s2 the Role of Liposomes in Drug Delivery and Diagnostic Imaging 2003

8/3/2019 s2 the Role of Liposomes in Drug Delivery and Diagnostic Imaging 2003

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THE ROLE OF LIPOSOMES IN DRUG DELIVERY AND DIAGNOSTICIMAGING: A REVIEWMARK . MATTEUCCI,VM , DONALD. THRALL,VM, PHD

Veterinary Radiology & Ultrasound, Val. 41, No. 2, 2000, p p 100-107.

IntroductionPPLICATIONS USING liposomes have grown significantly.A echnologic advances in the fields of materials, andchemical, and biophysical engineering have resulted in theability to prolong circulation of microparticles in the blood.Liposomes have been used in many scientific disciplinesand knowledge from basic research evolved into their in-

corporation into practical and clinical applications. Eventhough they have been extensively investigated for over 30years, it has only been in the last decade that their potentialis being realized in the targeted delivery of chemotherapeu-tics and in imaging applications. The literature containsmany reviews and books on liposomes and their applicationto medicine;'-' however, there are few reports discussingliposomes and their applications in veterinaryLiposome research has become very specialized andcrosses over to many interrelated disciples. Today lipo-somes have been incorporated in fields such as mathemat-ics, physics, biophysics, chemistry, colloid science, bio-chemistry, and biology.' This paper will not attempt to re-view the numerous uses of liposomes within all these fields.Instead, our goal is to familiarize the reader with the conceptand applications of liposomes in drug delivery systems andongoing research using liposomes in diagnostic imaging.Where relevant, specific use of liposomes in veterinary on-cology and imaging will be discussed.

Liposome Structure and CharacteristicsLiposomes are phospholipid vesicles first described in

1965 by Alec BanghamI7 when he discovered that phos-pholipids in water form closed vesicles. Liposomes werefirst used in the study of cellular membranes, as they are agood model for lipid bilayers. Liposomes are made fromneutral phospholipids such as lecithin and cholesterol. Suchlipids are amphipathic, meaning their structure containsboth a polar head region and nonpolar aliphatic chains.When placed in an appropriate environment and at specificconcentrations these lipids spontaneously form a spherical

From the Department of Anatomy, Physiological Sciences and Radiol-ogy, College of Veterinary Medicine,North Carolina State University 4700Hillsborough St. Raleigh, NC 27606.Address correspondence and reprint requests to Dr. T hrall.Submitted February 8, 1999; accepted for publication June 29, 19 99.

structure. The sphere is a closed bilayered vesicle. The hy-drophilic polar groups face the aqueous interior core, whilethe hydrophobic nonpolar portions are tucked into the inte-rior of the membrane bilayer (Fig. 1). This effectively re-sults in two potential compartments to entrap a substance.The hydrophilic aqueous core can trap a hydrophilic sub-stance and the hydrophobic interior of the membrane cantrap lipid soluble substances. Liposomes can be manufac-tured to contain multiple concentric bilayers resulting in avariety of compartments for substance entrapment.'The unique structural properties of liposomes allow dif-ferent methods to be used to entrap substances. Dependingon the substance used, it will either be trapped while lipo-somes form spontaneously or the substance can be loadedinto preformed liposomes. Substances with intermediatesolubility can be loaded into the aqueous interior of existingliposomes by the use of ionic gradients or molecular com-plexes. For example, the commercially available liposomalformulation of doxorubicin, Doxil,@* s loaded into pre-formed liposomes using an ionic gradient method whereasliposomes can be labeled with technetium using a molecularcomplex with g l~ tat hi on e.' ~,' ~The size of liposomes varies according to their intendeduse. Liposomes can be manufactured with a diameter rang-ing from several nanometers to several micrometers. Lipo-somes in most current applications have a diameter rangingfrom 50-150 nm. This range is a compromise between theefficiency of loading, liposome stability and the ability ofthe liposome to extravasate from the vasculature. For ex-ample, large liposomes can be loaded relatively easily buttend to remain in blood vessels. Because liposomes aremade using naturally occurring biodegradable substances,once they reach the target site they are metabolized andcleared making them safe and nontoxic.Liposome Types

The first liposomes, known as conventional liposomes,were composed of egg phosphatidylcholine and choles-terol.' When injected intravenously, plasma opsonins andlipoproteins coat the outer surface of these conventionalliposomes. Subsequently, reticuloendothelial cells quickly

*Sequus, Men lo Park, CA, USA .

