environment in the lungs, and thenature of these phospholipids to be less readily oxidizedthan their unsaturated counterparts

.

Recent studies of surfactant metabolism in vivoindicate alveolar surfactant phospholipids are in a complexdynamic equilibrium with the intracellular surfactant poolof alveolar type II cells. Physiologically, type IIpneumocytes synthesize the phospholipid and concentrate itin the form of lamellar bodies which are excreted in theaqueous alveolar subphase by exocytosis where thephospholipid becomes part of lung surfactant. The lamellarbodies undergo transformation into other characteristicstructures such as tubular myelin, certainly responsible forthe very fast adsorption rate at the air/water interface ofthe surfactant (Keough 1985). This surfactant is thenbelieved to be recycled by the alveolar macrophages. Forinstance, in three-day-old rabbits, the majority of theapproximately 5 umole phosphatidylcholine pool seems to berecycled back into type II cells and subsequently resecretedwith a turnover time around 10 hours (Jacobs et al. 1982).Therefore, this system can be considered as a process ofsynthesis, storage and secretion of surfactant components(Goerke 1974)

.


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32In addition to phospholipids, pulmonary surfactant

contains a protein component composed of three specificsurfactant apoproteins: SP-A is a higher MW glycoprotein(35,000), relatively hydrophilic and water soluble; and twolow MW apoproteins SP-B (8,000) and SP-C (5,000) which areextremely hydrophobic (Hawgood 1989). Metabolic experimentsshow the larger protein to be excreted into the alveolarlumen at the same rate as the lipids of pulmonarysurfactant.

DPPC is considered the major component responsible forpulmonary surfactant stability and surface activity (Kingand Clements 1971). However, clinical data indicate thatphosphatidylglycerol (PG) also plays a key role (Hallman etal. 1977). In addition, various studies have examined therole of the protein components. King and MacBeth (1979)found >71% binding of apoprotein to DPPC at roomtemperature, which greatly enhanced the adsorption(spreading) of DPPC at an air/water interface. Theapoprotein presence did not alter the capacity of DPPC tolower the surface tension below 5 dynes/cm. It has also beenshown that synthetic phospholipid mixtures with SP-B and SP-C alone or in combination adsorb rapidly to an air-liquidsurface and lower surface tension during prolonged dynamiccompression (Holm et al. 1990, Venkitaramam et al. 1991).

Immunoglobulin studies (LaForce 1976) indicate thepresence of both IgG and IgA in lavage supernatant, but


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33neither are found in the extracted lipid fraction. These mayspecifically be involved in the opsonization of particlesand therefore important for the recognition and uptake ofparticles by AM.

In addition, LaForce et al. (1973), LaForce (1976) andJuers et al. (1976) have shown alveolar lining material(ALM), the proposed pulmonary surfactant fraction extractedfrom bronchoalveolar lavage fluid, to be important in the invitro bactericidal capacity of AM.

It was the similarities between the composition ofpulmonary surfactant and a potential drug delivery system(liposomes) that prompted us to study them in associationwith the phagocytic ability of AM.

Background LiposomesPhospholipids

Phospholipid molecules consist of a glycerol backbonewhich is esterified with two fatty acid chains, and aphosphate group esterified with an aminoalcohol , alcohol orcarbohydrate. The molecules are clearly amphiphilic with ahydrophilic "head group" and a hydrophobic "tail". Manyearly studies discussing the physicochemical properties ofphospholipids, primarily organized as a monolayer at an airwater interface were reviewed by Bangham (1968).

There are two types of phospholipids, glycolipidsand sphingolipids the protypes being phosphatidylcholineand sphingomyelin, respectively (Figure 1). Although both


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A: PHOSPHOLIPID 34

Water-soluble head Water -insoluble tail

1 ICH 2 0-C CH (CH) CH2 n 3FATTY ACID

CH CH- I I-CFATTY ACID-CH (CH)m CH

X = -CH - CH -N-(CH2 2choline

-CH - CH - COO "21NH

serine

3 ) 3 OH OH

OH OHinositol

-CH 2-CH- CH ,- OH1

- CH - CH -NH +2 2 31

OHglycerolethanolamine

Figure 1: Structural representation of phospholipid (A) andsphingolipid (B). The circled area (top left)represents the hydrophilic headgroup with possibleX substituents (bottom box), and the shaded area(right) represents the hydrophobic, fatty acidportion of the molecule, with m and n denoting thelength of the hydrocarbon chain.


