THE ROLE OF LIPOSOMES IN DRUG DELIVERY AND DIAGNOSTIC IMAGING: A REVIEW

MARK MATTEUCCI, L. DVM, DONALD THRALL, E. DVM, PHD

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

Introduction
liposomes have grown significantly. Technologic advances in the fields of materials, and chemical, and biophysical engineering have resulted in the ability to prolong circulation of microparticles in the blood. Liposomes have been used in many scientific disciplines and knowledge from basic research evolved into their incorporation into practical and clinical applications. Even though they have been extensively investigated for over 30 years, it has only been in the last decade that their potential is being realized in the targeted delivery of chemotherapeutics and in imaging applications. The literature contains many reviews and books on liposomes and their application to medicine; however, there are few reports discussing liposomes and their applications in veterinary Liposome research has become very specialized and crosses over to many interrelated disciples. Today liposomes have been incorporated in fields such as mathematics, physics, biophysics, chemistry, colloid science, biochemistry, and biology.' This paper will not attempt to review the numerous uses of liposomes within all these fields. Instead, our goal is to familiarize the reader with the concept and applications of liposomes in drug delivery systems and ongoing research using liposomes in diagnostic imaging. Where relevant, specific use of liposomes in veterinary oncology and imaging will be discussed.
PPLICATIONS USING

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Liposome Structure and Characteristics

Liposomes are phospholipid vesicles first described in 1965 by Alec BanghamI7 when he discovered that phospholipids in water form closed vesicles. Liposomes were first used in the study of cellular membranes, as they are a good model for lipid bilayers. Liposomes are made from neutral phospholipids such as lecithin and cholesterol. Such lipids are amphipathic, meaning their structure contains both a polar head region and nonpolar aliphatic chains. When placed in an appropriate environment and at specific concentrations these lipids spontaneously form a spherical
From the Department of Anatomy, Physiological Sciences and Radiology, College of Veterinary Medicine, North Carolina State University 4700 Hillsborough St. Raleigh, NC 27606. Address correspondence and reprint requests to Dr. Thrall. Submitted February 8, 1999; accepted for publication June 29, 1999.

structure. The sphere is a closed bilayered vesicle. The hydrophilic polar groups face the aqueous interior core, while the hydrophobic nonpolar portions are tucked into the interior of the membrane bilayer (Fig. 1). This effectively results in two potential compartments to entrap a substance. The hydrophilic aqueous core can trap a hydrophilic substance and the hydrophobic interior of the membrane can trap lipid soluble substances. Liposomes can be manufactured to contain multiple concentric bilayers resulting in a variety of compartments for substance entrapment.' The unique structural properties of liposomes allow different methods to be used to entrap substances. Depending on the substance used, it will either be trapped while liposomes form spontaneously or the substance can be loaded into preformed liposomes. Substances with intermediate solubility can be loaded into the aqueous interior of existing liposomes by the use of ionic gradients or molecular complexes. For example, the commercially available liposomal formulation of doxorubicin, Doxil,@*is loaded into preformed liposomes using an ionic gradient method whereas liposomes can be labeled with technetium using a molecular complex with gl~tathione.'~,'~ The size of liposomes varies according to their intended use. Liposomes can be manufactured with a diameter ranging from several nanometers to several micrometers. Liposomes in most current applications have a diameter ranging from 50-150 nm. This range is a compromise between the efficiency of loading, liposome stability and the ability of the liposome to extravasate from the vasculature. For example, large liposomes can be loaded relatively easily but tend to remain in blood vessels. Because liposomes are made using naturally occurring biodegradable substances, once they reach the target site they are metabolized and cleared making them safe and nontoxic. Liposome Types The first liposomes, known as conventional liposomes, were composed of egg phosphatidylcholine and cholesterol.' When injected intravenously, plasma opsonins and lipoproteins coat the outer surface of these conventional liposomes. Subsequently, reticuloendothelial cells quickly
*Sequus, Menlo Park, CA, USA.

