CA2051439C - Liposomal targeting of ischemic tissue - Google Patents

Abstract

The invention relates in general to the field of biochemistry and medicine, and more particularly to the use of liposomes in the diagnosis and treatment of ischemic tissue. Liposomes of a size of less than 200 nanometers target ischemic tissue and preferentially deliver active agents to infarcted areas in the absence of antibodies bound to the liposomes to effect the delivery.

Description

WO 90/12595 z05~ PCl'/US90/02010 ' LIPOSOMAL TARGETING OF ISCHEMIC TISSUE

This invention relates in general to the field of biochemistry and medicine, and more particularly to the use of liposomes in the diagnosis and treatment of ischemic tissue.
5 Ischemia is a dericiency of blood in tissue, and is a significant medical problem. For example, heart disease is a leading cause of morbidity and ",o, ~alily and two related conditions which are of significant concern are myocardial ischemia (tissue anemia in the heart muscle as a result of obstruction of the blood supply such as by vasoconstriction), and m.vocardial infarct or ~0 infarction (an ischemic condition resulting in the localized death of heart muscle and caused by the particulate obstruction of the flow of arterial blood). While progress has been made in the treatment of ischemic tissue, there is much room for improvement.
One problem with potential antiischemic agents is the action of these agents 5 on other than ischemic tissue. For example, many potent coronary vasodilators are ineffective during myocardial ischemia because they dilate nonischemic coronary blood vessels as well as the ischemic vessels, which draws blood flow away from the ischemic zone. Additionally, many antiischemic compounds (e.g., calcium entr,v blockers) would be more effective if the agent could be targeted 20 directly to the ischemic region.
Thus, it has been a desideratum to provide a drug delivery system to selectively deliver a compound into an ischemic myocardial bed, that is, deliveran active agent preferenlially to infarcted heart tissue rather than nonischemictissue. In this regard, the terms "ischemic" or "ischemia" as used herein refer 25 to tissue in the state of traumatic tissue anemia and include infarcled tissue.
Phospholipid vesicles (liposomes) have been pursued in the hope that they would concer,l,~le in selected tissues and result in additional enhancement in the delivery of active agents from this tissue specificity. Accordingly, workershave attempted to employ liposomes for the delivery of active agents to 30 myocardial tissue. For example, in a publication by Caride and Zaret in Science 198, 735-738 (1977) multilamellar liposomes of approximately 1,000 nm in diameter with either net positive, negative or neutral charge were administered to mammals after the induction of embolic closed-chest interior wall myocardial ~0 5 ~ 4 3~

infarction. The liposomes were labeled with 99mTc-DTPA (Diethylene triamine pentaacetic acid). While this publication reports an accumulation of positive and neutral MLVs in infarcted myocardial tissue, free 99mTc-DTPA has been shown to accumulate in ischemic myocardium (ten times that of normal myocardium after a circulation time of one hour) and further data has shown that the accumulation of 99mTc-DTPA in infarcted myocardium observed in the subject reference was actually due to the release of vesicle contents in circulation and subsequent accumulation of free 99mTc-DTPA in the damaged myocardium.
The publication by Mueller et al in Circulation Research 49, 405-415 (1981) reports the use of a protein marker (131I- albumin) retained in 400 to 700 nanometer small, unilamellar liposomes. The results show a slight accumulation of positive liposomes in ischemic myocardium compared to normal myocardium (ischemic/normal equal 1.38:1) and no net accumulation in ischemic myocardium was seen with neutral liposomes (ratio 0.81:1).
An article in Cardiovascular Research 16, 516-523 (1982) Cole et al describes myocardial liposome uptake in the early stages of myocardial infarction and concluded that 75 to 125 nm liposomes show no evidence of preferential uptake by ischemic myocardium. The authors suggest that liposomes thus have limited potential as a means of drug delivery in myocardial infarction. Antibodies have also been covalently bound to liposomes in an attempt to deliver such vesicles preferentially to certain tissues, but the results have been less than successful in many instances.

