MXPA99004201A - Pressure-mediated intracellular delivery of molecules or microparticles - Google Patents

Abstract

The invention provides a method of enhancing the uptake by a cell of an atom, a molecule, or particle of<10&mgr;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

INTRA-CELLULAR DELIVERY MEDIATED BY PRESSURE OF MOLECULES OR MICROPARTICLES Background of the Invention This invention relates to the facilitation of the incorporation of extracellular substances by living cells. The study of cellular and molecular biology has clarified the existence of an intricate physical and biochemical environment within the membranes of living cells. These membranes provide a sophisticated semipermeable 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 conformation of their intracellular spaces, either by the passive exclusion of large substances and / or lipophobes that can not penetrate the lipid bilayer of the cellular limit, or by bidirectional, active, controlled movement , of molecules or particles through the cell membrane by specialized proteins. Some of these proteins can combine to form pores or channels to provide either a generalized passage to 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 physicians, and provides the basis for their ability to dissect the cellular processes of life and to devise pharmacological strategies to prevent, diagnose, or overcome the 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 been limited to potential uses of these materials in intracellular applications. In addition, many complex substances can be incorporated by cells only through a process of invagination of the cell membrane such as endocytosis or pinocytosis. In many cases, the endocytotic vesicles thus formed containing the extracellular substance are previously fused with lysosomes containing coarse enzymatic environments, which result in the digestive destruction of the swallowed matter. SUMMARY OF THE INVENTION The method of the invention involves the delivery of a substance to the extracellular environment of a cell or living cells, the creation of a closed space around the cell or cells, and the establishment of an elevated incubation pressure within the space closed enough to increase the incorporation of the substance by the cell or cells. The substance can vary in size from a small molecule to a microparticle, and can exert a range of effects, or serve a range of functions, on the cells by which it is incorporated. For example, the substance may be a small molecule (e.g., a drug), a sugar, a fatty acid or fatty acid derivative, or a protein (e.g., an antibody, an enzyme or a hormone). The cells can exist in vitro, in the culture of a tissue or organ, or can form a portion of a living organism, for example, a mammal such as a human. In accordance with the above, a purpose of this invention is to induce a higher degree of intracellular incorporation of microscopic or molecular species that would otherwise be expected or would be realized within a target cell or cell population. This method can therefore be used to facilitate the intracellular delivery of large quantities of a substance, which would normally be incorporated into cells only in small quantities, or allow the incorporation by a cell of a substance that would otherwise remain excluded by the cell membrane in the extracellular environment. This invention can also make more efficient the incorporation by a higher percentage of cells in a target population, and allow specific organs or specific tissues to be targeted. The intracellular delivery carried out using the method of the invention is also advantageous because it does not cause tissue damage (e.g., distension) or trauma, and because it is not potentially toxic to cells. In addition, in many cases, the intracellular delivery method of the invention ^^ provides a means of delivery for the cytoplasm of the cell without leading to lysosomal compartmentalization, whereby protection of the destruction of the substance delivered by lysosomal enzymes is provided. This method can also improve the movement of substances through intracellular barriers, in spaces such as nuclei ^ cell phones. Other features and advantages of the invention will be apparent from the detailed description, drawings, and claims. Brief Description of the Drawings Figure 1-A represents a supply system for the present invention. Figure 1-B shows the system of Figure 1-A attached to a free end of a blood vessel, prior to delivery of a solution containing a substance to be delivered to the blood vessel. Figure 1-C shows the system and blood vessel of Figure 1-33 during the delivery of the solution containing a substance to be delivered to the endothelium of the blood vessel. Figure 1-C shows the system and blood vessel of Figure 1-B during the delivery of a solution containing a substance to be delivered to the endothelium and to the outer surface of the vessel blood.