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(a) No PEG coating

(b) PEG-coated liposome

Cholesterol

FIG.1. The structure of liposomal delivery systems. (a) Drug-containing liposomes in the absence of a polyethylene glycol (PEG) coating. Hydrophobic drug is present in the liposome membrane and hydrophilic drug is present in the liposome aqueous interior. Protein opsonins are shown absorbing to the naked liposome surface. (b) PEG-coated liposome with entrapped drug in the liposome interior. Protein opsonins have significantly less absorption to the liposome 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 of liposomes in drug carriers systems, but this waned as they failed to live up to expectations for limited applications in vaccine formulations and in targeting the reticuloendothelial system in antiparasitic therapy. This inhibited the widespread use of liposomes because of the resultant short circulating half-life and led to a period of skepticism among scientists in the field of drug delivery. One of the most important breakthroughsin liposome research was the discovery of a polyethylene glycol (PEG) derivative as a liposome coating. The new formulation of PEG-coated liposomes results in greatly extended liposome circulation times. These liposomes have been termed second-generation or sterically stabilized liposomes because of

their ability to evade phagocytosis by the reticuloendothelial system.8 Sterically stabilized liposomes have the ability to remain in circulation up to 1OOX longer than conventional liposomes. This is due to the ability of the PEG coating to form a steric barrier by attracting water to the liposome surface (Figure 1). The liposome surface becomes hydrophobic, resulting in enhanced stability by inhibiting interactions with plasma proteins such as opsonins and lipoproteins. Thus, PEG coated liposomes have a decreased affinity for the reticuloendothelial system. The ability of PEG coated liposomes to avoid rapid reticuloendothelial system uptake has led to their use in drug delivery systems for sustained drug release and selective delivery of encapsulated substances to specific target sites. Despite the success and extensive use of PEGylation, in

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an attempt to achieve even better control over the properties of surface modified liposomes, alternatives to PEGylation are being actively developed. Synthetic polymers of the vinyl series, such as polyacryl amide and polyvinyl pyrrilidone, have been used for surface modification of liposomes. The use of copolymers such as PEG and polylactide glycolide (PEG-PLAGA) allows the ability to form a long circulating particle with an insoluble core and a water soluble shell.7 Research development is ongoing using different copolymers, crosslinking or polymerization of lipid bilayers, and molecular manipulation of the liposome surface to alter its behavior after injection. The most important biological consequence of these different surface modification substances and techniques is a significant increase in liposome circulation time and decrease in their reticuloendothelial system accumulation. Liposomes as Drug Carriers In a successful drug-carrier delivery system, the drug should be retained within a stable carrier, the carrier should be invisible to the reticuloendothelial system, and there should be a built in or controllable release mechanism for the drug. Ideally, the drug would localize directly and specifically at the intended targets, resulting in excellent therapeutic efficacy, with few or no adverse effects. Additionally, the drug would have a high therapeutic index. Especially in oncology, clinicians face toxicity problems with chemotherapeutic agents and the dose-limiting toxicity of drugs often results in suboptimal therapy. It is the ultimate goal of a drug delivery system to improve the therapeutic index and decrease side effects. Liposomes have been extensively investigated for their promise of being an ideal drug delivery system. Their amphilipathic properties make them versatile carriers of either water-soluble or lipid soluble drugs. The liposome bilayer is impermeable to large molecules such as enzymes and has a low permeability to charged molecules. The entrapped drug is protected from metabolism within the liposome interior and cannot be active or metabolized until released. Being able to have extended circulation times may favorably alter the ultimate rate and pathway of drug metabolism. It is the ability of liposomes to alter drug pharmokinetics by changing drug absorption, biodistribution, and clearance, which result in favorable effects at the target site. The ability to extend drug circulation times by encapsulation in a vesicle and the understanding of tumor microcirculatory anatomy has resulted in the passive targeting of drugs into solid tumors. Regions of solid tumor growth, infection and inflammation have capillaries with increased permeability.22 In solid tumors, angiogenesis has been shown to be an inherently leaky process with the new tumor microvessels being disorganized, having a tortuous and convoluted path compared to normal vasculature. In addition, tumor vessels are typified by venous lakes, regurgitant flow