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2a SummarY of the Invention In accordance with one aspect of the invention there is provided use, in the preparation of a composition for targeting a therapeutic or diagnostic agent to reversible ischemic tissue in a patient, of unilamellar liposomes of a size less than 200 nanometers and consisting essentially of cholesterol and a chemically pure neutral phospholipid consisting of distearoylphosphatidyl choline, and the agent and being substantially in the absence of antibodies bound to the liposomes.
In another aspect of the invention there is provided use of unilamellar liposomes, substantially in the absence of antibodies bound to the liposomes, of a size less than 200 nanometers and consisting essentially of cholesterol and a chemically pure neutral phospholipid consisting of distearoyl-phosphatidyl choline for containing a therapeutic or diagnostic agent to be targeted to reversible ischemic tissue in a patient.
In still another aspect of the invention there is provided unilamellar liposomes of a size less than 200 nanometers and consisting essentially of cholesterol and a chemically pure neutral phospholipid consisting of distearoylphosphatidyl choline and containing a therapeutic or diagnostic agent to reversible ischemic tissue and being substantially in the absence of antibodies bound to the liposomes, for use in targeting a therapeutic or diagnostic agent in an amount sufficient to preferentially target a quantity of the agent to reversible ischemic tissue.

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2b In an especially preferred embodiment of'another aspect of the invention there is provided the method for the delivery of a diagnostic agent to reversible ischemic tissue in a patient, comprising introducing into the patient's bloodstream an amount of unilamellar liposomes containing the agent, said liposomes being essentially neutral in charge, in the absence of antibodies bound to the liposomes to effect the delivery, the liposomes having a size of less than 200 nanometers and consisting essentially of cholesterol, which is 10 to 50% of total lipid, and a more than 97% chemically pure neutral phospholipid consisting of distearoylphosphatidyl choline to preferentially deliver the quantity of the agent to the ischemic tissue which is S to 10 times greater relative to a nonischemic region by the localization of liposomes in the ischemic tissue.
Ischemic tissue in a patient may be targeted by introducing into the patient's bloodstream an amount of liposomes, of a size of less than 200 nm (preferably unilamellar vesicles) and preferably characteri~ed by being comprised of chemically pure synthetic phospholipids, most preferably having aliphatic side chains of a length of at least 16 carbons, and containing a therapeutic or diagnostic agent, sufficient to preferentially deliver (i.e., target) a quantity of the agent to the ischemic tissue in the essential absence of antibodies bound to the liposomes to effect the delivery. The expression "chemically pure phospholipids" is meant to define phospholipids which are essentially free of deleterious detergent moieties and impurities which cause aggregation of small unilamellar vesicles (SUVs) formed therefrom, and which are more than 97% pure.

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2~95 ~051~9 ' Pcl'/us9o/o2o1o r,eferably, the SUVs have a diameter predominantly of from 50 to 100 nm, are essentially neutral in charge, and incorporate phospholipids having a side chain length of from 16 to 18 carl.on atoms. More preferably, the liposomes are prepared from ~li;,learoyl phosphatidylcholine and include cholesterol (most5 ~.referably in an amount of from 10 to 50% of total lipid) as a vesicle stabilizer.
The largeted ischemic tissue is pre~erably ischemic myocardial tissue such as reversibly infdrcled myocardial tissue, and the method has shown efficacy in ~aryeling active agents II,ereto. While I do not wish to be bound by any particular theory as to the targeLi"g of the liposomes to acute myocardial ,o ischemic tissue, it appears that the liposomes pass through capillaries with increased permeability (increased pore size) and can preferentially penetrate ischemic tissue, such as ischemic myocardium, relative to a nonischemic region.
A variety of diagnostic agents may be encapslJI~ted within the liposomes and used in the method of the invention. In the examples below, a radioactive 5 isotope of indium ("'In) is loaded into the liposomes and permit gamma imagingof acute myocardial ischemia. In addition, appropriate liposomal NMR contrast agents such as are described in U.S. Patent No. 4,728,575 may be administered for imaging myocardial ischemia by magnetic resonance techniques.
As to therapeutic agents, enzymes which catalyze the breakdown of 20 superoxide or oxygen radical species (e.g., superoxide dismutase) may be incorporated into a~ ~.ro,criale vesicles, as may therapeutic agents such as c~talase or glucose oxidase, or dihydro-pyridine compounds such as nicardipine.
The particular diagnostic or therapeutic agents which may be used with the invention will be apparent to those of skill in the art following disclosure of this 2s discovery, and do not conslil.Jte part of the invention per se In the illustrative examples, ra~iol~~elled liposomes are employed to delineate ische,nic tissue and thus demonstrate the ability of the method of the inventionto selectively deliver therapeutic or diagnostic agents to traumatized ischemic myocardium. With this ~lisclos'~re~ the use of additional diagnostic or therapeutic 30 agents in connection with the treatment of myocardial ischemia or infarct will be ap,uarenl to one of skill in the art.
Brief Description of the Drawing Figure 1 displays blood clearance data in dogs for "'In-labelled liposomes with time. Data are expressed as the radioactivity (CPM)/g blood. For each 35 point, 1~1=4. The values are expressed as mean + S.D.