Figure 2 illustrates the delivery to a portion of a blood vessel defining part of the limit of the pressurized confinement. Figure 3-A represents an alternative method of intracellular delivery to a blood vessel. Figure 3-B shows the blood vessel of Figure 3-A, where the blood vessel is pressurized mechanically. Figure 4-A represents a 2-balloon catheter adapted to deliver a solution containing a substance that is going to be delivered in a blood vessel. Figure 4-B shows the catheter of Figure 4-A with the balloons in an inflated state. Figure 4-C shows the catheter system that has a balloon and internal tubules to deliver a solution containing a substance to be delivered to the walls of a vessel blood. f Figure 5-A illustrates the supply of blood vessels in an organ. Figure 5-B shows the use of balloon catheters to pressurize an organ. Figure 6 shows a delivery system comprising a pressurization chamber. Figure 7-A shows the transfection efficiency as a function of the pressure applied to the transfected human saphenous vein according to the present invention. Figure 7-B illustrates the effect of a distension prevention liner on transfection efficiency for the human saphenous vein transfected with oligodeoxyribonucleotide labeled with fluorescein isothiocyanate. Figure 7-C illustrates the inhibition of interleukin-6 protein production after transfection of antisense oligodeoxyribonucleotide against interleukin-6 in the human saphenous vein. Figure 8-A illustrates the inhibition of interleukin-6 mRNA production after transfection of antisense oligodeoxyribonucleotide against interleukin-6 in the human saphenous vein of a first subject. Figure 8-B is a graph similar to that in Figure 8-A for a second subject. Figure 8-C is a graph similar to that of Figure 8-A for a third subject. Figure 9-A shows the transfection efficiency for in vivo transfection of the rabbit carotid artery as measured by fluorescence microscopy. Figure 9-B shows the luciferase activity for control, healthy, and atherosclerotic cells after in vivo transfection of the rabbit carotid artery with DNA encoding firefly luciferase. Figure 10 shows the transfection deficiencies for rat vascular smooth muscle cells transfected in vitro according to a method of the present invention. Figure 11 shows luciferase activities for rat kidneys flooded with plasmid DNA containing the gene coding for firefly luciferase. Figure 12-A shows the effect of pressure on transfection efficiency for rat aortic cells. Figure 12-B shows the expression of PCNA induced by ischemia in rat aortas transplanted with and without transfection mediated by pressure of the antisense oligodeoxyribonucleotide against PCNA. Figure 12-C shows the expression of cdc2 kinase induced by ischemia in rat aortas transplanted with and without transfection mediated by pressure of oligodeoxyribonucleotide cdc2 antisense kinase. Figure 12-D shows and illustrates the reduction in - lumenal narrowing of damaged-ischemic rat aortas, planted isotrans- 10, resulting from pressure-mediated transfection with antisense oligodeoxyribonucleotide against both PCNA and cdc2 kinase. Figure 13-A shows the transfection efficiencies for hearts of rats transfected ex vivo with oligodeoxyri-bonucleotide labeled with fluorescein isothiocyanate, with or ^ P without pressure. Figure 13-B shows the expression ICAM-1 in rat hearts transplanted with or without transfection mediated by pressure of antisense oligodeoxyribonucleotide against ICAM-1. Figure 13-C illustrates the induction of acceptance of the Long-term transplantation by means of pressure-mediated transfection of rat hearts transplanted with antisense oligodeoxyribonucleotide against ICAM-1. Figure 14-A shows wall thicknesses at six weeks and six months after transplant transplantation. treated (control) and transplanted transplants either with reverse antisense oligodeoxyribonucleotide (control) or with antisense oligodeoxyribonucleotide against both PCNA and cdc2 kinase. Figure 14-B shows results similar to those in Figure 14-A for veins transfected with oligodeoxyrribo-5 nucleotide bait E2F, compared to untreated grafts and control grafts transfected with scrambled oligodeoxyribonucleotide. Detailed Description - The types of substance that can be used with this The invention includes (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 which have a molecular weight of = 1,000), especially proteins and peptides, with the exception of nucleic acids, (4) polymers and j filaments (generally <10 μm in their largest dimension), (5) atoms and inorganic molecules, and (6) microscopic particles (generally <10 μm in its largest 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 chemistry, biochemistry, or the functional properties of the substance to be delivered. The substance may exist in solution in the extracellular environment, or may 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 enclosure boundary is substantially defined by a closure element, so that the target