and stasise2At the endothelial cell level the walls are not structurally complete and therefore are permeable to colloidal sized particles. Thus, circulating, liposomal encapsulated drug can be delivered to the interstitium of tumors. Once in the tumor interstitial space, the liposomes become heterogeneously distributed throughout the tumor by diffusion and/or hydraulic convection.22 It has been shown that not only does the PEG coating increase circulation times but also allows greater extravasation than conventional liposomes.22The end result is a several fold greater accumulation of drug in diseased tissue compared to normal tissue. Because liposomes and their associated drug are confined principally to the vascular space and tissues with increased vascular permeability, the volume of distribution? is dramatically lower than with free drug that is widely distributed to all tissues throughout the body. A reduction of drug concentration in normal tissues alters the toxicity profile of the liposomally encapsulated drug relative to free drug. By altering drug distribution, liposomal encapsulation can decrease many unwanted side effects and increase efficacy. The increased efficacy is due to the liposomes ability to increase drug concentration at its intended site of action and by decreasing the drug concentration in sensitive normal tissues resulting in an increased therapeutic index. This contrasts to intravenous administration of free drug where less than one percent of the injected dose reaches diseased tissue, whereas the rest damages healthy cells and tissues. It is not completely clear what happens to liposomes once they accumulate in tissue. Fluorescent microscopy has shown that once trapped in tissue the liposomes disintegrate, releasing drug into the surroundings.21 The exact mechanism of drug release in not completely understood but it is thought to be a result of bilayer chemical disintegration or mechanical rupture, enzymatic breakdown by lipases, or reversal of local concentration gradients causing drug leakage. Currently there are several liposome formulations undergoing testing in clinical trials and there are three liposome formulations commercially available for use in humans. The first liposoinal drug formulation to be approved for human use was the antifungal agent amphotericin B, Ambis0me.a-i: This formulation uses conventional liposomes and therefore allows more efficient drug delivery to macrophages and avoids the dose limiting nephrotoxic effects commonly observed when non-encapsulated amphotericin is used. It has been shown that using liposomal amphotericin B in dogs with blastomycosis resulted in effective treat-

?Volume of distribution at steady state provides an estimate of drug distribution that is independent of elimination processes and provides assessment of the systemic distribution characteristics of a drug. It is a function of a drugs affinity for and retention by, peripheral tissues. Because liposomes do not accumulate in normal tissues the volume of distribution is lower relative to free SNeXstar, Boulder, CO, USA.

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ment and nephrotoxic side effects were absent even at twice the recommended dose of free drug.16 The most extensively studied liposomally encapsulated anticancer drug formulation is liposomal doxorubicin, Doxil,@ which consists of doxorubicin encapsulated in sterically stabilized liposomes. By using PEG liposomes, favorable characteristics such as extended circulation time and altered drug biodistribution led to many favorable results in animal studies and human clinical trials. This led to Doxil's@FDA approval on November 21, 1995 for use in Kaposi s a r c ~ m a . ~In ~ ~ - ~ ~ , comparison to free drug or doxorubicin in conventional liposomes, Doxil@was shown to be superior in efficacy and the common side effects of cardiomyopathy and myelosuppression were decreased. The superior therapeutic index has been shown to be due to increased localization of Doxil@ into tumor tissue and sustained release of drug over several hours directly into the tumor interstitial space resulting in high drug concentrations while avoiding sensitive tissue such as myocardium.8,21 Prolonging circulation times increase the chances of extravasation at sites with a leaky vasculature, such as in many tumors. In human clinical trials, Doxil@was shown to have 11.4 times greater accumulation in soft tissue Kaposi sarcoma, and current research is focusing on other applications of Doxil@in various solid tumors.' Other approved liposomal drug formulations include DaunoXome@$ (duanorubicin) and ELA-MAX@" (lidocaine). Other liposomally encapsulated drugs such as cis-platinum, nystatin, retinoic acid, and numerous vaccines are currently in clinical trials in the United States and Europe and show promise of eventual marketability.

Liposomes as Drug Carriers in Veterinary Medicine

Liposomes 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 similar to that of free drug however side effects of myelosuppression, gastrointestinal upset and cardiotoxicity were significantly decreased. The dose limiting toxicity was a cutaneous toxicity similar to palmar-plantar erythrodysthesia seen in human patients." Cutaneous lesions ranged from mild erythema, hyperemia, edema and alopecia to severe crusting, ulceration and epidermal necrosis. Typical locations are areas of pressure points including paws, axilla and inguinal regions. The cause of palmar-plantar erythrodysthesia is poorly understood but it is speculated that lesions occur as a result of prolonged drug circulation times or greater accumulation of Doxil@in the skin to a greater degree than free drug. DoxiP was used to induce clinical remission in a dog with chemotherapy-resistant plasma cell myeloma. l3
SNeXstar, Boulder, CO, USA. lbiozone Labs, Pittsburgh, CA, USA.