WO 90/12595 20510~39 PCr/lJS90/02010 4 ,,_.
DE~AILED DESCRIPTION
Pl epa, alion of Liposomes The liposomes which are used in the invention are small unilamellar liposomes of a size of less than 200 nm, preferably having a dia,neter of from 50 to 100 5 nm. As noted above, the vesicles are preferal)ly comprised of chemically pure s~r,lhelic phospholipids having saturated aliphatic side chains and most prefefably are prepared from phospholipids such as distearoyl phosphatidylcholine. Cholesterol is advantageously incorporated into the liposomes to increase the stability of the vesicles which are used in the ~o disclosed process.
A wide variety of therapeutic or diagnostic agents may be incorporated in the inner aqueous space or the lipid bilayer of the liposomes by methods which will be apparent to one of skill in the art. In the following examples a chelating compound and an ionophore are employed for loading external cations for 5 r~iolabelling into the vesicles. The preferred ionophore is A23187, but other useful ionophores are polyethers such as lasalocid A(X-537A) and 5-bromo derivatives of lasalocid; cyclic depsipeptides such as beauvericin; and cyclic peptides such as valinomylin. The chelating agent is ,,,referably nitriloacetic acid (NTA) although other chelators may also be used.
20 The liposomes are prepared by dissolving the phospholipid and cholesterol in an appropriate organie solvent, such as chloroform, and evaporating the solvent to form a lipid film. If, as in the following examples, an ionophore is employed to load the diagnostic or therapeutic agent into the liposomes, this compound may be added to the lipid solution before evaporation.
25 The dried lipid film is then rehydrated in an ap~)ro~riale aqueous phase, such as phospl ,ate-buffered saline or other physiologically appropriate solution. Water soluble drugs or therapeutic agents may be contained in the hydrating solution, although if remote loading is desired a loading agent such as a chelating agent desc,il,ed above may be added to the hydrating solution to be encapsulated 30 within the inner aqueous space of the liposome.
Upon the addition of the hydrating solution, liposomes of varying size spontaneously form and enc~rslJIate a portion- of the aqueous phase.
Thered~er, the liposomes and suspending aqueous solution are subjected to a shear force such as sonication, or processed through a homogenizer according 35 to the method described in U.S. Patent No. 4,753,788; to produce vesicles within 4 ~ ~

the specified size. The liposomes are then processed to remove undesirable compounds from the suspending solution, for example, the chelating agent or unencapsulated drug, which may be accomplished through processes such as gel chromatography or ultra-filtration. If necessary, the product is then concentrated to remove excess buffer solution. Since the liposomes are smaller in size than 0.2 micron, they are then passed through a sterile 0.22 micron filter to remove any microorganisms which may be present in the suspension Thereafter, the liposomes are filled into sterilized glass containers and stoppered with a sterilized elastomer closure.

