PRESSURE-MEDIATED INTRACELLULAR DELIVERY OF MOLECULES OR MICROPARTICLES

Abstract: The invention provides a method of enhancing the uptake by a cell of an atom, a molecule, or particle of <10 mu m. The method involves (a) bringing into contact a cell and a liquid medium containing the atom, molecule, or particle, (b) maintaining the cell and the liquid medium in an enclosed space, and (c) subjecting the enclosed cell and liquid medium to an incubation pressure sufficient to enhance the uptake by the cell of the atom, molecule, or particle.

Description: PRESSURE-MEDIATED INTRACELLULA DELIVERY OF MOLECULES OR MICROP ARTICLES Background of the Invention This invention relates to facilitation of the uptake of extracellular substances by living cells. The study of cellular and molecular biology has elucidated the existence of an intricate physical and biochemical environment within the membranes of living cells. These membranes provide a sophisticated semi- permeable barrier that performs specialized functions in different cell types; the membranes of all living cells consist of lipid bilayers that serve to separate the microscopic aqueous spaces in which life takes place. All cells regulate the molecular make-up of their intracellular spaces, either by passive exclusion of large and/or lipophobic substances that cannot penetrate the lipid bilayer of the cell boundary, or by active controlled, bi-directional movement of molecules or particles across the cell membrane by specialized proteins. Some of these proteins can combine to form pores or channels to provide either generalized passage into the cell or allow entry only to specific elements. The introduction of atoms, molecules, and microscopic particles into cells has become an essential tool of biological scientists and medical clinicians, and provides the basis for their ability to dissect the cellular processes of life and to devise pharmacological strategies to prevent, diagnose, or defeat disease. The elaborate functional nature of the cell membrane often poses a difficult challenge to the delivery of charged or large molecules, and has therefore limited the potential uses of such materials in intracellular applications. In addition, many complex substances can be taken up by cells only through a process of cell membrane invagination such as endocytosis or pinocytosis. In most cases, the endocytotic vesicles thus formed that contain the previously extracellular substance then fuse with lysosomes containing harsh enzymatic environments, resulting in the digestive destruction of the engulfed matter. Summary of the Invention The method of the invention involves delivery of a substance to the extracellular environment of a living cell or cells, creation of an enclosed space around the cell or cells, and establishment of an elevated incubation pressure within the enclosed space sufficient to enhance uptake of the substance by the cell or cells. The substance can range in size from a small molecule to a microparticle, and can exert a range of effects on, or serve a range of functions in, the cells by which it is taken up. For example, the substance can be a small molecule (e.g., a drug), a sugar, a fatty acid or a fatty acid derivative, or a protein (e.g., an antibody, an enzyme, or a hormone). The cells can exist in vitro, in tissue or organ culture, or can form a portion of a living organism, e.g., a mammal such as a human. Accordingly, a purpose of this invention is to induce a greater degree of intracellular uptake of microscopic or molecular species than would otherwise be expected or realized within a target cell or population of cells. This method can therefore be used to facilitate the intracellular delivery of larger amounts of a substance, which would normally be taken up by cells only in smaller quantities, or to allow uptake by a cell of a substance that would otherwise remain excluded by the cell membrane in the extracellular environment. This invention can also yield more efficient uptake by a larger percentage of cells in a target population, and allow specific organs or tissues to be targeted. Intracellular delivery carried out using the method of the invention is also advantageous in not causing tissue damage (e.g., distension) or trauma, and in not being potentially toxic to cells. Furthermore, in many cases, the intracellular delivery method of the invention provides a means of delivery to the cell cytoplasm without entailing lysosomal compartmentalization, thereby providing protection from destruction of the delivered substance by lysosomal enzymes. This method can also enhance movement of substances across intracellular barriers, into spaces such as the cell nucleus. Other features and advantages of the invention will be apparent from the detailed description, the drawings, and the claims. Brief Description of the Drawings Fig. 1-A depicts a delivery system of the present invention. Fig. 1-B shows the system of Fig. 1-A attached to a free end of a blood vessel, before delivery of a solution containing a substance to be delivered to the blood vessel. Fig. 1-C shows