tissue (the tissue - which comprises the target cell) is subjected to isotropic pressure, 10 and does not distend or experience trauma. In another modality, part of the confinement limit is defined by a tissue. A protective element such as an inelastic liner is then placed around the tissue to prevent distension and trauma to the tissue. In particular, in a blood vessel, preferably it is ^ P defines a sealed enclosure between occlusions formed by inflatable balloons or bound 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, a confinement within an organ is defined by establishing occlusions within the organ's passages (eg, blood vessel), so that a space within the organ can be pressurized. The incubation pressure used to facilitate the The cellular incorporation is preferably maintained at a predetermined level during a predetermined incubation period. The incubation pressure depends on the application, including parameters such as the incubation period, the type of tissue, and the molecule to be delivered. The incubation pressure used can vary from, for example, 50 millimeters of mercury to 5 atmospheres above ambient pressure, for example, 300 millimeters of mercury to 1500 millimeters of mercury above ambient pressure, or from 0 to 2 atmospheres above room temperature; You can also use higher or lower pressures, provided that effective delivery is achieved and no compensatory damage is sustained by the target cell or cells. The incubation period used to facilitate cellular incorporation may vary, depending on parameters such as the incubation pressure, the type of target tissue, ^ P the molecule being delivered, and the desired dosage. The duration of the incubation pressure may vary, for example, from about one second to four hours. Preferably, the duration of the pressure 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, urinary genital 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 the complete organ (eg, a heart), or to a complete organism. In one embodiment a solution containing a substance to be delivered is flooded in a target region (eg, a kidney) of a patient, and the patient is pressurized in a pressurization chamber. The applications of the present invention may include the treatment of allografts (grafts derived from a - different subject than the patient of the transplant) and sininjertos (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, chemical label, and then chasing the substance, following the administration increased by pressure, by middle of the label. Alternatively, the measurement of ^ P biological activity can be carried out. Similarly, optimization of supply conditions can be achieved via the quantification of the supply of an easily labeled material that is chemically and / or physically similar to the desired substance. As discussed above, a system for delivering a molecule in a cell using the methods of the invention includes a closure element to define at least part of a limit of a sealed enclosure, and an enclosure element. pressurization to establish an incubation pressure inside the enclosure. The closure contains the target cell and an extracellular environment of the cell. A delivery element, such as a catheter or syringe, is used to deliver the molecule to the extracellular environment, directly or indirectly (for example, by intravenous injection). The confinement limit is defined by the confinement element, and possibly by a tissue. The convenient closure element includes, depending on the embodiment, a pressurization chamber, a waterproof liner or bag, and an occlusion element for occluding a passage in a fabric. A pressurization chamber is particularly suitable for the treatment of grafts or whole organisms, while other devices are very suitable for the intraoperative treatment of tissue. In one embodiment, the confinement limit is substantially defined by the confinement element. The pressure is applied to the target tissue uniformly from all directions, and the target tissue is not subjected to a risk of trauma. In another embodiment, part of the tissue is defined by a tissue (e.g., the target tissue). The tissue that forms part of the closure of the enclosure is pressed from only one side, and can be distended. A protective element such as an inelastic lining is placed around the tissue to prevent distension and trauma to the tissue. The methods and delivery systems of the invention are illustrated in more detail as follows: Figures 1-A to 1-C illustrate methods of pressurized delivery of molecules to the cells of a blood vessel. Figure 1-A is a side view of a delivery system 11 of the present invention. The system 11 comprises a container 10 for holding a solution 40 containing a substance to be delivered, and a supply element for expelling the solution 40 from the container 10. The supply element comprises a plunger 12 and a supply pipe 14. Opposite the plunger 12, the container 10 opens towards the tube 14. Attached to the tube 14 are , listed in order of