ExamPle 1 SUVs have been prepared by formulating an organic solution of distearoyl phosphatidylcholine and cholesterol in a 2:1 molar ratio, evaporating the solution to dryness to form a lipid film, and further drying the lipid under vacuum. In order to permit the subsequent loading of the lllIn into the vesicles, a divalent ionophore (A23187) was added to the lipid solution before evaporation. In a typical preparation, 20 ~moles distearoyl phosphatidylcholine, ~moles cholesterol and 0.04 ~moles A23187 were dissolved in chloroform, dried to a think film at 60~C
under a stream of nitrogen and then dried in vacuo overnight.

5a The dried lipid film was then rehydrated with an appropriate aqueous phase, for example, phosphate-buffered saline solution ~.9~ NaCl and 5 mM sodium phosphate, pH 7.4) containing a chelating agent for loading the lllIn3+, and a shear force applied to form the SUVs. In a typical preparation, the dried lipids were rehydrated with phosphate-buffered saline containing 1 mM nitrilotriacetic acid (NTA) as a chelating agent, and the mixture was sonicated at approximately 65~C until the suspension cleared (approximately five minutes) and then centrifuges at 400 g. Unencapsulated NTA was removed from the vesicles by filtering the mixture through a Sephadex G-50 (Trade-mark) column. The liposomes were determined by a Nicomp Model 270 (Trade-mark) submicron particle size analyzer to have a mean diameter less than 100 nm.

ExamPle 2 A 2:1 molar ratio mixture of distearoyl phosphatidylcholine and cholesterol was dissolved in chloroform, along with the ionophore A23187. These components were thoroughly mixed until completely dissolved. This solution 20S~9 PCr/~lS90/0201~

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was then placed in a rotary evaporator to remove the chloroform and deposit a lipid film on the surface of the evaporator flask. Alternatively, other known drying methods could be used to form the lipid film or powder.
The chelating l"~t~rial (NTA) was then mixed in phosphate buffered saline 5 and the resulting solution was added to the lipid film. The liposomes thus formed were processed through a homogenizer, according to the process taught in U.S. Patent No. 4,753,788 to Gamble, to produce vesicles having a mean diameter not to exceed 100 nanometers. The liposomes were then passed through a gel chromatography column to remove the chelating agent which ,0 remained in the suspending solution outside the liposomes. The product was then concenl,a~ed through a hollow fiber concentrator to remove excess buffer and to concenlr~te the liposome suspension.
Thereafter, the liposomes were filtered through a 0.22 micron sterile filter an~transferred to sterilized glass contai,1ers in a class 100 hood and stoppered with 15 a sterilized elastomer closure. Throughout this process, appropriate QA/QC
procedures were employed to ensure sterile processing conditions.

Vesicle Loading As noted above, the vesicles may be loaded with amphiphilic agents during lipid film forlnalio,1, with aqueous-soluble agents during hydration, or by other 20 known loading procedures. Since the targeting of the liposomes to myocardial tissue is best demonstrated by the delivery of radioactive agents, the gamma-imaging agent '1'indium3+ was loaded into the liposomes immediately prior to use.

Example 3 25 Loading has been accomplished by using incubation mixtures consisting of 500 ~l of vesicles, 35 ~l of 3.4 ~IM InCI3 in 104 mM sodium citrate (pH 7.4), and 1-50 ~l of 1"1n3+, depending on the required activity. The volume of PBS
equal to twice that of the "'indium3+ addition was included in the incubation mixture. Incubation time and temperature may be selected according to 30 published procedures such as Mauk et al. Analytical Biochemistry 94, 302-307 (1979). Generally, the loading is performed by incubation at 60 to 80~C for 15 to 60 minutes. The incubation is terminated by cooling the sample followed by the addition of 10 mM EDTA in PBS. Up to 90% of the added "1indium can be 7 ~ 4 3~
incorporated into the preformed liposomes by this method, and the liposomes produce specific activities of up to 300 ~Ci/mg lipid.