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35

B: SPHINGOLIPID

Water-soluble head

CH

Water -insoluble tail

OH

CH-CH = CH-(CH ) CH 3FATTV ACID

Figure 1: (continued)


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36groups contain the characteristic phosphorylcholine headgroup, they are distinctly different in their hydrophobictails. Whereas the phosphatidylcholines have two long chainhydrocarbons of nearly equal length attached to carbon twoand three of the connecting glycerol backbone, sphingomyelinhas an acyl chain of 16 to 24 carbons attached to the secondcarbon linked by an amide bond, and a paraffinic residue ofthe sphingosine base, which contributes only 13 to 15 carbonatoms to the nonpolar region. Synthetic derivatives of thesephospholipids can be made with varying degrees of saturationand chain lengths

.

Phospholipids belong to a class of lipid compoundsincluding surfactants, which are known to undergo lyotropicmesomorphism, or they exist in various hydration statescalled liquid crystals. According to the classification ofSmall (1968), phospholipids belong to the Class II:Insoluble Swelling Amphiphiles . They are insoluble in water,but water can penetrate the hydrophilic phospholipidheadgroups. Due to their amphipathic nature, they can existin different types of liquid crystalline organization uponhydration (Luzzati et al. 1968). The form occupieduponhydration, depends on the physical structure of themolecule, including micelles, bilayers and hexagonal arrays(Figure 2) (Cullis and DeKrujff 1979). If the molecule has asingle hydrophobic tail (i.e. lysophosphatidylcholine) orthe headgroup is small in relation to the tails, it will


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37

Phospholipid Molecular shape Phase

Lysophos

-

pholipidsV

inverted cone micellarPhosphatidyl-cholinePhosphatidyl -serinePhosphatidyl -glycerol

cylindrical

ftftffff....bilayer

Sphingomyeline

stacked cylinder....

interdigitated bilayer

Phosphatidyl -ethanolamine(unsaturated)Phosphatidyl serine(pH < 4.0)

ZA

hexagonal (H. )

Figure 2: Phospholipids with correspondshapes and polymorphic phasesbilayer, (C) interdigitated,ing dynamic molecular; (A) micellar, (B)(D) hexagonal.


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33assume the structure of a micelle (Figure 2A) , if there aretwo chains of similar length in balance with the headgroup(i.e. PC) a lamellar structure will result (Figure 2B), ifthe chains are of sufficiently different length (i.e.sphingomyelin (SPM) ) interdigitation of the bilayer (Figure2C) can occur, and if the headgroup is large in relation tothe tails (i.e. phosphatidylethanolamine (PE)) a hexagonalphase (Figure 2D) will form. In addition, bilayer tohexagonal phase transitions can be induced by binding ofdivalent cations to acidic phospholipids such asphosphatidylserine (PS) (Harlos and Eibl 1980, reviewed byCrowe and Crowe 1984).

When bilayer forming phospholipids swell upon contactwith water they spontaneously rearrange to form closedconcentric bilayers of phospholipid enclosing an aqueousspace (Figure 3) with the hydrophobic tails opposing eachother and the hydrophilic head groups facing the surroundingaqueous bulk phase. This derives from the opposinghydrophobic interactions of the hydrocarbon tails that forcelipids to assemble and hydrophilic tendencies of theheadgroups seeking to be solvated by water. For pure lipidmolecules, this leads to the formation of an extendedbilayer that must close and form a vesicle to preventexposure of the hydrocarbon chains to water at the edges ofthe bilayer. Since the molecules are essentially insolubleand do not form micelles, there is no equilibrium between


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40individually dissolved molecules, micellar aggregates andbilayers (Lasic 1988).