proximity to the container 10, a stopcock 16, a pressure meter 18, a retracted liner 20, and a notch 22. The liner 20 is preferably impermeable and inelastic. The notch 22 is near the distal open end 30 of the tube 14. The stopcock 16 is initially in a closed position, preventing the solution 40 from passing from the container 10 to the tube 14. Figure 1-B shows a blood vessel 24 attached to the system 11. The open end 30 is positioned at a proximal end of the blood vessel 24. The notch 22 fits within the proximal end of the tissue 24. The liner 20 is pulled down to cover the tissue 24. A tie or ligature 26 -A is entangled around the liner 20 and the fabric 24 at the point where they are attached to the tube 14, to prevent the fabric 24 from slipping off the open end 30. When the stopcock 16 is turned to an open position, the solution 40 enters the tube 14 and the liner 20, extracting all gases and liquids present through the open end 28 of the liner 20. After extraction, a tie wrap 26-B is placed over the distal open end 28 of the liner 20 to form a hermetic seal or to water, as shown in Figures 1-C and 1-C. Figure 1-C illustrates the delivery directed to the endothelium of the blood vessel 24, while Figure 1-C illustrates the supply to the endothelium and the outer surface of the blood vessel 24. In Figure 1-C, the tie wrapping 26- B is placed around the liner 20 and the fabric 24. The binding wrap 26-B occludes the blood vessel 24. The stopcock 16 is turned to its open position, and the plunger 12 is pushed, so that the solution 40 it is supplied in vessel 24 under a supply pressure. The supply pressure is allowed to rise until an incubation pressure is reached, and the stopcock 16 is closed. The blood vessels 24 are allowed to incubate during an incubation period, after which the binding wrapper 26- B is released to release the pressure (not shown). The limit of the sealed closure is defined by the walls of the vessel 24 and by an enclosure element. The sealed enclosure contains the target (endothelial) cells of the blood vessel 24, and its extracellular environment. If the stopcock 16 is in a closed position, the closure member comprises a tube 14, the stopcock 16, and the ligature 26-B. If the stopcock 16 is in an open position, the closure element comprises the ligature 26-B, the tube 14, the plunger 12, and parts of the walls of the container 10. The closure element defines at least part of the limit of the encierro. In the embodiment shown in Figure 1-C, part of the confinement boundary is defined by blood vessel 24. By applying pressure only inside the blood vessel 24 it could cause blood vessel 24 to relax and experience trauma. The liner 20 acts as a protective element, preventing the blood vessel 24 from distending. In a configuration such as that of Figure 1-C, it is thus important that the liner 20 is inelastic. It is also possible to place a tie wrapping 26-B around the liner 20 only, as illustrated in Figure 1-C. In this case, the sealed closure containing the target cells of the blood vessel 24 and its extracellular environment is substantially defined by an enclosing element. If the stopcock 16 is in a closed position, the closure member comprises the liner 20, the tube 14, the stopcock 16, and the ligature 26-B '. If the stopcock 16 is in an open position, the closure member comprises the liner 20, the tube 14, the ligature 26-B ', the plunger 12 and parts of the walls of the container 10. In the embodiment shown in Figure 1-C, the confinement boundary is substantially defined by the encerrment element. The pressure around blood vessel 24 is uniform, and thus blood vessel 24 does not experience trauma. Since the liner 20 acts as part of the enclosing element, it is important that the liner 20 is impermeable. The liner 20 need not necessarily be inelastic in the configuration of Figure 1-C ', however, since the use of an elastic liner would not lead to trauma to the blood vessel 24. Figure 2 illustrates a live method of delivery of molecules to a blood vessel connected to the circulatory system of a patient. The tube 14 is inserted into the lumen of a vessel 224 that is still connected to the body of a living animal. A liner 220 is wrapped around the vessel 224, and a fastener 228 (e.g., a hot seal) joins the two flanges of the sheet 220 to form a tube. The liner 220 acts as a protective element, preventing distention of the cup 224. Two tie wraps 226-A and 226-B are wrapped around the liner 220. The tie wraps 226-A and 226-B act as elements of occlusion, occluding vessel 224. Occlusions 226-A and 226-B, and vessel walls 224 between occlusions 226-A and 226-B define a sealed enclosure 230 that contains the target cells of vessel 224 and its environment extracellular Solution 40 is injected into the sealed enclosure, and segment 230 is allowed to incubate during an incubation period. After the incubation period, the 226-A and 226-B occlusions are removed, and the blood is allowed to flow through the vessel 224.