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VOL. 41, No. 2 ROLEOF LIPOSOMES 101

(a) No PEG coating

(b) PEG-coated liposome CholesterolB

FIG.1. T he structure of liposomal delivery systems. (a) Drug-containing liposomes in the absen ce of a polyethylene glycol (PE G) coating. Hydrophobicdrug is present in the lipos ome mem brane and hydrophilic drug is present in the liposome aqueous interior. Protein opson ins are shown absorbing to thenaked liposom e surface. (b) PEG-coated liposom e with entrapped d rug in the liposom e interior. Protein op sonins have significantly less absorption to theliposome surface. Reprinted from Drugs 1997;54(Suppl. 4):8-14. Pg. 9, Fig. 1.phagocytize and remove these liposomes from circulation.Initially there was widespread enthusiasm for the use ofliposomes in drug carriers systems, but this waned as theyfailed to live up to expectations for limited applications invaccine formulations and in targeting the reticuloendothelialsystem in antiparasitic therapy. This inhibited the wide-spread use of liposomes because of the resultant short cir-culating half-life and led to a period of skepticism amongscientists in the field of drug delivery.One of the most important breakthroughs n liposomeresearch was the discovery of a polyethylene glycol (PEG)derivative as a liposome coating. The new formulation ofPEG-coated liposomes results in greatly extended liposomecirculation times. These liposomes have been termed sec-ond-generation or sterically stabilized liposomes because of

their ability to evade phagocytosis by the reticuloendothelialsystem.8 Sterically stabilized liposomes have the ability toremain in circulation up to 1OOX longer than conventionalliposomes. This is due to the ability of the PEG coating toform a steric barrier by attracting water to the liposomesurface (Figure 1). The liposome surface becomes hydro-phobic, resulting in enhanced stability by inhibiting inter-actions with plasma proteins such as opsonins and lipopro-teins. Thus, PEG coated liposomes have a decreased affinityfor the reticuloendothelial system. The ability of PEGcoated liposomes to avoid rapid reticuloendothelial systemuptake has led to their use in drug delivery systems forsustained drug release and selective delivery of encapsu-lated substances to specific target sites.Despite the success and extensive use of PEGylation, in


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an attempt to achiev e even better control over the propertiesof surface modified liposomes, alternatives to PEGylationare being actively developed. Synthetic polymers of thevinyl series, such as polyacryl amide and polyvinyl pyrrili-done, have been used for surface modification of liposom es.The use of copolymers such as PEG and polylactide gly-colide (PEG-PLAG A) allows the ability to form a long cir-culating particle with an insoluble core and a water solubleshell.7 Research development is ongoing using different co-polymers, crosslinking or polymerization of lipid bilayers,and molecular manipulation of the liposome surface to alterits behavior after injection. The m ost important biologicalconsequence of these different surface modification sub-stances and techniques is a significant increase in liposomecirculation time and decrease in their reticuloendothelialsystem accumulation.