LiposomesThe early characterizations of phospholipid vesicles

were done by Bangham and Home in 1964, who observed thatpurified phospholipid mixtures extracted from biologicalmembranes, spontaneously swell in aqueous salt solutions to

form liquid crystals. These lamellar structures were shownto be able to entrap ions in water and release them atvariable rates, suggesting that each consisted of completelyclosed phospholipid bilayers forming selective permeabilitybarriers (Bangham et al. 1965b). In 1968 these dilutephospholipid dispersions were termed "liposomes" by Sessaand Weissmann.

Bangham' s group characterized the diffusion of water(Bangham et al . 1967) and ions (Bangham et al. 1965 a,b)across these multilamellar vesicles. They were found to beosmotically sensitive, with permeability characteristics forsimple univalent cations, anions, and water, qualitativelysimilar to those occurring across biological membranes; i.e.freely permeable to water, CI", and I" but less so to F",N0 3_, S0 4 2 " and HP0 4 2 ". In particular, CI" and I", are byseveral orders of magnitude more free to diffuse than simpleunivalent cations and uncharged molecules. Likewise, cationsdo not diffuse through positively charged multilamellarvesicles whereas anions diffuse freely. Due to their osmotic


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41sensitivity, these bilayers are predicted to be impermeableto small polar solutes (Bangham et al. 1972).

There are two basic types of lipid dispersions:multilamellar vesicles (MLV) and small unilamellar vesicles(SUV). MLV, first described by Bangham and Home (1964),consist of vesicles in which phospholipid is organized intoconcentric bimolecular lamellae, each separated from itsneighbor by an interspersed water lamella. Each lamella is atopologically closed surface, with a single liposomecomposed of many concentric vesicles . A typical aqueousdispersion of this type contains multilamellar liposomeswhich vary in size in the micron range. The second type, SUVor small unilamellar vesicles, described by Huang (1969),are spherical vesicles, homogeneous in size, consisting of asingle continuous lipid bilayer enclosing a volume ofaqueous solution. These have been shown to be metastablewith time, undergoing fusion into larger unilamellarvesicles, or into MLV (Suurkuursk et al. 1976, Lasic 1988).

Through the years, many methods have been establishedto prepare liposomes (Szoka and Papahadjopoulos 1980, Martin1990, New 1990). The original method described by Bangham etal. (1965b) is also the simplest and involves the depositionof a phospholipid film after evaporation of organic solvent,phospholipid film reconstitution with an aqueous buffer,then gentle shaking until the lipid is hydrated and has


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42formed MLV (Figure 4). Sonication or extrusion can be usedto obtain a smaller and/or more uniform preparation.Sonication is difficult to standardize (Huang 1969),whereas, pressure extrusion through polycarbonate filterswith a defined pore size is easy to employ and givesreproducible size distribution (Olson et al. 1979).Encapsulated water soluble compounds can be separated fromnon-encapsulated by dialysis, gel filtration orcentrifugation (Olson et al. 1979).

The type of phospholipid used in liposome preparationcan influence the intracellular volume and hence, theentrapped volume of MLV. Charge repulsion via incorporationof a charged phospholipid such as phosphatidylglycerol in

the liposome membrane is a useful means for increasing theencapsulated volume due to a wider spacing distance betweenbilayers (Bangham 1968, Gregoriadis et al. 1977). This canalso improve the physical stability of the liposome due toreduction of aggregation and fusion. In addition, lipophilicdrugs can be anchored into the liposomal lipid phase(Gregoriadis 1973, Juliano and Stamp 1978).

The type of phospholipid used also has a great effecton liposome stability. In particular, incorporation of 30 to50 mol% cholesterol has been shown to stabilize bilayers ofunsaturated phospholipids (Ladbrooke et al. 1968). Certainphospholipids are inherently unstable due to theirpropensity to undergo oxidation (ie., unsaturated PC).