The liposomal formulation allowed delivery of a higher cumulative dose of drug without cardiotoxicity than could be achieved with free drug. Cis-platinum is contraindicated in cats as intravenous administration at doses used in dogs causes acute respiratory failure. A sterically stabilized liposomal formulation of cisplatinum was used safely in cats without evidence of respiratory crisis. l1 The favorable alterations in biodistribution of sterically stabilized liposomes are the impetus for the development of more liposomal drug formulations that increase efficacy and reduce toxicity. For example, preliminary testing of a sterically stabilized cis-platinum formulation is being done in cats.29 Liposomes encapsulated with the bacterial cell wall component muramyl dipeptide (MDP) or muramyl tripeptidephosphatidylethanolamine (MTP-PE) have been investigated as a method to activate macrophages against drug resistant tumor cells in animals with spontaneous tumars, 12,14,15 When injected intravenously these substances are rapidly removed from circulation, however encapsulation enhances delivery to the reticuloendothelial system. In mononuclear cells, the slow release of the drug induces antitumor activity so that activated macrophages selectively kill neoplastic cells and do not affect non-neoplastic cells.' In studies using canine osteosarcoma, liposome encapsulated MTP-PE was found to delay the time to development of metastasis and prolong survival compared to dogs receiving empty liposomes. 12228 When liposome encapsulated MTP-PE was used in dogs post splenectomy due to hemangiosarcoma, patients had increased serum TNF-a and IL-6 levels and significantly prolonged disease free interval and overall survival compared to dogs receiving empty liposomes. l4 These results illustrate the potential benefits of encapsulating immunomodulators into liposomes to increase antimetastatic activity and overall survival.

Targeted Liposome Delivery

As discussed,the usefulness of liposomes for passive targeting of tumors is related to the altered vascular permeability in tumors compared to normal tissue and the ability to prolong liposome circulation so that the particles can extravasate from the leaky vasculature.21'22 extension of An this concept is targeted delivery of liposomes. This has been investigated by using surface ligands or by altering the permeability of the vasculature of the tumor using hyperthermia. Ligand coated liposomes would be especially useful in targeting hematogenous tumors or metastatic cells in the blood or lymph where passive targeting cannot be done. 194,8 Techniques used to attach the ligand to sterically stabilized liposomes include covalent attachment of antibody to the liposome surface, covalent attachment of antibody to liposome surface by end-functionalized PEG, and noncovalent binding of biotinylated antibody using an avidin linker.4 By

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using monoclonal antibodies attached to sterically stabilized liposomes, it has been possible to specifically target cancers, both in vitro and in V ~ V O In~a ~ ~ model of . murine squamous lung carcinoma there was increased localization of antibody labeled DoxiP within the tumor compared to free drug and unlabeled Doxi1.a Histopathologically there was a reduction in both the number and size of tumor foci in mice receiving the antibody labeled Doxila with some mice having no lung tumors. The apparent increase in drug efficacy is speculated to be the result of extravasation of liposomes through leaky vessels into the tumor interstitium and subsequent binding of the surface ligand to the targeted epitope on the tumor cells.8 This results in sustained release of entrapped drug locally in the direct vicinity of the targeted tumor cells. This approach also provides an opportunity for diffusion of drug throughout the tumor and producing cytotoxicity of even cells that lack the specific epitope. Also, with some monoclonal antibody-liposomes the entire package may be internalized by tumor cells resulting in increased cytot~xicity.~ Hyperthermia is used in both humans and animals for cancer treatment. Hyperthermia may sensitize tumor cells to radiation and increase cytotoxicity based on its ability to kill cells without regard for some factors that render cells radiation resistant, such as cell cycle phase and energy stat~~~ Hyperthermia also increases tumor blood flow and vascular ~ e r m e a b i l i t yTherefore it is logical to use hy.~~ perthermia in an attempt to selectively enhance vascular delivery of substances to the tumor. Because of the knowledge about the changes in tumor microenvironment from hyperthermia, drug delivery using liposomes in conjunction with hyperthermia has been investigated.34935 a study In using a murine tumor model, accumulation of Doxilm was 15 fold higher compared to free drug after tumor heating, showing that hyperthermia has a marked effect on promoting extravasation of intact ~ i p o s o m e s . ~ ~ Advances in liposome engineering have led to the development of thermosensitive liposomes. They are designed to change from a gel to liquid phase at a specific temperature and subsequently release the encapsulated drug. Hyperthermia and thermosensitive liposomes have a synergistic effect. In an in vitro study it was found that hyperthermia enhanced antitumor effects of thermosensitive liposome encapsulated cisplatin on human osteosarcoma cells. Hyperthermia caused release of free drug from the liposomes resulting in greater intracellular accumulation of cisplatinum.37 Similarly, in a study using a murine mammary adenocarcinoma model there was a 47 fold increase in thermosensitive liposome encapsulated doxorubicin after heating and that release of doxorubicin could occur with the reapplication of heat many hours after initial injection.38 These results suggest that hyperthermia can be used to selectively enhance both the delivery and release of drugs from 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, and control the release of a drug from thermosensitive liposomes. Studies using more relevant spontaneous animal tumors are being conducted.# If hyperthermia can significantly increase the delivery of liposomes there are numerous applications using liposomes that could lead to more effective cancer treatment in humans and animals.39