Liposomes as Drug CarriersIn a successful drug-carrier delivery system, the drugshould be retained within a stable carrier, the carrier shouldbe invisible to the reticuloendothelial system, and thereshould be a built in or controllable release mechanism forthe drug. Ideally, the drug would localize directly and spe-cifically at the intended targets, resulting in excellent thera-peutic efficacy, with few or no adverse effects. Addition-ally, the drug would have a high therapeutic index. Espe-cially in oncology, clinicians face toxicity problems withchemotherapeutic agents and the dose-limiting toxicity ofdrugs often results in suboptimal therapy. It is the ultimategoal of a drug delivery system to improve the therapeuticindex and decrease side effects.Liposomes have been extensively investigated for theirpromise of being an ideal drug delivery system. Their am-philipathic properties make them versatile carriers of eitherwater-soluble or lipid solub le drugs. The liposome bilayer isimpermeable to large molecules such as enzymes and has alow permeability to charged molecules. The entrapped drugis protected from metabolism within the liposome interiorand cannot be active or metabolized until released. Beingable to have extended circulation times may favorably alterthe ultimate rate and pathway of drug metabolism. It is theability of liposomes to alter drug pharmokinetics by chang-ing drug absorption, biodistribution, and clearance, whichresult in favorable effects at the target site.The ability to extend drug circulation times by encapsu-lation in a vesicle and the understanding of tumor micro-circulatory anatomy has resulted in the passive targeting ofdrugs into solid tumors. Regions of solid tumor growth,infection and inflammation have capillaries with increasedpermeability.22 In solid tumors, angiogenesis has beenshown to be an inherently leaky process with the new tumormicrovessels being disorganized, having a tortuous and con-voluted path compared to norm al vasculature. In addition,tumor vessels are typified by v enous lakes, regurgitant flow

and stasise2At the endothelial cell level the walls are notstructurally complete and therefore are permeable to colloi-dal sized particles. Thus, circulating, liposomal encapsu-lated drug can be delivered to the interstitium of tumors.Once in the tumor interstitial space, the liposomes becomeheterogeneously distributed throughout the tumor by diffu-sion and/or hydraulic convection.22 It has been shown thatnot only d oes the PEG coating increase circulation times butalso allows greater extravasation than conventional lipo-somes.22The end result is a several fold greater accumula-tion of drug in diseased tissue compared to normal tissue.Because liposom es and their associated drug are confinedprincipally to the vascular space and tissues with increasedvascular permeability, the volume of distribution? is dra-matically lower than with free drug that is widely distrib-uted to all tissues throughout the body. A reduction of drugconcentration in normal tissues alters the toxicity profile ofthe liposomally encapsulated drug relative to free drug. Byaltering drug distribution, liposomal encapsulation can de-crease many unwanted side effects and increase efficacy.The increased efficacy is due to the liposomes ability toincrease drug concentration at its intended site of action andby decreasing the drug concentration in sensitive normaltissues resulting in an increased therapeutic index. This con-trasts to intravenous administration of free drug where lessthan one percent of the injected dose reaches diseased tis-sue, whereas the rest damages healthy cells and tissues. It is not completely clear what happens to liposom es oncethey accumulate in tissue. Fluorescent microscopy hasshown that once trapped in tissue the liposomes disinte-grate, releasing drug in to the surrou ndings.21 The ex actmechan ism of drug release in not completely understo od butit is thought to be a result of bilayer chem ical disintegrationor mechanical rupture, enzymatic breakdown by lipases, orreversal of local concentration gradients causing drug leak-age.Currently there are several liposome formulations under-going testing in clinical trials and there are three liposomeformulations commercially available for use in humans.The first liposoinal drug formulation to be approved forhuman use was the antifungal agent amphotericin B, Am -bis0me.a-i: This formulation uses conventional liposomesand therefore allows more efficient drug delivery to mac-rophages and avoids the dose limiting nephrotoxic effectscommonly observed when non-encapsulated amphotericinis used. It has been shown that using liposomal amphoteri-cin B in dogs with blastomycosis resulted in effective treat-

?Volume of distribution at steady state provides an estimate of drugdistribution that is independent of elimination processes and provides as-sessment of the systemic distribution characteristics of a drug. It is afunction of a drugs affinity for and retention by, peripheral tissues. Be-cause liposom es do not accumulate in normal tissues the volume of dis-tribution is lower relative to freeSNeXstar, Boulder, CO , USA.