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43

))

^7

Thin Lipid Film Dispersion in AqueousMedium

SizingExtrusion

Figure 4: Preparation of multilamellar vesicles, and sizingby extrusion through a polycarbonate membrane.


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44Incorporation of an antioxidant or the use of fullysaturated phospholipids can inhibit this effect (Allen1981)

.

In vivo , liposomes are susceptible to release ofencapsulated materials when they come in contact with plasmacomponents. This happens in a non-linear fashion presumablydue to a rearrangement of membrane lipids and adsorbedproteins to form their most stable configuration (Hunt1982). According to Damen et al. (1980, 1981), both highdensity lipoprotein and non-lipoprotein components of humanand rat plasma cooperate in the destructive action of plasmaon phosphatidylcholine liposomes

.

In general, the permeability of liposomes to entrapped

solutes has also been shown to increase when they interactwith various cell types (Szoka et al. 1979, van Renswoude etal. 1979, van Renswoude and Hoekstra 1981, Hoekstra et al.1981, Margolis et al . 1982).

Therefore, the success of liposomes as vehicles for thetargeted delivery of specific drugs depends on theircompatibility with the encapsulated drug, their stabilityunder physiological conditions and their ability to interactwith specific target sites. This will depend on the typesand amounts of phospholipids used for the preparation andwill require the characterization of a wide variety ofphospholipid mixtures in conjunction with biologicallyrelevant ions and molecules.


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45Phvsicochemical Parameters of Liposomes

Thermotropic Phase TransitionsIn addition to lyotropic mesomorphism, phospholipids

also undergo thermotropic mesomorphism or temperatureinduced phase changes. This is similar to the thermotropicbehavior of the soaps. When a soap is heated to a certaintemperature (sometimes several hundreds of degrees below thefinal melting point), the hydrocarbon chain portion "melts",the all planar trans configuration breaks up, and the chainsnow contain gauche isomers (Chapman 1958) . Phospholipidsexhibit this same property. The capillary melting pointsrange from 200C for phosphatidylethanolamine to 230C forphosphatidylcholine. However, in addition to the capillarymelting point, a temperature dependent endothermictransition occurs around 120C and again at ca. 135C (Byrneand Chapman 1964, Chapman and Collin 1965) (Table 2). Thistransition does not involve the change of state from solidcrystal to "normal" lipid usually implied by the term"melting", but rather a shift from a crystalline gel to a"liquid crystal". In other words, above the transitiontemperature, the lipid chains are "melted and fluid" , whereasbelow the transition temperature the chains are organized ina crystalline manner (Chapman and Salsbury 1966)

.

One of the first to characterize the gel to liquidcrystal transition (Tc ) in phospholipids was Chapman (1968)who related the temperature of this phase change to a


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46TABLE 2

Phase Transitions of Anhydrous PhospholipidsPhospholipid Liquid Crystal (C) Melting Point (C)

236-237234-237219-234159-160195-207175-185170-182190-198

209-211209-210213-216205-207

from Dervichian (1964) and references therein.

dependence on phospholipid chain length and degree ofsaturation. The phase transition was found to be primarilyconcerned with the hydrocarbon chains since the space takenup by glycerol and the polar group remained essentiallyunchanged when the phase transition occured (Chapman et al.1967) . The glycerol and polar groups retain a fairly regularorganization although the polar group does have considerablemobility, but the fatty acid chains "melt" and acquireconsiderably more mobility, with the methyl end of the chainhaving greatest motion (Chapman and Salsbury 1966) . Only one

DMPC 105DPPC 90DSPC 90DSPS 120DMPE 86DPPE 135DSPE DSPC(unsaturated)SPM

C24Natural from brain

180-190iii

165-175


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48of additional liquid crystalline forms between the firsttransition temperature and the capillary melting point(Chapman et al. 1967). As the amount of water increases, thetransition temperature is decreased until it reaches alimiting value corresponding to the maximum uptake of boundwater by the phospholipid gel (20% or more water by weight)(Ladbrooke and Chapman 1969). In this excess of water, thehydrated phospholipid transforms from a gel phase to asmectic mesophase or liquid crystal at temperatures abovethis transition temperature (Ladbrooke and Chapman 1969,Ladbrooke et al. 1968) called the gel to liquid crystallinetransition.