Liposomes in Imaging
Diagnostic medical imaging is a relatively new but fast growing area of research and application of liposome techn~logy.,~~ Adopting concepts learned from use of liposomes in drug delivery systems, the ability of sterically stabilized liposomes to remain in circulation led to the concept of loading the vesicles with iodine based contrast media for computed tomographic imaging and with paramagnetic substances for magnetic resonance imaging. Liposoma1 agents are also used in ultrasound imaging and in targeted delivery of radionuclides in nuclear medicine. The goal of giving a patient contrast medium is to produce a detectable difference between tissues and thus help differentiate normal from abnormal. The role of liposomes in imaging is to provide selective targeting of only the normal or pathologic tissue and to be retained long enough to permit satisfactory imaging.41 Liposomal contrast media for computed tomography have been developed based on the properties of the carrier to alter the biodistribution of the entrapped compound.41By prolonging circulation times and the ability to target the reticuloendothelial system, various liposomally encapsulated iodinated contrast media have been shown to produce selective enhancement of normal liver and spleen.42More importantly, in comparison to free contrast media, which is quickly cleared from circulation, the liposomally encapsulated contrast media allow for persistence of tissue opacification for several hours. Thus, more accurate and if necessary multiple studies can be obtained following a single injection!l Sterically stabilized liposomes with encapsulated contrast medium have been used in a rabbit model as blood pool agents as they have decreased affinity for the reticuloendothelial system. Because of the protective mechanism of the polyethylene glycol coating, blood remained opacified for more than twenty-four hours after inje~tion.~~ Contrast media are used in magnetic resonance imaging to help differentiate anatomic structures and also to evaluate function. In magnetic resonance, contrast media acts by

#Matteucci ML, Anyarambhatla GR, et al. Hyperthermia effect on uptake of technetium-99m labeled liposomes in feline sarcomas. Not yet submitted for publication.

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shortening the relaxation times (T, and T,) of water protons in the surrounding environment resulting in an increase (T, agents) or decrease (T2 agents) in intensity of tissue signal.2344Liposomes are being investigated as carrier agents for paramagnetic substances for magnetic resonance imaging. Soluble metal ions, metal ion chelates, metal oxides, and nitroxides are some of the various paramagnetic liposoma1 formulations being i n ~ e s t i g a t e d . The, ~ ~ ~ ~ contrast media were initially entrapped in the core of the liposome; however, it is thought that for optimal signal, the paramagnetic substance should be freely exposed for interaction with water molecule^.^.^^^^.^^ Therefore, more recent investigations have concentrated on gadolinium and manganese chelates on the liposome s ~ r f a c e .In studies using animal ~,~~ models of various tumors, liposomal contrast media have shown promise in allowing differentiation of normal tissue to tumor and also in metastasis A relative difference in signal between normal tissue and tumor has been observed and this is thought to be due to the lack of normal phagocytes in tumors resulting in a region of decreased signal intensity. Also blood pool effects and clearance of the contrast medium by normal hepatocytes affects the signal of tissues. Imaging of lymph nodes is important in determining potential for metastatic spread of tumors. Liposomes with a paramagnetic or radionuclide label have been used to detect nodal metastasis. Compared with normal nodes, lymph nodes containing tumor infiltrate will have less macrophages that absorb the particulate substance.2 Thus the affected node will show a filling defect. Sterically stabilized gadolinium-labeled liposomes have been shown to allow more rapid visualization of lymph nodes, which is a major advantage of liposomal MRI agents compared to conventional agents which accumulate slowly in the lymph node., Diagnostic liposomes' role in musculoskeletal magnetic resonance imaging has also been investigated. Intraarticular injection of gadolinium improves the visualization of the articular surface by offsetting the low differences in intrinsic contrast of joint structures. However, because of intracartilaginous diffusion of the contrast medium in the cartilage layer, exact visualization of the cartilage surface with respect to early arthrotic change and chondral defects is limited. Entrapping the gadolinium in liposomes resulted in good demarcation between the joint space and hyaline cartilage surface allowing for early detection of small articular cartilage lesions in an animal Further investigation is needed comparing liposomally encapsulated contrast media for magnetic resonance imaging to traditional nonliposomal formulations to determine the true benefit of encapsulating paramagnetic substances. In nuclear medicine 67Ga, l 1 'In, and 99Tc are commonly used for imaging. Even though all three have been developed and used as liposomally encapsulated imaging agents, 99Tc is currently the most commonly investigated for its