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ment and nephrotoxic side effects were absent even at twicethe recommended dose of free drug.16The most extensively studied liposomally encapsulatedanticancer drug formulation is liposomal doxorubicin,Doxil,@which con sists of doxorubicin encapsulated in steri-cally stabilized liposomes. By using PEG liposomes, favor-able characteristics such as extended circulation time andaltered drug biodistribution led to many favorable results inanimal studies and human clinical trials. This led to Dox-il's@ FDA approval on November 21, 1995 for use in Ka-posi s a r c ~ m a . ~ , ~ ~ - ~ ~n comparison to free drug or doxoru-bicin in conventional lip osomes, D oxil@ was show n to besuperior in efficacy and the common side effects of cardio-myopathy and myelosuppression were decreased. The su-perior therapeutic index has been shown to be due to in-creased localization of Do xil@ into tum or tissue and sus-tained release of drug over several hours directly into thetumor interstitial space resulting in high d rug concentrationswhile avoiding sensitive tissue such as myocardium.8,21Prolonging circulation times increase the chances of extrav-asation at sites with a leaky vasculature, such as in manytumors. In human clinical trials, Doxil@was shown to have11.4 times greater accumulation in soft tissue Kaposi sar-coma, and current research is focusing on other application sof Doxil@ n various solid tumors.'Other approved liposomal drug formulations includeD a u n o X o m e @ $ ( d u a n o r u b i c i n ) a n d E L A - M A X @ "(lidocaine). Other liposomally encapsulated drugs such ascis-platinum, nystatin, retinoic acid, and numerous vaccinesare currently in clinical trials in the United States and Eu-rope and show promise of eventual marketability.Liposomes as Drug Carriers in Veterinary MedicineLiposomes have been used in veterinary medicine a?well. In phase I dose escalation trials in dogs using Doxil,@the maximally tolerated dose was determined to be similarto that of free drug however side effects of myelosuppres-sion, gastrointestinal upset and cardiotoxicity were signifi-cantly decreased. The d ose limiting toxicity was a cutaneoustoxicity similar to palmar-plantar erythrodysthesia seen inhuman patients." Cutaneous lesions ranged from mild er-ythema, hyperemia, edema and alopecia to severe crusting,ulceration and epidermal necrosis. Typical locations are ar-

eas of pressure points including paws, axilla and inguinalregions. The cause of palmar-plantar erythrodysthesia ispoorly understood but it is speculated that lesions occur asa result of prolonged drug circulation times or greater ac-cumulation of Doxil@ n the skin to a greater degree thanfree drug. Do xi P was used to induce clinical remission in adog with chemotherapy-resistant plasma cell myeloma. 3SNeXstar, Boulder, CO, USA.lbiozone Labs, Pittsburgh,CA, USA.

The liposomal formulation allowed delivery of a higher cu-mulative dose of drug without cardiotoxicity than could beachieved with free drug.Cis-platinum is contraindicated in cats as intravenous ad-ministration at doses used in dogs causes acute respiratoryfailure. A sterically stabilized liposomal formulation of cis-platinum was used safely in cats without evidence of respi-ratory crisis. 1 The favorable alterations in biodistributionof sterically stabilized liposomes are the impetus for thedevelopment of more liposomal drug formulations that in-crease efficacy and reduce toxicity. For example, prelimi-nary testing of a sterically stabilized cis-platinum formula-tion is being done in cats.29Liposomes encapsulated with the bacterial cell wall com-ponent muramyl dipeptide (MDP) or muramyl tripeptide-phosphatidylethanolamine (MTP-PE) have been investi-gated as a method to activate macrophages against drugresistant tumor cells in animals with spontaneous tu-mars,12,14,15 When injected intravenously these substancesare rapidly removed from circulation, however encapsula-tion enhances delivery to the reticuloendothelial system. Inmononuclear cells, the slow release of the drug inducesantitumor activity so that activated macrophages selectivelykill neoplastic cells and do not affect non-neoplastic cells.'In studies using canine osteosarcoma, liposome encapsu-lated MTP-PE was found to delay the time to developmentof metastasis and prolong survival compared to dogs receiv-ing empty liposomes.12228 When liposome encapsulatedMTP-PE w as used in dogs post splenectomy due to heman-giosarcoma, patients had increased serum TNF-a and IL-6levels and significantly prolonged disease free interval andoverall survival compared to dogs receiving empty lipo-somes. 4 These results illustrate the potential benefits ofencapsulating immunomodulators into liposomes to in-crease antimetastatic activity and overall survival.