Figures 3-A and 3-B illustrate the delivery system having different delivery and pressurization elements, used to deliver molecules to the blood vessel shown in Figure 2. A rigid tubular wrap 250 is placed around the sheet 220, and a screw 252 is placed around the wrapper 250, as illustrated in Figure 3-B. The sheath 250 is circumferentially flexible, so that the diameter of the tube that forms is vare, but is axially rigid, of Thus, although its diameter changes, it remains substantially tubular. An adjusting screw 254 adjusts the screw 252, pulling the wrapper 250 just enough, creating pressure within the vessel 224. This pressure is maintained during an incubation period, after which the screw 254 is unscrewed. Figures 4-A to 4-D illustrate a method of provide a solution containing a substance to be delivered to the lumen of a blood vessel 324 through a catheter 314. The catheter 314 is inserted into the vessel 324. The catheter 314 is closed at its end 316. The catheter 314 has two balloons 332-A and 332-B, and a 330 supply port between the balloons 332-A and 332-B. Initially, balloons 332-A and 332-B are deflated, as shown in Figure 4-A. After catheter 314 is inserted into vessel 324, balloons 332-A and 332-B are inflated, as shown in Figure 3-B. The balloons 332-A and 332-B occlude the glass 324 and create a sealed enclosure 334 within the vessel 324. A solution 340 containing a substance to be delivered is supplied to the enclosure 334 through the port 330. The solution 340 is supplied under pressure, so that the enclosure 334 becomes pressurized. After an incubation period, the balloons 332-A and 332-B deflate and the target closure 334 is depressurized. Figure 4-C shows an alternative delivery system of the present invention, in which a balloon mounted on a The catheter has miniature tubules to deliver 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 the holes 452 in the segment of the catheter 314 within the balloon 432. When a pressurized solution 440 is delivered through the catheter 314, solution 440 leaves holes 452, travels through tubules 450, and reaches walls of vessel 324. Figure 5-A illustrates the use of system 11 to supply blood conduits (vessels and / or atrium). and ventricles) of an organ 124 such as a heart. A protective liner 120 is wrapped around the organ 124. The organ 124 has an artery 112 that carries blood therein and a vein 114 that carries blood outwardly. The tube 14 is inserted into the lumen of the artery 112, and the liner 120 is wrapped around the artery 112 and the vein 114. The tie wrap 126-A fits around the liner 120 in artery 112, and the wrapper Attachment 126-B fits around liner 120 in vein 114, to prevent spillage of fluid out of organ 124. Tether wrap 126-A allows tube 14 to enter artery 112, and wrap tight enough to seal artery 112 and prevent spillage. The solution 40 containing a substance to be delivered is injected, and the organ 124 allows it to be incubated. After the incubation period, the binding wrap 126-A and 126-B are removed, and the blood is allowed to flow through the organ 124 once again. Figure 5-B illustrates the use of balloon catheters to seal an entrance and exit of an organ (eg, 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 that is directed away from the organ 524. The balloons 552 and 562 are initially deflated (not illustrated). As soon as catheters 550, 560 are inserted into their respective blood vessels, balloons 552, 562 are inflated and occlusions are established in conduits 512, 514, respectively. A solution 540 containing a substance to be supplied is supplied to the organ 524 under a supply pressure. Figure 6 illustrates the use of a pressurization chamber to facilitate the delivery of substances to cells. A support element such as a plate 610 contains a tissue 624 containing target cells. A solution 640 containing the substance is placed in the dish 610. The dish 610 is placed in a pressure chamber 650. The chamber 650 is closed and sealed, and a pressurized gas (eg, C02) is introduced into the chamber 650 through a duct 660. Solution 640 and tissue 624 are maintained under an incubation pressure during an incubation period. The tissue 624 may comprise an entire organ. The pressurization chamber such as that shown in Figure 6 is particularly suitable for a delivery method in which the entire organism is pressurized. In such a method, a solution containing a substance to be delivered is preferably flooded in the blood vessels and / or organs (eg, kidney) of a patient. The patient is placed in the pressurization chamber. The pressurization chamber is then maintained under an incubation pressure, during an incubation period. There are several possible mechanisms that underlie the increased permeability of cell membrane molecules under pressure. The increase in membrane permeability requires