the system and blood vessel of Fig. 1-B during delivery of a solution containing a substance to be delivered to the endothelium of the blood vessel. Fig. 1-C shows the system and blood vessel of Fig. 1-B during delivery of a solution containing a substance to be delivered to the endothelium and outside surface of the blood vessel. Fig. 2 illustrates delivery to a portion of a blood vessel defining part of the boundary of the pressurized enclosure. Fig. 3-A depicts an alternative method of intracellular delivery to a blood vessel. Fig. 3-B shows the blood vessel of Fig. 3-A, wherein the blood vessel is pressurized mechanically. Fig. 4-A depicts a two-balloon catheter adapted to deliver a solution containing a substance to be delivered into a blood vessel. Fig. 4-B shows the catheter of Fig. 4- A with the balloons in an inflated state. Fig. 4-C shows a catheter system having a balloon and inner tubules for delivering a solution containing a substance to be delivered to the walls of a blood vessel. Fig. 5- A illustrates delivery to blood vessels in an organ. Fig. 5-B shows the use of balloon-catheters for pressurizing an organ. Fig. 6 shows a delivery system comprising a pressurization chamber. Fig. 7 -A shows transfection efficiency as a function of applied pressure for human saphenous vein transfected according to the present invention. Fig. 7-B illustrates the effect of a distension-preventing sheath on transfection efficiency for FITC-ODN transfected human saphenous vein. Fig. 7-C illustrates inhibition of IL-6 protein production following transfection of IL-6 antisense ODN into human saphenous vein. Fig. 8-A illustrates inhibition of IL-6 mRNA production following transfection of IL-6 antisense ODN into human saphenous vein of a first subject. Fig. 8-B is a graph similar to that in Fig. 8-A, for a second subject. Fig. 8-C is a graph similar to that in Fig. 8-A, for a third subject. Fig. 9-A shows transfection efficiency for in vivo transfection of rabbit carotid artery measured by fluorescence microscopy. Fig. 9-B shows luciferase activity for control, healthy, and atherosclerotic cells following in vivo transfection of rabbit carotid artery with DNA encoding firefly luciferase. Fig. 10 shows transfection efficiencies for rat vascular smooth muscle cells transfected in vitro according to a method of the present invention. Fig. 11 shows luciferase activities for rat kidneys perfused with plasmid DNA containing the gene encoding firefly luciferase. Fig. 12-A shows the effect of pressure on transfection efficiency for rat aorta cells. Fig. 12-B shows ischemia- induced PCNA expression in transplanted rat aortae with and without pressure-mediated transfection of antisense-PCNA ODN. Fig. 12-C shows ischemia-induced cdc2 kinase expression in transplanted rat aortae with and without pressure-mediated transfection of antisense-cdc2 kinase ODN. Fig. 12-D shows illustrates the reduction in lumenal narrowing of isotransplanted, ischemic-injured rat aortae, resulting from pressure-mediated transfection with antisense ODN against both PCNA and cdc2 kinase. Fig. 13-A shows transfection efficiencies for rat hearts transfected ex vivo with FITC-ODN, with and without pressure. Fig. 13-B shows ICAM-1 expression in transplanted rat hearts with and without pressure-mediated transfection of antisense-IC AM- 1 ODN. Fig. 13-C illustrates the induction of long-term graft acceptance by pressure-mediated transfection of transplanted rat hearts with antisense ODN against ICAM-1. Fig. 14- A shows wall thicknesses at 6 weeks and 6 months after transplantation for untreated (control) grafts, and grafts transfected with either reverse antisense (control) ODN or with antisense ODN against both PCNA and cdc2 kinase. Fig. 14-B shows results similar to those in Fig. 14-A for veins transfected with E2F decoy ODN, as compared to untreated grafts and control grafts transfected with scrambled ODN. Detailed Description The types of substances that can be used with this invention include (1) charged atoms or ions, (2) neutral or charged small molecules (e.g., molecules having a molecular weight of < 1 ,000), with the exception of small nucleic acids, (3) large molecules (e.g., molecules having a molecular weight of > 1,000), especially proteins and peptides, with the exception of nucleic acids, (4) polymers and filaments (generally < 10 μm in greatest dimension), (5) inorganic atoms and molecules, and (6) microscopic particles (generally <10 μm in greatest dimension). These substances can be delivered to the extracellular environment of the cell in a wide range of concentrations, preferably, but not limited to, 1 nM to 1 mM, depending on the type of substance and its intended use. The exact concentrations to be used in each application of the invention can be determined by the user based on his or her knowledge of the chemistry, biochemistry, or functional properties of the substance to be delivered. The substance can exist in solution in the extracellular environment, or it can comprise a suspension or colloid. A sealed enclosure containing the tissue and the extracellular