Targeted Liposome DeliveryAs discussed,the usefulness of liposomes for passive tar-geting of tumors is related to the altered vascular perme-ability in tumors compared to normal tissue and the abilityto prolong liposome circulation so that the particles canextravasate from the leaky vasculature.21'22An extension ofthis concept is targeted delivery of liposomes. This has beeninvestigated by using surface ligands or by altering the per-

meability of the vasculature of the tumor using hyperther-mia.Ligand coated liposomes would be especially useful intargeting hematogenous tumors or metastatic cells in theblood or lym ph where passive targeting cannot be done.194,8Techniques used to attach the ligand to sterically stabilizedliposomes include covalent attachment of antibody to theliposome surface, covalent attachment of antibody to lipo-some surface by end-functionalized PEG, and noncovalentbinding of b iotinylated antibod y using an avidin linker.4 By


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using monoclonal antibodies attached to sterically stabilizedliposomes, it has been possible to specifically target can-cers, both in vitro and in V ~ V O . ~ ~ ~ n a murine model ofsquamous lung carcinoma there was increased localizationof antibody labeled DoxiP within the tumor compared tofree drug and unlabeled Doxi1.a Histopathologically therewas a reduction in both the number and size of tumor fociin mice receiving the antibody labeled Doxila with somemice having no lung tumors. The apparent increase in drugefficacy is speculated to be the result of extravasation ofliposomes through leaky vessels into the tumor interstitiumand subsequent binding of the surface ligand to the targetedepitope on the tumor cells.8 This results in sustained releaseof entrapped drug locally in the direct vicinity of the tar-geted tumor cells. This approach also provides an opportu-nity for diffusion of drug throughout the tumor and produc-ing cytotoxicity of even cells that lack the specific epitope.Also, with some monoclonal antibody-liposomes the entirepackage may be internalized by tumor cells resulting inincreased cytot ~xi cit y.~Hyperthermia is used in both humans and animals forcancer treatment. Hyperthermia may sensitize tumor cells toradiation and increase cytotoxicity based on its ability to killcells without regard for some factors that render cells ra-diation resistant, such as cell cycle phase and energy sta-t~~~ Hyperthermia also increases tumor blood flow andvascular ~ermeability.~~herefore it is logical to use hy-perthermia in an attempt to selectively enhance vasculardelivery of substances to the tumor. Because of the knowl-edge about the changes in tumor microenvironment fromhyperthermia, drug delivery using liposomes in conjunctionwith hyperthermia has been investigated.34935n a studyusing a murine tumor model, accumulation of Doxilm was15 fold higher compared to free drug after tumor heating,showing that hyperthermia has a marked effect on promot-ing extravasation of intact ~i p oso mes .~~Advances in liposome engineering have led to the devel-opment of thermosensitive liposomes. They are designed tochange from a gel to liquid phase at a specific temperatureand subsequently release the encapsulated drug. Hyperther-mia and thermosensitive liposomes have a synergistic ef-fect. In an in vitro study it was found that hyperthermiaenhanced antitumor effects of thermosensitive liposome en-capsulated cisplatin on human osteosarcoma cells. Hyper-thermia caused release of free drug from the liposomes re-sulting in greater intracellular accumulation of cis-platinum.37 Similarly, in a study using a murine mammaryadenocarcinoma model there was a 47 fold increase in ther-mosensitive liposome encapsulated doxorubicin after heat-ing and that release of doxorubicin could occur with thereapplication of heat many hours after initial injection.38These results suggest that hyperthermia can be used to se-lectively enhance both the delivery and release of drugsfrom thermosensitive liposomes. Therefore, it may be pos-

sible to use hyperthermia to make a drug more effective,increase the quantity of a drug delivered to the target, andcontrol the release of a drug from thermosensitive lipo-somes. Studies using more relevant spontaneous animal tu-mors are being conducted.# If hyperthermia can signifi-cantly increase the delivery of liposomes there are numer-ous applications using liposomes that could lead to moreeffective cancer treatment in humans and animals.39