increased pressure within the cell and / or the extracellular environment, but not necessarily a pressure gradient across the cell membrane. It is possible that the proteins that form the transmembrane channels change conformation at high pressure, and thus allow the passage of the molecules through the channels and into the cytoplasm. The exact pressures, incubation periods and concentrations used depend on the type of target tissue. For example, as described further below, an incubation period of about 5 minutes at low pressure (-0.5 atmospheres) is sufficient to achieve an almost maximum transfection efficiency in the human saphenous vein., while an incubation period of more than one hour at high pressure (~ 2 atmospheres) is required to achieve a transfection efficiency of 80-90% in rat aortas. For rat hearts, an incubation period of 30 to 45 minutes at 2 atmospheres 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 of the order of atmospheres. Suitable incubation periods and pressures for a given tissue type can easily be determined by the experienced technician. In the absence of limitations imposed by surgical procedures, it is generally preferred that the pressurized enclosure walls do not include living tissue, since the parts forming the tissue of the enclosing wall are subjected to mechanical stress. Some surgical procedures, such as the treatment of blood vessels connected to the circulatory system during the procedure (see Figures 3-A and 3-B), require that at least parts of the walls of the confinement be defined by the tissue. In this case, it is important that a protective element be used to prevent tissue distention. Ex vivo treated grafts are generally preferably treated by incubation in a pressurized chamber or equivalent pressurized enclosure. The methods of the invention are further illustrated below in experiments showing the intracellular delivery of nucleic acid molecules. The transfection efficiencies of the 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, immunosorbent test of linked enzyme; FITC, fluorescein isothiocyanate; FITC-ODN, oligodeoxyribonucleotide labeled with FITC; 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 net pressures applied ^ P to the samples, above the ambient (atmospheric) pressure. Example 1 The human saphenous vein was transfected with fluorescein isothiocyanate-labeled oligodeoxyribonucleotide. according to a method similar to that illustrated in Figure 1-C. Figure 7-A shows the efficiency of the transfection, as a function of the applied pressure, for various concentrations of oligodeoxyribonucleotide. Efficiency was measured as a percentage of the intimal and total mean cells found by have nuclear localization of oligodeoxyribonucleotide labeled with fluorescein isothiocyanate via fluorescence microscopy. The pressures vary from 50 to 760 millimeters of mercury, and the concentrations of oligodeoxyribonucleotide in physiological saline vary from 5 to 100 μM. n = 6. The effect of the stretch prevention liner on the efficiency of transfection was evaluated using a method similar to that illustrated in Figure 1-C, at an oligodeoxyribonucleotide concentration of 20 μM and at pressures ranging from 50 to 760 millimeters of mercury. Figure 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 interleukin-6 production by the antisense oligodeoxy ribonucleotide in the culture of the total organ. The vein segments were incubated in a culture medium for 24 hours after transfection. The transitions were carried out at 5 mM, 10 mM, and 100 mM for 10 minutes according to a method similar to that illustrated in Figure 1-C. Figure 7-C shows reductions in the interleukin-6 protein detected via the enzyme linked immunosorbent assay in culture medium for antisense transfected cultures. The reduction levels shown are in relation to the levels found in control cultures of non-transfected and transfected veins of non-specific oligodeoxyribonucleotide. n = 6. EXAMPLE 2 The quantitative reverse transcription polymerase chain reaction was used to measure the reduction in interleukin-6 mRNA resulting from transfection of the antisense oligodeoxyribonucleotide made in accordance with the present invention. Three samples of human saphenous vein were transfected as illustrated in Figure 1-C, and levels of mRNA were compared in transfected antisense vein segments with levels in segments transfected by antisense oligodeoxyribonucleotide (control, reverse and untreated). The results of the three samples are shown in Figure 8-A, 8-B, and 8-C, respectively. Reductions in mRNA levels indicate frequency specific efficacy in the treatment of oligodeoxyribonucleotide anti-sense. EXAMPLE 3 Rabbit carotid arteries were transfected in vivo with fluorescein-labeled oligodeoxyribonucleotide labeled with a method similar to that illustrated in FIG.