environment is defined, and the incubation pressure is established, within the sealed enclosure. In a preferred embodiment, the boundary of the enclosure is defined substantially by an enclosing means, so that target tissue (tissue comprising the target cell) is subjected to isotropic pressure, and does not distend or experience trauma. In another embodiment, part of the enclosure boundary is defined by a tissue. A protective means such as an inelastic sheath is then placed around the tissue to prevent distension and trauma in the tissue. In particular, in a blood vessel, a sealed enclosure is preferably defined between occlusions formed by inflatable balloons or tie wraps. A solution containing a substance to be delivered is delivered to the enclosure through a catheter having a delivery outlet between the occlusions. More generally, an enclosure is defined within an organ by establishing occlusions within organ conduits (e.g., blood vessels), such that a space within the organ can be pressurized. The incubation pressure used to facilitate cellular uptake is preferably maintained at a predetermined level for a predetermined incubation period. The incubation pressure depends on the application, including parameters such as the incubation period, the tissue type, and the molecule being delivered. The incubation pressure used can range from, for example, 50 mm Hg to 5 atmospheres above ambient pressure, for example, 300 mm Hg to 1500 mm Hg above ambient pressure, or 0 to 2 atmospheres above ambient temperature; higher or lower pressures can also be used, provided that effective delivery is achieved and that a counter veiling injury is not sustained by the target cell or cells. The incubation period used to facilitate cellular uptake can vary, depending on parameters such as the incubation pressure, the target tissue type, the molecule being delivered, and the desired dosage. The duration of the incubation pressure can range, for example, from approximately 1 second to four hours. Preferably, the pressure duration is between 20 seconds and 30 minutes, more preferably between 60 seconds and 10 minutes. Suitable mammalian target tissues include blood vessel tissue (in particular, veins used as grafts in arteries), heart, bone marrow, connective tissue, liver, kidney, genital-urinary system tissue, bones, muscles, gastrointestinal organs, and endocrine and exocrine organs. The methods of the present invention can be applied to parts of an organ, to a whole organ (e.g., a heart), or to a whole organism. In one embodiment a solution containing a substance to be delivered is perfused into a target region (e.g., a kidney) of a patient, and the patient is subject to pressure in a pressurization chamber. Applications of the present invention also include the treatment of allografts (grafts derived from a different subject than the transplant patient) and syngrafts (grafts derived from the transplant patient). The conditions for each application of the invention can be determined by labeling the substance with a radioactive, fluorescent, or chemical tag, and then following the substance, following pressure-enhanced administration, via the label. Alternatively, measurement of biological activity can be carried out. Similarly, optimization of delivery conditions can be achieved via quantification of delivery of an easily-labeled material that is chemically and/or physically similar to the desired substance. As is discussed above, a system for delivering a molecule into a cell using the methods of the invention includes an enclosing means for defining at least part of a boundary of a sealed enclosure, and a pressurization means for establishing an incubation pressure within the enclosure. The enclosure contains the target cell and an extracellular environment of the cell. A delivery means, such as a catheter or syringe is used to deliver the molecule to the extracellular environment, directly or indirectly (e.g., by intravenous injection). The enclosure boundary is defined by the enclosing means, and possibly by tissue. Suitable enclosing means include, depending on the embodiment, a pressurization chamber, an impermeable sheath or bag, and an occlusion means for occluding a passage in a tissue. A pressurization chamber is particularly suited for the treatment of grafts or entire organisms, while other devices are well suited for intraoperative treatment of tissue. In one embodiment, the enclosure boundary is defined substantially by the enclosing means. Pressure is then applied to the target tissue uniformly from all directions, and the target tissue is not subjected to a risk of suffering trauma. In another embodiment, part of the boundary is defined by a tissue (e.g., the target tissue). The tissue forming part of the enclosure boundary is subjected to pressure from one side only, and can become distended. A protective means such as an inelastic sheath is placed around the tissue to prevent distension and trauma in the tissue. The methods and delivery systems of the invention are illustrated in further detail as follows. Figs. 1-A through 1-C