potential because of its more favorable imaging characteristics and availability.2340 Early efforts using liposomes in scintigraphy were complicated by poor labeling efficiency and unstable product resulting in poor image quality and nonspecific distribution of the radiotracer. A recently described technique of labeling liposomes with 99Tc using the lipophilic chelator hexamethylpropyleneamine oxime (HMPAO) to carry 99Tc inside preformed liposomes containing reduced glutathione has resulted in a very stable product with >95% labeling efficiency.'' When these liposomes were used in rats for detection of infection and cancer, it was found that the liposomes were more stable than other formulations as little radioactivity was seen in the kidneys or bladder.40 Thus, increased tumor to muscle and abscess to muscle ratios were demonstrated using the reduced glutathione/HMPAO labeling method. Additionally, the 99Tc label is retained at the site of liposome deposition for prolonged periods without rapid metabolism resulting in improved image quality. In human trials "'In labeled liposomes provided for detection of various carcinomas, melanoma, sarcoma, and lymphoma.40 99Tc-labeled liposomes were shown to accumulate in areas of abscessation in a rat model and were useful in detecting deep-seated infections in humans.50953 Accumulation of liposomes at sites of inflammation and arthritis has also been shown.40 Sterically stabilized liposomes have been investigated as blood-pool imaging agents because of their ability to circulate for prolonged period of time. Current1y~'Tc-labeled red blood cells are used for blood pool imaging. Because of the superior labeling efficiency and increased safety of 99Tc-labelled liposomes, these diagnostic liposomes may prove to replace red-blood cells for gated blood pool imaging and other applications such as detection of sites of gastrointestinal bleeding and deep vein t h r o m b o ~ i s . ~ ~ Liposomes for ultrasonography are prepared by incorporating gas bubbles into the liposomes or by forming a bubble directly inside the liposome as a result of a chemical reaction producing carbon dioxide.40 These vesicles are not truly liposomes as the aqueous core has been replaced by gas. The gas is an efficient reflector of sound and this can be used to produce Doppler and color signal enhan~ement.~' a study using rabbits and pigs, gasIn filled lipid bilayers were used to stabilize and create uniformly sized bubbles small enough to avoid rapid clearance by the lungs.52 These vesicles allowed prolonged myocardial enhancement and improved visualization of intracardiac structures such as the endomyocardial border, papillary muscles and valves. As these results show promise, human trials are sure to follow. Currently there is investigation of liposomal encapsulation of gases other than air, such as perfluorate gases, that are more stable and offer a stronger longer lasting effect since they are less soluble in water.51

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Summary This manuscript is not intended as a comprehensive overview of he large filed of liposome technology and all its applications. H ~ our intent was ~to present current ~ ~ ~ data, which are active, cutting-edge research. Because of

their unique properties liposomes will continue to be investigated in drug delivery and imaging systems, and very likely will be incorporated into our discipline of veterinary medicine as the clinical applications of liposomes continue ~ , to expand.