Figure 2. Transfection efficiency was measured at four hours, four days, and seven days after transfection. The percentages of nuclei positive to fluorescein isothiocyanate as a function of time are shown in Figure 9-A. n = 2-3. Expression of the gene for firefly luciferase was then measured in vivo from the transfection of the rabbit carotid artery with a plasmid DNA construct containing the luciferase gene. Healthy arteries (normal) and arteriosclerotic vessels (CHOL / inj) were transfected under pressure, as shown in Figure 2. The control artery (CTRL) was exposed to the plasmid carrying the luciferase gene in the absence of pressure. The arteries were harvested on day five and the 5 tissue homogenates were tested to determine the activity of the luciferase. The test results are shown in Figure 9-B. n = 2-4. Example 4 - Rat vascular smooth muscle (VSMC) cells are transfected in vi tro with oligodeoxyribonucleotide labeled with fluorescein isothiocyanate, as shown in Figure 6. The cells were exposed either at atmospheric pressure (net pressure 0 atmospheres) or at two atmospheres for 45 minutes. Figure 10 shows the transfection efficiencies for concentrations of oligodeoxyribonucleotide labeled with fluorescein ^ P isothiocyanate of 1 mM and 80 mM. n = 4. Example 5 Figure 11 illustrates the effect of pressure on transfection efficiency for flooded rat kidney cells in vivo with plasmid DNA containing the gene for firefly luciferase, as illustrated in FIG. Figure 6. Rats were exposed at atmospheric pressure (0 atmospheres) or at 2 atmospheres for 30 minutes after perfusion of the kidney. Kidneys were harvested three days after transfection and tissue homogenates were tested to determine the activity of luciferase. n = 7. Example 6 Figure 12-A shows the effect of pressure on transfection efficiency for rat aortic cells. The aortas were harvested from donor rats and incubated at 4 ° C for 24 hours in physiological solution to induce ischemic damage, in a manner similar to that illustrated in Figure 1-C. The incubation solutions contained 40 μm oligodeoxyribonucleotide labeled with fluorescein isothiocyanate. Incubations were performed at 0 atmospheres and at 2 atmospheres above atmospheric pressure. After incubation, the tissue was isotransplanted in rat aortas and harvested 24 hours after transplantation. The nuclear localization of the FITC was assessed by fluorescence microscopy of sections stained with fluorescent DNA intercalator dye. Transfection effi ciency is expressed as cells that display nuclear localization of oligodeoxyribonucleotide labeled with fluorescein isothiocyanate as a percentage of total cells. n = 3, p < 0.005. Figure 12-B shows the expression of PCNA induced by ischemia in a transplanted rat aorta and without transfection mediated by pressure of antisense oligodeoxyribonucleotide against PCNA. The transfection procedure was similar to that illustrated in Figure 1-C. Ischemic lesions were induced by incubations of 24 hours at 4 ° C, either in saline (control), or in saline containing 40 μM of oligodeoxyribonucleotide. A pressure of two atmospheres above the atmospheric pressure was applied during the incubation both to the control and to the samples treated by oligodeoxyribonucleotide. The tissue was harvested 6 days after the isotransplant, and the PCNA protein levels in the tissue homogenates were measured by enzyme-linked immunosorbent assay. n = 7, P = 0.02. Figure 12-C shows the expression of cdc2 kinase induced by ischemia in rat aortas transplanted with and without transfection mediated by anti-sense oligodeoxyribonucleotide pressure against cdc2 kinase. Transfection, transplantation, and harvesting procedures were similar to those described above in relation to Figure 12-B. Protein levels for the cdc2 kinase were measured by the enzyme-linked immunosorbent assay. Figure 12-B illustrates the reduction in lumenal narrowing of isotransplanted rat aortas, with ischemic damage, resulting from pressure-mediated transfection with anti-sense oligodeso-xiribonucleotide against both PCNA and cdc2 kinase. Ischemic damage was induced by 24 hours of incubation at 4 ° C in either saline (control) or oligodeoxyribonucleotide anti-sense-PCNA / anti-sense kinase cdc2 solution (40 μM each). A pressure of two atmospheres above the ambient pressure was applied to all tissues (including control). Blocking the expression of the regulatory genes of the two cell cycle reduced neointimal hyperplasia and luminal narrowing in ischemically damaged isografts, as measured by computerized image analysis. n = 5 12, P = 0.03. Example 7 Figure 13-A shows transfection efficiencies for rat hearts transfected ex vivo with fluorescein