In the ordered gel state below a characteristictemperature Tc , the hydrocarbon chains are in an all-transconfiguration. On increasing temperature, an endothermicphase transition occurs during which there is a trans-gaucherotational isomerization along the chains resulting in alateral expansion and a decrease in the thickness of thebilayer (Wilkinson and Nagale 1981).

This transition temperature occurs at a temperaturecharacteristic of both the nature (chain length andsaturation) of the hydrocarbon chains (Table 3) and thepolar head group (Table 4) of the molecule and can rangefrom approximately -20C to about 60C (refer to Szoka andPapahadjopoulos 1980 for a list of phospholipids with chargeand Tc ). If water is added to DPPC in the anhydrous state,


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50to a degree approximating physiologic conditions, the Tcdecreases sharply from 100C to ca. 42C. Thus < 42C, thelipid is in a gel state, while > 42C it is in a fluidstate. On the other hand, unsaturated phosphatidylcholine inthe presence of water undergoes a transition around -20C.Thus at biological temperatures, phospholipids with highlyunsaturated chains can be expected to be in a highly mobileand fluid state.

Multilamellar dispersions of homogeneous phospholipidsexhibit two reversible phase transitions: the major chainmelting transition characterized by a sharp symmetric first-order endothermic transition, and an additional broadertransition of lower enthalpy occurring 5 to 10C below themajor transition. The distance between the 2 transitionsdecreases with increasing chain length. Structural changesaccompanying the pretransition are unclear. Thepretransition of DPPC has been associated with the motion ofthe phospholipid polar headgroups by Ladbrooke and Chapman(1969), the co-operative movement of the rigid acyl sidechains in a transition between crystal forms below theirmelting temperature (Hinz and Sturtevant 1972a, b) and, as athird possibility, tilting of the hydrocarbon chains beforemelting (Chapman et al . 1974). Interestingly enough, thepretransition is negated when mixtures of phospholipids arestudied.


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52proceed in an endothermic direction when the temperature israised, and exothermic peaks will be observed only if theprocess is kinetically rather than thermodynamically limited(Mabrey-Gaud 1981). Since the shift from gel to liquidcrystal is endothermic (i.e. relatively large quantities ofheat must be absorbed in order to break the bonds that holdthe molecules in the rigid gel structure), a sharp peak inheat absorption at a given temperature signals the change ofstate that in fact has occurred (Chapman 1975) (Figure 5).

The DSC apparatus consists of two cells: one for thesample and the other for an inert reference material, whichcan be heated at a programmed rate controlled to maintainzero temperature difference between the cells. If the sampleis in solution or suspension, the reference material will bethe corresponding solvent. When a thermally initiatedprocess takes place in the sample cell the control systemresponds by supplying either more or less heat to the samplecell to hold its temperature equal to the reference cell.Data output, of either excess heat or the correspondingexcess power, is presented as a function of temperature(Mabrey and Sturtevant 1978).

The point of departure from the baseline, the onsettemperature as seen in Figure 5, is normally taken as thetemperature of transition. Since the peak maximumcorresponds to the temperature at which change is occurringat the maximum rate and the point at which the curve returns


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3.75 -

53

2.5

1.25 -

A: HEPC heating thermogram

Paak frami 49. 53toi 58. 28Onaat- 51. 17

J/gm. - 89. 41Paak- 52. 68

35.00 40-00 45.00 50.00

Temperature (C)55.00

Figure 5: Representative endothermic phase transition forheating (A) and cooling (B) of hydrogenated eggphosphatidylcholine.