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Vet Clin North Am Small Anim Pract 1998;28:269-295. 10. Vail DM, Kravis LD, Cooley AJ, Chun R, MacEwen EG. Preclinical trial of Doxorubicin entrapped in sterically stabilized liposomes in dogs with spontaneously arising malignant tumors. Cancer Chemotherapy and Pharmacology 1997;39:4lO-416. 11. Thamm DH, Vail DM. Preclinical evaluation of a sterically stabilized liposome-encapsulated cisplatin in clinically normal cats. AJVR 1998;59:286-290. 12. MacEwen EG, Kurzman ID, Rosenthal RC, et al. Therapy for osteosarcoma in dogs with intravenous injection of liposome-encapsulated muramyl tripeptide. Journal of National Cancer Institute 1989;81:935-938. 13. Kisseberth WC, MacEwen EG, Helfand SC, Vail DM, London CL, Keller E. Response to liposome-encapsulated doxorubicin (TLC D-99) in a dog with myeloma. JVIM 1995;9:425-428. 14. Vail DM, MacEwen EG, Kurzman ID, et al. Liposome-encapsulated muramyl tripeptide phosphatidylethanolamine adjuvant immunotherapy for splenic hemangiosarcoma in the dog: a randomized multi-institutional clinical trial. Clinical Cancer Research 1995;l:1165-1 170. 15. Fox LE, MacEwen EG, Kurzman ID, et al. Liposome-encapsulated muramyl tripeptide phosphatidylethanolamine for the treatment of feline mammary adenocarcinoma-a multicenter randomized double blind study. Cancer Biotherapy 1995;lO:125-130. 16. Krawiec DR, McKieman BC, Twardock AR, et al. Use of an amphotericin b lipid complex for treatment of blastomycosisin dogs. JAVMA 1996;209:2073-2075. 17. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. Journal of Molecular Biology 1965;13:238-252. 18. Lasic DD. Doxorubicin in sterically stabilized liposomes. Nature 1996;380:561-562. 19. Phillips WT, Rudolph AS, Goins B, Timmons JH, Klipper R, Blumhardt R. A simple method for producing a technetium-99m-labeledliposome which is stable in vivo. Nuclear Medicine Biology 1992;5:539-547. 20. Papahadjopoulos D, Allen TM, Gabizon A, et al. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proceedings of the national academy of science. USA. 1991;88:11460. 21. Wu NZ, Da D, Rudoll TL, Needham D, Whorton AR, Dewhirst MW. Increased microvascular permeability contributes to preferential accumulation of stealth liposomes in tumor tissue. Cancer Research 1993; 5313765-3770. 22. Gerlowski LE, Jain RK. Microvascular permeability of normal and neoplastic tissues. Microvascular Research 1986;31:288-305, 23. Huang SK, Mayhew E, Gilani S, Lasic DD, Martin FJ, Papahadjopoulos D. Pharmacokinetics and therapeutics of sterically stabilized liposomes in mice bearing c-26 colon carcinoma. Cancer Research 1992; 52:6774-678 1. 24. Gabizon A, Catane R, Uziely B, et al. 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Adjuvant therapy for osteosarcoma in dogs: Results of randomized clinical trials using combined liposome encapsulated muramyl tripeptide and cisplatin. Clinical Cancer Research 1995;1:1595. 29. Thamm DH, Vail DM. Evaluation of SPI-77, a sterically stabilized (Stealth) liposome-encapsulated cisplatin in cats. In: Veterinary Cancer Society 16" Annual Conference, Pacific Grove, 1996;74. 30. Allen TM. Antibody-mediated targeting of long-circulating (Stealth) liposomes. Journal of Liposome Research 1994;4 1-7. 31. Ahmad I, Allen TM. Antibody-mediatedspecific binding and cytotoxicity of liposomes-entrapped doxorubicin to lung cancer cells in vitro. Cancer Research 1992;52:4817. 32. Ahmad I, Longnecker M, Samuel J, Allen TM. Antibody-targeted delivery of doxorubicin entrapped in sterically stabilized liposomes can erradicate lung cancer in mice. Cancer Research 1993;53:1484 33. Dewhirst MW. Future directions in hyperthermia biology. International Journal of Hyperthermia 1994;10:339-345. 34. 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Dewhirst MW, Prosnitz L, Thrall DE, et al. Hyperthermic treatment of malignant disease: current status and a view toward the future. Seminars in Oncology 1997;24(6):616-625. 40. Torchilin VP. Handbook of Targeted Delivery Of Imaging Agents. Boca Raton, CRC Press, 1995.

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