Liposomes in ImagingDiagnostic medical imaging is a relatively new but fastgrowing area of research and application of liposome tech-n~logy. ,~~dopting concepts learned from use of lipo-somes in drug delivery systems, the ability of stericallystabilized liposomes to remain in circulation led to the con-cept of loading the vesicles with iodine based contrast me-dia for computed tomographic imaging and with paramag-

netic substances for magnetic resonance imaging. Liposo-ma1 agents are also used in ultrasound imaging and intargeted delivery of radionuclides in nuclear medicine. Thegoal of giving a patient contrast medium is to produce adetectable difference between tissues and thus help differ-entiate normal from abnormal. The role of liposomes inimaging is to provide selective targeting of only the normalor pathologic tissue and to be retained long enough to per-mit satisfactory imaging.41Liposomal contrast media for computed tomographyhave been developed based on the properties of the carrierto alter the biodistribution of the entrapped compound.41Byprolonging circulation times and the ability to target thereticuloendothelial system, various liposomally encapsu-lated iodinated contrast media have been shown to produceselective enhancement of normal liver and spleen.42Moreimportantly, in comparison to free contrast media, which isquickly cleared from circulation, the liposomally encapsu-lated contrast media allow for persistence of tissue opacifi-cation for several hours. Thus, more accurate and if neces-sary multiple studies can be obtained following a singleinjection!l Sterically stabilized liposomes with encapsu-lated contrast medium have been used in a rabbit model asblood pool agents as they have decreased affinity for thereticuloendothelial system. Because of the protectivemechanism of the polyethylene glycol coating, blood re-mained opacified for more than twenty-four hours after in-j e ~ t i o n . ~ ~Contrast media are used in magnetic resonance imagingto help differentiate anatomic structures and also to evaluatefunction. In magnetic resonance, contrast media acts by

#Matteucci ML, Anyarambhatla GR, et al. Hype rthermia effect on up-take of technetium-99m labeled liposomes in feline sarcomas. Not yetsubmitted for publication.


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shortening the relaxation times (T, and T,) of water protonsin the surrounding environment resulting in an ncrease (T,agents) or decrease (T2 agents) in intensity of tissue sig-nal.2344Liposomes are being investigated as carrier agentsfor paramagnetic substances for magnetic resonance imag-ing. Soluble metal ions, metal ion chelates, metal oxides,and nitroxides are some of the various paramagnetic lipo-soma1 fo rm ulatio ns b ein g i n ~ e s t i g a t e d . ~ ~ , ~ ~he contrastmedia were initially entrapped in the core of the liposome;however, it is thought that for optimal signal, the paramag-netic substance should be freely exposed for interactionwith water molecule^.^.^^^^.^^ Therefore, more recent inves-tigations have concentrated on gadolinium and manganesechelates o n th e lip oso me s ~ r f a c e . ~ , ~ ~n studies using animalmodels of various tumors, liposomal contrast media haveshown promise in allowing differentiation of normal tissueto tumor and also in metastasis A relativedifference in signal between normal tissue and tumor hasbeen observed and this is thought to be due to the lack ofnormal phagocytes in tumors resulting in a region of de-creased signal intensity. Also blood pool effects and clear-ance of the contrast medium by normal hepatocytes affectsthe signal of tissues.Imaging of lymph nodes is important in determining po-tential for metastatic spread of tumors. Liposomes with aparamagnetic or radionuclide label have been used to detectnodal metastasis. Compared with normal nodes, lymphnodes containing tumor infiltrate will have less macro-phages that absorb the particulate su bstance.2 Thus the af-fected node will show a filling defect. Sterically stabilizedgadolinium-labeled liposomes have been shown to allowmore rapid visualization of lymph nodes, which is a majoradvantage of liposomal MRI agents compared to conven-tional agents which accumulate slowly in the lymph node.,Diagnostic liposomes' role in musculoskeletal magneticresonance imaging has also been investigated. Intraarticularinjection of gadolinium improves the visualization of thearticular surface by offsetting the low differences in intrin-sic contrast of joint structures. However, because of intra-cartilaginous diffusion of the contrast medium in the carti-lage layer, exact visualization of the cartilage surface withrespect to early arthrotic change and chondral defects islimited. Entrapping the gadolinium in liposomes resulted ingood demarcation between the joint space and hyaline car-tilage surface allowing for early detection of small articularcartilage lesions in an animalFurther investigation is needed comparing liposomallyencapsulated contrast media for magnetic resonance imag-ing to traditional nonliposomal formulations to determinethe true benefit of encapsulating paramagnetic substances.In nuclear medicine 67Ga, l 1 In, and 99Tc are commonlyused for imaging. Even though all three have been devel-oped and used as liposomally encapsulated imaging agents,99Tc is currently the most com monly investigated for its