illustrate methods of pressurized delivery of molecules to cells of a blood vessel. Fig. 1-A is a side view of a delivery system 11 of the present invention. System 11 comprises a reservoir 10 for holding a solution 40 containing a substance to be delivered, and a delivery means for expelling solution 40 from reservoir 10. The delivery means comprises a plunger 12 and a delivery tube 14. Opposite plunger 12, reservoir 10 opens into tube 14. Attached to tube 14 are, listed in order of proximity to reservoir 10, a stopcock 16, a pressure gauge 18, a retracted sheath 20, and a notch 22. Sheath 20 is preferably impermeable and inelastic. Notch 22 is next to a distal open end 30 of tube 14. Stopcock 16 is initially in a closed position, preventing solution 40 from passing from reservoir 10 to tube 14. Fig. 1-B shows a blood vessel 24 attached to system 11. Open end 30 is placed into a proximal end of blood vessel 24. Notch 22 fits inside a proximal end of tissue 24. Sheath 20 is pulled down to cover tissue 24. A tie or ligature 26A is wrapped around sheath 20 and tissue 24 at the point where they are attached to tube 14, to prevent tissue 24 from slipping from open end 30. When stopcock 16 is turned to an open position, the solution 40 enters tube 14 and sheath 20, flushing out all gases and liquids present through open end 28 of sheath 20. After the flushing, a tie wrap 26B is placed over distal open end 28 of sheath 20 to form a water-tight seal, as shown in Figs. 1-C and 1-C. Fig. 1-C illustrates delivery targeted to the endothelium of blood vessel 24, while Fig. 1-C illustrates delivery to the endothelium and to the outside surface of blood vessel 24. In Fig. 1-C, tie wrap 26B is placed around sheath 20 and tissue 24. Tie wrap 26B occludes blood vessel 24. Stopcock 16 is turned to its open position, and plunger 12 is pushed, such that the solution 40 is delivered into vessel 24 under a delivery pressure. The delivery pressure is allowed to increase until an incubation pressure is reached, and stopcock 16 is closed. Blood vessel 24 is allowed to incubate for an incubation period, after which tie wrap 26B is untied to release the pressure (not illustrated). The boundary of a sealed enclosure is defined by the walls of vessel 24 and by an enclosing means. The sealed enclosure contains the target (endothelial) cells of blood vessel 24, and their extracellular environment. If stopcock 16 is in a closed position, the enclosing means comprises tube 14, stopcock 16, and ligature 26B. If stopcock 16 is in an open position, the enclosing means comprises ligature 26B, tube 14, plunger 12, and parts of the walls of reservoir 10. The enclosing means defines at least part of the boundary of the enclosure. In the embodiment shown in Fig. 1-C, part of the boundary of the enclosure is defined by blood vessel 24. Applying pressure only to the inside of blood vessel 24 could cause blood vessel 24 to distend and experience trauma. Sheath 20 acts as a protective means, preventing blood vessel 24 from distending. In an arrangement such as the one in Fig. 1-C, it is thus important that sheath 20 be inelastic. It is also possible to place tie wrap 26B around sheath 20 only, as illustrated in Fig. 1-C. In this case, the sealed enclosure containing the target cells of blood vessel 24 and their extracellular environment is defined substantially by an enclosing means. If stopcock 16 is in a closed position, the enclosing means comprises sheath 20, tube 14, stopcock 16, and ligature 26B\ If stopcock 16 is in an open position, the enclosing means comprises sheath 20, tube 14, ligature 26B', plunger 12, and parts of the walls of reservoir 10. In the embodiment shown in Fig. 1-C, the boundary of the enclosure is defined substantially by the enclosing means. The pressure around blood vessel 24 is uniform, and thus blood vessel 24 does not experience trauma. Since sheath 20 acts as part of the enclosing means, it is important that sheath 20 be impermeable. Sheath 20 need not necessarily be inelastic in the arrangement of Fig. 1-C, however, since the use of an elastic sheath would not lead to trauma in blood vessel 24. Fig. 2 illustrates a method of in vivo delivery of molecules to a blood vessel connected to the circulatory system of a patient. Tube 14 is inserted into the lumen of a vessel 224 which is still connected to the body of a living animal. A sheath 220 wraps around vessel 224, and a fastener 228 (e.g., a heat seal) attaches the two flaps of sheet 220 to form a tube. Sheath 220 acts as a protective means, preventing the distension of vessel 224. Two tie wraps 226A and 226B wrap around sheath 220. Tie wraps 226A and 226B act as occluding means, occluding vessel 224. Occlusions 226A and 226B, and the walls of vessel 224 between occlusions 226A and 226B define a sealed enclosure 230 containing the target cells of vessel 224 and their extracellular environment. The solution 40 is injected into the sealed enclosure, and segment 230 is allowed to incubate for an incubation period. After the incubation period, occlusions 226A and 226B are removed, and blood is allowed