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3.75

2.5

1.25

54B: HEPC cooling thermogram

Pack fromi 43. 02toi 51.97OnsQt- 49. 32

J/gm. 72. 49

35.00 40.00

Poak- 49. 08

45.00 50.00Temperature (C)

55.00

Figure 5: (continued)


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55to the baseline is influenced by instrumental factors,neither of these points are useful in determining thetemperature range of the transition. In order to distinguishbetween an isothermal transition and a change taking placeover a finite temperature range, the transition region canbe defined by the difference between onset temperature onheating and cooling . For a pure lecithin this differenceshould be less than one degree Celsius, indicating a trulyreversible isothermal transition (Phillips et al. 1970).

Ladbrooke and Chapman (19 69), showed the importance ofwater on the mesomorphic behavior of phospholipids andemphasized the need to maintain a constant amount of waterin samples for thermal analysis. However, when the lipidcontains 50% weight water or more, the phase transitions andthermal spectrum are insensitive to variations in the actualwater content of the phospholipid.

Factors Affecting Thermotropic Transitions of PhospholipidsThe thermogram can also be affected by pH, divalent

ions, and vesicular structure (packing constraints of theacyl chains) (Szoka and Papahadjopoulos 1980) . This isparticularly true for acidic phospholipids.

Effect of pH. The phase behavior of phosphatidylcholineis little affected by pH between 3 and 13, correspondingwith the respective pKa's for the ionizable groups(Kimelberg and Mayhew 1978) . In contrast tophosphatidylcholine, the phase behavior of phospholipids


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56

with glycerol or serine headgroups is pH sensitive and thussensitive to the charge on the polar headgroup (Table 5)

.

This has been ascribed to a decrease in the repulsive forcesbetween adjacent negatively charged phosphate groups(Jacobson and Papahadjopoulos 1975, Findlay and Barton 1978,van Dijck et al. 1978).

TABLE 5Effect of pH on T of DPPG and DPPS

Phospholipid pH ReferenceDPPG 1.1 57.0 Cevc et al. (1980)Watts et al. (1978)

7.0 41.5 van Dijck et al.(1978)Jacobson andPapahadjopoulos(1975)

DPPS 1.0 62.0 Cevc et al. (1981)7.0 54.0 Cevc et al. (1981)13.0 32.0 Cevc et al. (1981)

Effect of ions. Generally, the transition of neutralphospholipids is little affected by monovalent cationsincluding sodium and potassium even at concentrations of 1M. The presence of 1 M magnesium increases the transitiontemperature of DPPC slightly, while calcium concentrations


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58Vesicular structure. In addition to phase separations

with negatively charged phospholipids, the addition ofcations to DPPS or DPPG can induce a change in vesicularstructure from a bilayer to a hexagonal phase. This changein state can also produce changes in Tc as seen in Table 6

.

TABLE 6Effect of Vesicular Structure on Tc

Phospholipid Conditions Tc (C) ReferenceDMPC

DPPS

pH 7.0 23IM CaCl 2pH 4.6 81 Harlos

and Eibl1980pH 7.0 54>1.5M LiCl 2pH 7.5 90 Seddon et al

.

1984

Thermal Transitions in Phospholipid Mixturesand Phase DiagramsThe phase behavior of mixed phospholipid and water

systems was first reported by Ladbrooke and Chapman (1969)and was studied in more detail by Phillips et al. (1970)using calorimetric methods. In contrast to purephospholipids which exhibit sharp, highly cooperative phasetransitions, mixtures of phospholipids containing differenthydrocarbon chains melt over a much broader temperaturerange. The shape of the transition as well as its positionis dependent on lipid composition, and a significant


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59asymmetry is apparent at compositions other than equimolar(Mabrey and Sturtevant 1976)

.

The equilibrium between two component lipids in thesame two-dimensional bilayer plane at various temperaturesare frequently described in terms of a phase diagram. Suchdiagrams be can constructed from the phase transition curvesof binary lipid mixtures obtained using high sensitivityDSC. The phase diagram is constructed based on the onsettemperature of the cooling and heating curves (Chapman etal. 1974) of a series of phase transition curves for the twocomponent mixtures at various molar ratios, generallyplotted as a function of the relative concentration of thehigher melting component. However, the onset and completiontemperatures can also give information on the miscibility ofphospholipid mixtures as described by Mabrey and Sturtevant(1976) .