ExamPle 4 Vials produced in the process of Example 2 each contained 4.7 ml of the liposome suspension t25 mg liposomes per ml) and contained small unilamellar liposomes having a diameter predominantly of from 50 to 100 nm. The liposomes in these vials were loaded according to the following process. 0.2 ml of 0.1 M
sodium citrate for injection was added to each vial and mixed well. Following standard radiopharma-ceutical procedures to calculate radiopharmaceutical dosages, an amount of lllindium chloride solution sufficient to yield the prescribed dose of lllIn3+ at time of injection was then added. This mixture was then incubated at 80~C for 30 minutes, followed by cooling to room temperature.
0.1 ml of 0.1 M sodium edetate for injection was then added to stop the liposome loading by chelating any excess lllIn. During this process, the radioactive loading efficiency was tested by withdrawing 0.5 ml of the liposome solution prior to terminating the liposome loading procedure, and transferring the solution to a 1.5 ml centrifuge vial containing 0.5 g Chelex 100 (Trade-mark). The contents of the centrifuge vial were incubated for 5 minutes at room temperature, with occasional mixing, and the total radioactivity of the centrifuges vial was determined using a dose calibrator.

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7a 0.5 ml of O.lM sodium citrate for injection was then added to the centrifuge vial and then mixed. The vial was then centrifuged for 5 minutes at moderate speed to compact the Chelex 100 (Trade-mark). 0.5 ml of the supernatant was removed with an appropriate syringe and the radioactivity of the supernatant determined. Loading efficiency was calculated by dividing twice the supernatant radioactivity by the total radioactivity, times 100 to yield percent loading efficiency. In all instances, the loading efficiency was greater than 90%.

Tarqetinq to Ischemic MYocardial Tissue Example 5 An example of the preferential delivery of the small unilamellar liposomes of the invention to ischemic myocardial in the absence of antibody targeting tissue is demonstrated by the use of the labeled liposomes produced in accordance with the procedures in Examples 2 and 4. The liposomes were administered to animals and found to target such tissue.
All animals used were mongrel dogs of either sex (16-20 kg, N=4). The animals were anesthetized before surgery using 30 mg/kg sodium pentobarbital as an i.v.
injection. Polyethylene catheters were inserted into a femoral artery and vein for measurement of blood pressure and heart rate, for blood sampling, and for injection of liposomes. The trachea was cannulated and the animal was artifically respired with a Harbard (Trade-mark) respirator using room air. Eucapnia was maintained and monitored with a Godart-Statham (Trade-mark) capnograph. A left thoracotomy was performed at the fifth intercostal space, a partial pericardiotomy .~,. .~

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exposed the heart, and a pericardial cradle was formed. Approximately one cm of the left anterior descending coronary artery (LAD) was isolated just distal to its first major branch and a silk ligature was loosely placed around the vessel. Aortic pressure and heart rate were measured using a Statham P23AA
(Trade-mark) transducer and recorded on a Beckman R-411 (Trade-mark) recorder. Blood samples obtained from the femoral artery catheter were analyzed electrometrically for blood gases and pH tRadiometer BMS 3 (Trade-mark) blood gas analyzer).
At this time, 6 mg/kg of lllIn labelled liposomes prepared as in Examples Z and 3 were injected i.v.
After injection of the liposomes into the animals, arterial blood samples were taken 1, 2, 3, 4, 5, 10 minutes and 1, 2, 3, 4, 5, 6 hours post injection and the blood radioactivity was determined later. Ten minutes after liposome injection, the LAD was occluded via the surgical silk snare and the occlusion was continued for 2 hours. At this time, the occlusion was released and the reperfusion was allowed to continue for 4 hours.