to flow through vessel 224. Figs. 3-A and 3-B illustrate a delivery system having distinct delivery and pressurization elements, used to deliver molecules to the blood vessel shown in Fig. 2. A rigid tubular wrap 250 is placed around sheet 220, and a vise 252 is placed around wrap 250, as illustrated in Fig. 3-B. Wrap 250 is circumferentially flexible, so that the diameter of the tube it forms is variable, but it is rigid axially, so that even when its diameter changes, it still remains substantially tubular. A tightening screw 254 tightens vise 252, pulling wrap 250 tight, creating pressure within vessel 224. This pressure is maintained for an incubation period, after which screw 254 is unscrewed. Figs. 4- A through 4-D illustrate a method of delivering a solution containing a substance to be delivered to the lumen of a blood vessel 324 through a catheter 314. Catheter 314 is inserted into vessel 324. Catheter 314 is closed at its end 316. Catheter 314 has two balloons 332A and 332B, and a delivery port 330 between balloons 332A and 332B. Initially, balloons 332A and 332B are deflated, as shown in Fig. 4-A. After catheter 314 is inserted into vessel 324, balloons 332 A and 332B are inflated, as shown in Fig. 3-B. Balloons 332A and 332B occlude vessel 324 and create a sealed enclosure 334 within vessel 324. A solution 340 containing a substance to be delivered is delivered to enclosure 334 through port 330. Solution 340 is delivered under pressure, such that enclosure 334 becomes pressurized. Following an incubation period, balloons 332A and 332B are deflated and target enclosure 334 is depressurized. Fig. 4-C shows an alternative delivery system of the present invention, in which a balloon mounted on a catheter has miniature tubules for delivering a solution containing a substance to be delivered to the walls of the vessel. A balloon 432 has tubules 450 that are directly connected to holes 452 in the segment of catheter 314 within balloon 432. When a pressurized solution 440 is delivered through catheter 314, solution 440 exits holes 452, travels through tubules 450, and reaches the walls of vessel 324. Fig. 5-A illustrates the use of system 11 for delivery to blood conduits (vessels and/or atria and ventricles) of an organ 124 such as a heart. A protective sheath 120 is wrapped around organ 124. Organ 124 has an artery 112 which carries blood into it and a vein 114 which carries blood away. Tube 14 is inserted into the lumen of artery 112, and sheath 120 is wrapped around artery 112 and vein 114. Tie wrap 126A is tightened around sheath 120 at artery 112, and tie wrap 126B is tightened around sheath 120 at vein 114, to prevent leakage of fluid out of organ 124. Tie wrap 126A allows tube 14 to enter artery 112, yet wraps tightly enough to seal artery 112 from leakage. A solution 40 containing a substance to be delivered is injected, and organ 124 is allowed to incubate. After the incubation period, tie wraps 126A and 126B are removed, and blood is allowed to flow through organ 124 once more. Fig. 5-B illustrate the use of balloon-catheters for sealing an inlet and an outlet of an organ (e.g., a gastrointestinal organ). A catheter 550 with a balloon 552 is inserted into a first organ conduit 512 in communication with an organ 524, and another catheter 560 with a balloon 562 is inserted into a second organ conduit 514 leading away from organ 524. Initially balloons 552 and 562 are deflated (not illustrated). Once catheters 550, 560 are inserted into their respective blood vessels, balloons 552, 562 are inflated and establish occlusions in conduits 512, 514, respectively. A solution 540 containing a substance to be delivered is delivered to organ 524 under a delivery pressure. Fig. 6 illustrates the use of a pressurization chamber to facilitate delivery of substances to cells. A holding means such as a dish 610 contains a tissue 624 containing target cells. A solution 640 containing the substance is placed in dish 610. Dish 610 is placed in a pressure chamber 650. Chamber 650 is closed and sealed, and a pressurized gas (e.g., C0 2 ) is introduced into chamber 650 through a duct 660. Solution 640 and tissue 624 are maintained under an incubation pressure for an incubation period. Tissue 624 can comprise an entire organ. A pressurization chamber such as the one shown in Fig. 6 is particularly suited to a delivery method in which an entire organism is pressurized. In such a method, a solution containing a substance to be delivered is preferably perfused into blood vessels and/or organs (e.g., kidney) of a patient. The patient is placed in the pressurization chamber. The pressurization chamber is then maintained under an incubation pressure, for an incubation period. There are several possible mechanisms underlying the increased permeability to molecules of cell membranes under pressure. The increase in membrane permeability requires an increased pressure within the cell and/or extracellular environment, but not necessarily a pressure gradient across the cell membrane. It is possible that proteins forming transmembrane channels change