The simplest mixing behavior of binary lipid mixturesin the bilayer is exhibited by the isomorphous system inwhich the two component lipids are completely miscible overthe entire composition range in both the gel and liquidcrystalline phases (Mabrey and Sturtevant 1978, Melchior andSteim 1979) . Based on the onset of cooling and heating, thephase diagram for this type of mixture gives rise to acigar-like enclosed region (Figure 6) with threeidentifiable regions. The area above the upper curve (i.e.the onset temperature of heating) , represents the point


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60above which the phospholipid is in a more fluid state called"liquidus"; the area below the lower curve (i.e., the onsettemperature of cooling) represents the point below which thephospholipid is in a more solid or gel state called"solidus"; and the enclosed area (region of calorimetricpeak) contains both phases in equilibrium. The phaseboundaries of such a system are defined by smooth andcontinuous solidus and liquidus curves . The composition andamounts of liquid and solid phases at any temperature can beobtained directly from the phase diagram.

For binary mixtures of diacyl phospholipids, if thecomponent lipids are very similar in both structural andpacking properties, then one component lipid can be replacedisomorphously by the other in the lamella in both the geland liquid crystalline states. For example, when the chainsof two components are similar (e.g., nCu and nC18 ), co-crystallization and ideal mixing of the componentphospholipids continuously in the bilayer plane occurs(Chapman 1976); consequently, lipid dispersions of thesebinary mixtures will display phase diagrams with a cigarshape. In addition, saturated phospholipids of the sameheadgroup, differing by 2 methylene units in acyl chains,will exhibit complete miscibility in all proportions. Theseisomorphous systems are often detected for different diacylphospholipids with the same polar headgroup and with a smalldifference in acyl chain lengths as seen for the mixtures in


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61

uoLJCC

|LJQ_UJ

(/J 10% PC and narrow transitions were seen as longas the chain lengths were the same.

Use of DSC in Liposome FormulationDifferential scanning calorimetry has been shown to be

potentially useful in studying phospholipid interactionswith drugs and other macromolecules (Juliano and Stamp 1978)especially if the drug is sufficiently hydrophobic andcreates an easily quantifiable change in Tc or transitionendotherm. These include Cortisol esters (Fildes and Oliver1978), morphine derivatives and antidepressants (Cater et


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64al. 1974, Knight and Shaw, 1979) and various small molecules(Jain and Wu 1977) .

Other compounds causing a thermogram change includeproteins (Papahadjopoulos et al. 1975a, Melchior and Steim1979), sterols (Ladbrooke et al. 1968) and metal ions(Hauser et al. 1969) .

When phospholipids react with proteins, a lowertemperature reversible Tc due to phospholipid and a highertemperature transition due to protein are observed. Thisimplies the polar lipid-protein interaction is not extensive(Melchior and Steim 1979).

It has been shown that some substances can stabilizephospholipid membranes by eliminating the gel to liquidcrystalline phase transition. This includes the sterols:cholesterol (Ladbrooke et al. 1968, Papahadjopoulos et al.1973), a-tocopherol (Ortiz et al. 1987) and vitamin D 3(Castelli et al. 1990). Thus, DSC may be a very useful toolfor the characterization of liposome formulations

.

Knowledge of Tc is practically indispensible inliposome formulation since the ability of phospholipids toform liposomes increases markedly above the transitiontemperature where dried phospholipid is hydrated easiest.Only those phospholipids with Tc in water near or below roomtemperature spontaneously form bilayers. Fully saturatedphospholipids with high transition temperatures do not formbilayers unless the temperature is raised above Tc .


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66Electrophoresis involves the motion of dissolved or

suspended material under the influence of an appliedelectric field. It is one of four related electrokineticphenomena, the others being electro-osmosis, streamingpotential, and sedimentation potential. All four involve therelative movement between the rigid and mobile parts of anelectric double layer (Figure 7).