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8a At the end of the experiment, blood gas and hemodynamic variables were again determined and then the heart was removed. The aorta was perfused at a pressure of 100 mm Hg with saline to clear the coronary vessels of blood. The left ventricular free wall was then cut into 6 transmural pieces from the ischemic zone and 6 from the nonischemic zone. These pieces were then divided into subepicardial and subendocardial halves. Samples of the lever and gracilis muscle were also taken. The radioactivity was then determined in both blood and tissue samples using a Hewlett-Packard (Trade-mark) gamma counter.
All data were analyzed using a paired T-test.
The tissue and blood clearance data were expressed as the counts per minute (CPM) of radioactivity per gram of tissue or blood. All data are presented as mean S.D.

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WO 90/1259~ ~Q5145~9 Pcr/usgo/02o1o Hemodynamic data are shown in Table 1. All values were within the normal range for dogs. No differences existed for any of these variables during the course of the experiment. No changes in blood gases were seen during the - experiment.
5 The blood clearance data for the liposomes are shown in Fig. 1. As can be seen there is a fast initia! clearance followed by a slower clearance phase.
The data are expressed as the CPM radioactivity (CPM)/g blood. It is apparent that more than half of the liposomes were cleared from the blood at the end of the experiment.
Data for myocardial tissue clearance of liposomes are shown in Table 2.
The data are expressed as the CPM/g tissue. The ischemic region in all animals co"la..1ed significantly more radioactivity compared to its paired nonischemic region. This cli~rence was 5-10 fold. Within the ischemic zone, the subendocardium col ,lai,1ed twice the radioactivity contained in the 15 subepicardium and this difference was significant. The liver was actively clearing liposomes with the CPM/g cleared being 12.34 x 104 + 5.49 x 104 PM/g and skeletal muscle cleared an amount similar to the nonischemic region of the hear~0.11 x 104 + 0.01 x 104 CPMtg.

Summary Biodistribution of Labelled Liposomes in Canine Ischemia 20 Animals: 16-20 kg dogs - 4 studied Average total lipid dose: 6 mg/kg x 18 kg = 108 mg lipid Average total radioactivity (calculated from average blood level at injection):
1300 x 105 cpm Average biodistribution at 6 hrs: cpm/gm (x 104) ischemic subendocardium 2.2 + 1.1 ischemic sube~ car~ium 1.6 + 0.9 nonischemic subendocardium 0.26 + 0.10 nonischemic subepicardium 0.27 + 0.14 Blood 2.0 + 0.5 Skeletal muscle 0.11 + 0.01 Liver 12.3 + 5.5 Myocardial ischemia and infarction are characterized among other things by an increase in capillary permeability. This increased permeability may allow 205~4 ~ ~ PCl'/US90/02010 1 o .,.,...~
selective drug delivery to the ischemic region by using appropriately sized liposomes as delivery vehicles. In the present study, the ischemic region loc5,~ ;o,l of radioactivity (and presumably liposomes) was 5-10 times greater compared to the nonischemic myocardium. While I do not wish to be bound 5 by any particular theory, it appears that the liposomes were localized in the ischemic zone due to increased capillary permeability. Interestingly, the ischemic subendocardium tended to localize more liposomes compared to the ischemic subepicardium. This may reflect the fact that the subendocardium is usually more at risk during ischemia. The nonischemic subepicardial-subendocardial 10 dir~ere"ce in loc~ tion of liposomes was not significantly different.
The high liver clearance of liposomes is not surprising as this organ is one of the major sites of blood borne particulate removal. This also indicates that the "'In was bound to liposomes, as ~t'ln that is free would probably not be cleared by the liver. The estimated labelling efficiency was 70-80%. The nonischemic myocardium and skeletal muscle had relatively low liposome localization.
From the desc,iplio" set forth above, it will be apparent that liposomes having a size of less than about 200 nanometers, preferal,ly 60 to 100 nm will prefere"~ially target active agents such as diagr~ostic or therapeutic agents to20 an ischemic myGcardial region, and in particular permit the selective localization of the liposomes into the ischemic subendocardium which is typically more at risk, thus facilitating drug delivery to the region of greatest ischemic severity.

WO 90/1259~ 51~ 9 PCI/US90/02010 ., . ,, ~, ', ~, .