potential because of its more favorable imaging character-istics and availability.2340 arly efforts using liposomes inscintigraphy were complicated by poor labeling efficiencyand unstable product resulting in poor image quality andnonspecific distribution of the radiotracer. A recently de-scribed technique of labeling liposo mes with 99Tc usingthe lipophilic chelator hexamethylpropyleneamine oxime(HMPA O) to carry 99Tc inside preformed liposomes con-taining reduced glutathione has resulted in a very stableproduct with >95% labeling efficiency.'' When these lipo-somes were used in rats for detection of infection and can-cer, it was found that the liposomes were more stable thanother formulations as little radioactivity was seen in thekidneys o r bladder.40 Thus, increased tumo r to mu scle andabscess to muscle ratios were demonstrated using the re-duced glutathione/HMPAO labeling m ethod. Additionally,the 99Tc abel is retained at the site of liposome depositionfor prolonged periods without rapid metabolism resulting inimproved image quality.In human trials "'In labeled liposom es provided for de-tection of various carcinomas, melanoma, sarcoma, andlymphoma.40 99Tc-labeled liposomes w ere shown to accu-mulate in areas of abscessation in a rat model and wereuseful in detecting deep-seated infections in humans.50953Accumulation of liposomes at sites of inflammation andarthritis has also been shown .40 Sterically stabilized lipo-somes have been investigated as blood-pool imaging agentsbecause of their ability to circulate for prolonged period oftime. Current1y~'Tc-labeled red blood cells are used forblood pool imaging. Because of the superior labeling effi-ciency and increased safety of 99Tc-labelled liposome s,these diagnostic liposomes may prove to replace red-bloodcells for gated blood pool imaging and other applicationssuch as detection of sites of gastrointestinal bleeding andd eep vein t h r o m b o ~ i s . ~ ~Liposomes for ultrasonography are prepared by incor-porating gas bubbles into the liposomes or by forminga bubble directly inside the liposome as a result of a chemi-cal reaction producing carbon dio xide.40 These vesiclesare not truly liposomes as the aqueous core has been re-placed by gas. The gas is an efficient reflector of soundand this can be used to produce Doppler and color sig-na l enhan~ement .~ 'n a study using rabbits and pigs, gas-filled lipid bilayers were used to stabilize and create uni-formly sized bubbles small enough to avoid rapid clearanceby the lungs.52 These vesicles allowed prolonged myo-cardial enhancement and improved visualization of intra-cardiac structures such as the endomyocardial border, pap-illary muscles and valves. As these results show promise,human trials are sure to follow. Currently there is investi-gation of liposomal encapsulation of gases other than air,such as perfluorate gases, that are more stable and offer astronger longer lasting effect since they are less soluble inwater.51


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SummaryThis m anuscript is not intended as a comprehensive over-

view of he large filed of liposome technology and all itsapplications. H ~ ~ ~ ~ ~ ~ ,ur intent was to present currentdata, which are active, cutting-edge research. Because of

their unique properties liposomes will continue to be inves-tigated in drug delivery and imaging systems, and verylikely will be incorporated into our discipline of veterinarymedicine as the clinical applications of liposomes continueto expand.

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