conformation at high pressure, and thus allow the passage of molecules through the channels and into the cytoplasm. The exact pressures, incubation periods and concentrations used depend on the target tissue type. For example, as is described further below, an incubation period of approximately 5 minutes at low pressure (-0.5 atm) is sufficient for achieving a near-maximal transfection efficiency in human saphenous vein, while an incubation period of over one hour at high pressure (~2 atm) is required for achieving a transfection efficiency of 80-90% in rat aortae. For rat hearts, an incubation period of 30 to 45 minutes at 2 atm is necessary for a transfection efficiency above 50%. In general, the incubation period necessary to achieve a given transfection efficiency in different tissue types varies from minutes to hours, at incubation pressures on the order of atmospheres. Suitable incubation periods and pressures for a given tissue type can be readily determined by the skilled artisan. In the absence of limitations imposed by surgical procedures, it is in general preferred that the walls of the pressurized enclosure do not include living tissue, since tissue forming parts of the enclosure wall is subject to mechanical stress. Some surgical procedures, such as the treatment of blood vessels connected to the circulatory system during the procedure (see Figs. 3-A and 3-B), require that at least parts of the enclosure walls be defined by tissue. In such a case, it is important that a protective means be used to prevent distension of the tissue. Grafts treated ex-vivo are, in general, preferably treated by incubation in a pressurized chamber or an equivalent pressurized enclosure. The methods of the invention are illustrated further below in experiments showing intracellular delivery of nucleic acid molecules. Transfection efficiencies of methods of the present invention were evaluated for various incubation pressures, incubation periods, and tissue types. Some of the abbreviations used in the following discussion are: CTRL, control; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; FITC- ODN, FITC-labeled oligodeoxyribonucleotide; IL-6, interleukin-6; ODN, oligodeoxyribonucleotide; PCR, polymerase chain reaction; VSMC, vascular smooth muscle cell. The letter "n" refers to the number of subjects evaluated for each data point. The pressures given are the net pressures applied to the samples, above ambient (atmospheric) pressure. Example 1 Human saphenous vein was transfected with FITC-ODN, according to a method similar to that illustrated in Fig. 1-C. Fig. 7-A shows transfection efficiency, as a function of applied pressure, for several ODN concentrations. Efficiency was measured as percentage of total intimal and medial cells found to have nuclear localization of FITC-ODN via fluorescence microscopy. Pressures ranged from 50 to 760 mm Hg, and ODN concentrations in physiologic saline solution ranged from 5 to 100 μM. n=6. The effect of a distension-preventing sheath on transfection efficiency was evaluated using a method similar to that illustrated in Fig. 1-C, at an ODN concentration of 20 μM and at pressures ranging from 50 to 760 mm Hg. Fig. 7-B shows the results of the experiment. n=6. The transfection efficiency of a method of the present invention was investigated in vitro by measuring the inhibition of IL-6 production by antisense ODN in whole organ culture. Vein segments were incubated in growth medium for 24 hours after transfection. Transfections were performed at 5 mM, 10 mM, and 100 mM for 10 minutes, according to a method similar to that illustrated in Fig. 1-C. Fig. 7-C shows reductions in IL-6 protein detected via ELISA in growth medium for antisense-transfected cultures. The reduction levels shown are relative to the levels found in control cultures of untransfected and non-specific ODN-transfected veins. n=6. Example 2 Quantitative reverse transcription PCR was used to measure the reduction in IL-6 mRNA resulting from antisense-ODN transfection performed according to the present invention. Three specimens of human saphenous vein were transfected as illustrated in Fig. 1-C, and mRNA levels in antisense- transfected vein segments were compared to levels in untreated and reverse antisense (control) ODN-transfected segments. Results for the three specimens are shown in Figs. 8-A, 8-B, and 8-C, respectively. Reductions in mRNA levels indicate sequence-specific efficacy of antisense-ODN treatment. Example 3 Rabbit carotid arteries were transfected in vivo with FITC-ODN, according to a method similar to that illustrated in Fig. 2. Transfection efficiency was measured at 4 hours, 4 days, and 7 days after transfection. The percentages of FITC-positive nuclei as a function of time are shown in Fig. 9- A. n=2-3. Expression of the gene for firefly luciferase was measured after in vivo transfection of rabbit carotid artery with a plasmid DNA construct containing the luciferase gene. Healthy arteries (Normal) and atherosclerotic vessels (CHOL/inj) were transfected under pressure, as shown in Fig. 2. Control (CTRL) artery was exposed