The electric double layer arises due to the acquisitionof a surface electric charge when a substance is broughtinto contact with a polar (i.e., aqueous) medium and themixing tendency of thermal motion. It is comprised of thecharged surface and a neutralizing excess of counter-ionsover co-ions distributed in a diffuse manner in the medium(Figure 7). The situation is that of a double layer ofcharge: one localized on the surface of the plane and theother in a diffuse region extending into the solution. Thequantity which is a measure of both the degree of surfacecharge and the distance the effect extends into the solutionis the "zeta potential". Ions or molecules close to theparticle's surface are not free to migrate in the liquiduntil they pass the plane (or surface) of shear.

If an electric field is applied tangentially along thecharged surface a force is exerted on both plates of theelectric double layer. The charged surface (plus attachedmaterial) tends to move in the appropriate direction, whilethe ions in the mobile part of the double layer show a net


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67

ZETA POTENTIAL

ELECTRICPOTENTIALSURROUNDINGTHE PARTICLE

DISTANCEPLANE OF SHEAR

Figure 7: The electric double layer, represented by aspherical particle with a net negative surfacecharge suspended in aqueous medium and surroundedby a layer of opposite charge. Below the particleis a diagram indicating the electrical potentialas a function of distance from the surface of theparticle. The potential at the plane of shear isthe zeta potential.


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68migration in the opposite direction carrying solvent alongwith them, thus causing its flow. Conversely, a potentialgradient is created if the charged surface and the diffusepart of the double layer are made to move relative to oneanother.

Electrokinetic phenomena are only directly related tothe nature of the mobile part of the electric double layerand may, therefore, only be interpreted in terms of zetapotential or charge density at the surface of shear. Theelectrophoretic charge or zeta potential does not representthe actual charge on the surface of the particle, as eachwill move in the electric field with an associated cloud ofcounterions which are in equilibrium with those present inthe bulk aqueous phase. The actual mobility therefore willdepend on the charge existing at the plane of shear betweenthe particles and the solution.

There is a direct relationship between the zetapotential and the electrophoretic mobility (the velocity ofthe particle per unit electric field) of colloidal particlesaccording to the Smoluchowski equation.

iiie= DCe/4fl-T7

where /je = electrophoretic mobility (/x/sec per volt/cm) , D=dielectric constant of medium, e= strength of appliedelectric field (V/cm) , rj = viscosity of medium (poise) , andC = zeta potential in mV. For the derivation of this


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69equation one can refer to Overbeek and Wiersema (1967) andWiersma et al. (1966).

This equation can be simplified to:Me = e/T)

where e now equals permittivity. Permittivity is related tothe dielectric constant by ( x/e vacuu)

For an aqueous medium at 25C, the dielectric constantof water is 78.8, and the viscosity is .00899 poise, thusthe zeta potential is related to the electrophoreticmobility by: C = 12.85 pe millivoltswith fie expressed in micron/sec per volt/cm.At 37C this becomes: C = 10.35 fie

Factors Affecting ElectrophoresisParticle size. The Smoluchowski equation is applicable

to particles in which the double layer is effectively flat(i.e. large Ka, where Ka is the ratio of the radius ofparticle curvature to double layer thickness) , includingcolloids and cells (Shaw 1969) . Generally this includes allparticles > 10 nm. Accordingly, the electrophoretic mobilityof a non-conducting particle for which Ka is large at allpoints on the surface should be independent of its size andshape provided that the zeta potential is constant.

Phospholipid bilayers were previously shown to conformto double layer theory, with the Smoluchowski equation beinguseful for the conversion of the measured electrophoreticmobility to the zeta potential (McLaughlin et al. 1981)

.


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7

Viscosity . The viscosity of water decreases withincreasing temperature as seen in Table 8. In addition,various other molecules such as sugars can profoundly affectviscosity as seen in Table 9. This can affect the measured/xe, particularly at high temperatures.

TABLE 8Effect of Temperature on Dielectric Constantand Viscosity of Water

Temperature r\ (cp) e20 1.002 80.125 0.8993 78.830 0.798 76.5435 75.04

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