Hemodynamic data for liposome treated animals before and after LAD occlusion and reperfusion.

Before After Occlusion Occlusion + Reperfusion Systolic Blood 137 + 22 136 + 22 Pressure (mm Hg) Diastolic Blood 116 + 27 100 + 20 Pressure (mm Hg) Heart Rate 160 + 28 182 + 21 (Beats/min) All values are mean + S.D. (N=4) Localization of 1~1n-labelled liposomes in the ischemic and nonischemic myocardium.
Ischemic Region Nonischemic Region (x104) (x1 o4) Subepi- Subendo- Subepi- Subendo-cardium cardium cardium cardium Radioactivity- 1.55 2 22* 0.27** 0.26**
(CPM/g) + + + +
0.92 .- 1.14 0.14 0.10 All values are mean + S.D. (N=4) * Significantly different from its respective subepicardial region value (P 0.05) ** Significantly different from its respective ischemic region value (P 0.05)

Claims (12)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Use, in the preparation of a composition for targeting a therapeutic or diagnostic agent to reversible ischemic tissue in a patient, of unilamellar liposomes of a size less than 200 nanometers and consisting essentially of cholesterol and a chemically pure neutral phospholipid consisting of distearoylphosphatidyl choline, and the agent and being substantially in the absence of antibodies bound to the liposomes.

2. The use according to claim 1, in which the liposomes are used in an amount sufficient, when introduced into the bloodstream of the patient, to preferentially deliver a quantity of the agent to the ischemic tissue.

3. The use according to claim 1 or 2, in which the ischemic tissue is myocardial tissue and the agent is preferentially delivered to the ischemic tissue rather than to the nonischemic myocardium.

4. The use according to claim 1, 2 or 3, in which the liposomes have a size of from about 50 to about 100 nanometers.

5. The use according to claim 1, 2, 3 or 4, in which the chemically pure phospholipids are synthetic phospholipids.

6. Use of unilamellar liposomes, substantially in the absence of antibodies bound to the liposomes of a size less than 200 nanometers and consisting essentially of cholesterol and a chemically pure neutral phospholipid consisting of distearoylphosphatidyl choline for containing a therapeutic or diagnostic agent to be targeted to reversible ischemic tissue in a patient.

7. The use according to claim 6, in which the ischemic tissue is myocardial tissue and wherein the liposomes are used in an amount sufficient, when introduced into the bloodstream of the patient, to preferentially deliver a quantity of the agent to ischemic myocardial tissue rather than to nonischemic myocardium.

8. The use according to claim 6 or 7, in which the chemically pure phospholipids are synthetic phospholipids.

9. The method for the delivery of a diagnostic agent to reversible ischemic tissue in a patient, comprising introducing into the patient's bloodstream an amount of unilamellar liposomes containing the agent, said liposomes being essentially neutral in charge, in the absence of antibodies bound to the liposomes to effect the delivery, the liposomes having a size of less than 200 nanometers and consisting essentially of cholesterol, which is 10 to 50% of total lipid, and a more than 97% chemically pure neutral phospholipid consisting of distearoylphosphatidyl choline to preferentially deliver the quantity of the agent to the ischemic tissue which is 5 to 10 times greater relative to a nonischemic region by the localization of liposomes in the ischemic tissue.

10. The method of claim 9, in which the ischemic tissue is myocardial tissue and the agent is preferentially delivered to the ischemic tissue rather than to the nonischemic myocardium.

11. The method of claim 9 or 10, in which the liposomes have a size of from about 50 to about 100 nanometers.

12. Unilamellar liposomes of a size less than 200 nanometers and consisting essentially of cholesterol and a chemically pure neutral phospholipid consisting of distearoylphosphatidyl choline and containing a therapeutic or diagnostic agent to reversible ischemic tissue and being substantially in the absence of antibodies bound to the liposomes, for use in targeting a therapeutic or diagnostic agent in an amount sufficient to preferentially target a quantity of the agent to reversible ischemic tissue.