to the plasmid carrying the luciferase gene in the absence of pressure. Arteries were harvested at day 5 and tissue homogenates were assayed for luciferase activity. Assay results are shown in Fig. 9-B. n=2-4. Example 4 Rat vascular smooth muscle cells (VSMC) were transfected in vitro with FITC-ODN, as shown in Fig. 6. Cells were exposed to either atmospheric pressure (0 atm net pressure) or to 2 atm for 45 minutes. Fig. 10 shows transfection efficiencies for FITC-ODN concentrations of 1 mM and 80 mM. n=4. Example 5 Fig. 11 illustrates the effect of pressure on transfection efficiency for rat kidney cells perfused in vivo with plasmid DNA containing the gene for firefly luciferase, as illustrated in Fig. 6. Rats were exposed to atmospheric pressure (0 atm) or 2 atm for 30 minutes after kidney perfusion. Kidneys were harvested 3 days after transfection and tissue homogenates were assayed for luciferase activity. n=7. Example 6 Fig. 12-A shows the effect of pressure on transfection efficiency for rat aorta cells. Aortae were harvested from donor rats and incubated at 4°C for 24 hours in physiologic solution to induce ischemic injury, in a manner similar to that illustrated in Fig. 1-C. The incubation solutions contained 40 μm FITC-ODN. Incubations were performed at 0 atm and 2 atm above atmospheric pressure. Following incubation, the tissue was isotransplanted into rat aortae and harvested 24 hours after transplantation. Nuclear localization of FITC was assessed by fluorescence microscopy of sections co-stained with a fluorescent DNA-intercalating dye. Transfection efficiency is expressed as cells displaying nuclear localization of FITC-ODN as a percent of total cells. n = 3, p < 0.005. Fig. 12-B shows ischemia-induced PCNA expression in transplanted rat aortae with and without pressure-mediated transfection of antisense-PCNA ODN. The transfection procedure was similar to that illustrated in Fig. 1-C. Ischemic injuries were induced by 24 hour incubations at 4°C, either in saline solution (control), or in saline solution containing 40 μM ODN. A pressure of 2 atm above atmospheric pressure was applied during incubation to both control and ODN-treated samples. Tissue was harvested six days after isotransplantation, and PCNA protein levels in tissue homogenates were measured by ELISA. n = 7, p = 0.02. Fig. 12-C shows ischemia-induced cdc2 kinase expression in transplanted rat aortae with and without pressure-mediated transfection of antisense-cdc2 kinase ODN. The transfection, transplantation, and harvesting procedures were similar to those described above in relation to Fig. 12-B. Protein levels for cdc2 kinase were measured by ELISA. Fig. 12-D illustrates the reduction in lumenal narrowing of isotransplanted, ischemic-injured rat aortae, resulting from pressure-mediated transfection with antisense ODN against both PCNA and cdc2 kinase. Ischemic injury was induced by 24 hours of incubation at 4°C in either saline solution (control), or antisense-PCNA/antisense-cdc2 kinase ODN solution (40 μM each). A pressure of 2 atm above ambient pressure was applied to all tissues (including control). Blockade of expression of the two cell cycle regulatory genes reduced neointimal hyperplasia and lumenal narrowing in ischemically injured isografts, as measured by computerized image analysis. n = 12, p = 0.03. Example 7 Fig. 13-A shows transfection efficiencies for rat hearts transfected ex vivo with FITC-ODN, with and without pressure. A FITC-ODN solution (80 μM) was perfused into the coronary arteries of donor hearts after aortic crossclamping. The hearts were submerged in FITC-ODN solution and exposed to either 0 atm or 2 atm above ambient pressure for 45 minutes at 4°C, as shown in Fig. 6. The hearts were then heterotopically transplanted into the abdominal aorta and vena cava of recipient rats. Nuclear localization of FITC was assessed 24 hours after transplantation by fluorescent microscopy of sections co-stained with a fluorescent DNA-intercalating dye. Transfection efficiency is expressed as cells displaying nuclear localization of FITC-ODN as a percent of total cells, n = 3, p < 0.005. Fig. 13-B shows ICAM-1 expression in transplanted rat hearts, with and without pressure-mediated transfection of antisense-ICAM-1 ODN. Either saline (control) or antisense-ICAM-1 -ODN solution (80 μM) was perfused into the coronary arteries of donor PVG strain hearts after aortic crossclamping. The hearts were then submerged in FITC-ODN solution and exposed to 2 atm above ambient pressure for 45 minutes at 4°C, as illustrated in Fig. 6. Tissue was harvested 3 days after heterotopic transplantation into ACI recipients. ICAM-1 positive area was measured by image analysis of sections stained immunohistochemically for ICAM-1. n = 3-6, p = 0.04. Fig. 13-C illustrates the induction of long-term graft acceptance by pressure-mediated transfection of transplanted rat hearts with antisense ODN against ICAM-1. PVG strain rat hearts were harvested and transfected ex-vivo with either antisense-ICAM-1 ODN solution (80 μm) or with saline solution (control), as described above in relation to Fig. 13-B. The hearts were then heterotopically transplanted into ACI strain recipients. All animals were systemically administered anti-LFA-1 antibody for 6 days following transplantation, to block the ligand for ICAM-1. No further immunosuppression was administered. Tolerance is reported as percentage of treated animals found to have long term acceptance of their allografts. Graft acceptance was defined by presence of heart beat in the graft for >100 days, control n = 12, antisense-treated n = 27, p = 0.003. Example 8 Figs. 14-A and 14-B show inhibition of neointimal hyperplasia in rabbit jugular veins grafted into carotid arteries, following pressure-mediated transfection with ODN designed to block up regulation of cell cycle regulatory genes. Fig. 14-A shows wall thicknesses at 6 weeks and 6 months after transplantation for untreated (control) grafts, and grafts transfected with either reverse antisense (control) ODN or with antisense ODN against both PCNA and cdc2 kinase. Neointima formation was inhibited for up to 6 months, while medial hypertrophy allows adaptive wall thickening to reduce wall stress in the high-pressure arterial environment. Fig. 14-B shows results similar to those in Fig. 14-A for veins transfected with E2F decoy ODN, as compared to untreated grafts and control grafts transfected with scrambled ODN. n = 6, p < 0.005. Although the above descriptions contain many specificities, these should not be construed as limitations on the scope of the invention, but rather as illustrations of particular embodiments thereof. For example, a time- varying incubation pressure can be used in general. Many potential designs for enclosing means, protective means, and/or occluding means can be readily devised by one skilled in the art, depending on the application. Various incubation pressures, periods, and active agent dosages leading to desirable or near-maximal transfection efficiencies can be readily determined for different tissue types. What is claimed is:

Claims: 1. A method of enhancing the uptake by a cell of an atom, a molecule, or a particle of maximum dimension of <10 μm, said method comprising bringing into contact said cell and a liquid medium comprising said atom, molecule, or particle, maintaining said cell and said liquid medium in an enclosed space, and subjecting said enclosed cell and liquid medium to an incubation pressure sufficient to enhance uptake by said cell of said atom, molecule, or particle. 2. The method of claim 1, wherein said cell is contained within a mammalian blood vessel that is sealed at two locations to provide said enclosed space. 3. The method of claim 1, wherein said molecule is a therapeutic, charged organic molecule having a molecular weight of less than 1 ,000. 4. The method of claim 1 , wherein said molecule is a protein or a peptide having a molecular weight of greater than 1,000. 5. The method of claim 1 , wherein said incubation pressure is between 50 mm Hg and 5 atmospheres above ambient pressure. 6. The method of claim 50, wherein said incubation pressure is between 200 mm Hg and 2.5 atmospheres above ambient pressure. 7. The method of claim 1, wherein said cell is contained in a mammalian blood vessel or organ, and the incubation pressure equilibrates between the exterior and interior of the vessel or organ so that there is substantially no pressure gradient between said exterior and interior. 8. A system for delivering a substance into a cell, said system comprising: an enclosing means for defining at least part of a boundary of a sealed enclosure, said sealed enclosure containing said cell and an extracellular environment of said cell, said extracellular environment containing said substance; and a pressurization means for establishing an incubation pressure within said enclosure; whereby an establishment of said incubation pressure facilitates uptake of said substance by said cell. 9. The system of claim 8, further comprising a delivery means for delivering said substance to said extracellular environment. 10. The system of claim 8, wherein said enclosing means comprises an impermeable sheath. 11. The system of claim 8, wherein said enclosing means comprises an occlusion means for occluding a passage in a tissue. 12. The system of claim 8, wherein said enclosing means comprises a pressurization chamber. 13. The system of claim 8, wherein said boundary is defined substantially by said enclosing means. 14. The system of claim 8, wherein part of said boundary is defined by a tissue. 15. The system of claim 14, further comprising a protective means adapted to be placed around said tissue, for preventing a trauma in said tissue. 16. The system of claim 15, wherein said protective means comprises an inelastic sheath.

Inventors: DZAU VICTOR J, GIBBONS GARY H, MANN MICHEAL J, DZAU, VICTOR, J., GIBBONS, GARY, H., MANN, MICHEAL, J.
Applicants: UNIV LELAND STANFORD JUNIOR, THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIO
Application year: 1997
Main classification: C12M3
IPC codes: A61K31, A61K45, A61K48, A61M25, A61M37, A61P43, C12M1, C12M3, C12N5, C12N15