isothiocyanate labeled oligodeoxyrri- - bonucleotide, with and without pressure. A solution of oligodeoxyribonucleotide labeled with fluorescein isothiocyanate (80 μM) was flooded in the coronary arteries of donor hearts after aortic cross-stapling. The hearts were immersed in a solution of oligodeoxyribonucleotide labeled with isothiocyanate of fluorescein and were exposed to either 0 atmospheres or 2 atmospheres above ambient pressure for 45 minutes at 4 ° C, as shown in Figure 6. Hearts were transplanted heterotopically into the abdominal aorta and vein Cava of receiving rats. Nuclear localization of fluorescein isothiocyanate was evaluated 24 hours after transplantation by fluorescent microscopy of sections stained with fluorescent DNA intercalator dye. The efficiency of the transfection is expressed as the cells that display nuclear localization of oligodeoxyribonucleotide labeled with fluorescein isothiocyanate as a percentage of the total cells. n = 3, p < . 0.005. Figure 13-B shows the expression ICAM-1 in transplanted rat hearts, with and without transfection mediated by anti-sense oligodeoxyribonucleotide pressure against ICAM-1. Either saline (control) or oligodeoxyribonucleotide anti-sense against ICAM-1 (80 μM) was immersed in the coronary arteries of hearts of the PVG strain of donors after aortic cross-stapling. The hearts were immersed in an oligodeoxyribonucleotide solution labeled with fluorescein isothiocyanate and exposed to two atmospheres above the ambient pressure for 45 minutes at 4 ° C, as illustrated in Figure 6. The tissue was harvested three days after heterotopic transplantation. in ACI receivers. The positive ICAM-1 area was measured by image analysis of sections stained immunohistochemically for ICAM-1. n = 3-6, p = 0.04. Figure 13-C illustrates the induction of long-term graft acceptance by pressure-mediated transfection of rat hearts transplanted with anti-sense oligodeoxyribonucleotide against ICAM-1. The rat hearts of the PVG strain were harvested and transfected ex-vivo with anti-sense oligodeoxyribonucleotide solution of ICAM-1 (80 μm) or with saline (control), as described above in relation to Figure 13-B . The hearts were heterotopically transplanted into recipients of the ACI strain. All animals were systemically administered anti-LFA-1 antibody for 6 days after transplantation, to block the ligand for ICAM-1. No further immunosuppression was administered. Tolerance was reported as the percentage of treated animals that were found to have long-term acceptance of their allografts. Graft acceptance is defined by the presence of heart beats in the graft during > 100 days, control n = 12, treated with anti-sense n = 27, P = 0.003. Example 8 - Figures 14-A and 14-B show the inhibition of Neointimal hyperplasia in rabbit jugular veins grafted to carotid arteries, after pressure-mediated transfection with oligodeoxyribonucleotide designed to block the regulation of cell cycle regulatory genes. Figure 14-A shows the thickness of the wall at six weeks and six months after transplant for grafts ^ P untreated (control), and transfected grafts, either with oligodeoxyribonucleotide (control) anti-reverse sense or with anti-sense oligodeoxyribonucleotide against both PCNA and cdc2 kinase. Neointima formation was inhibited during to six months, while medial hypertrophy allowed the thickness of the adaptive wall to reduce wall tension in the high-pressure arterial environment. Figure 14-B shows results similar to those in Figure 14-A for veins transfected with oligodeoxyribonucleotide bait E2F, compared to untreated grafts and control grafts transfected with scrambled oligodeoxyribonucleotide. n = 6, P <; 0.005. Although the foregoing description contains many specific aspects, this should not be considered as limitations of the scope of the invention, but as illustrations of particular modalities thereof. For example, an incubation pressure that varies with time can generally be used. One skilled in the art can easily devise many potential designs for closure means, protection means, and / or occlusion means, depending on the application. Various incubation pressures, periods and dosages of active agents leading to desirable or near maximal transfection efficiencies can be easily determined for different types of fabrics.

Claims (16)

1. CLAIMS 1. A method of increasing the admission by ^ F of a cell of an atom, a molecule, or a maximum dimension particle of < 10 μm, said method comprising contacting said cell and a liquid medium comprising said atom, molecule, or particle, maintaining said cell and said liquid medium in an enclosed space, and t subjecting said enclosed cell and liquid medium to a
2. 10 enough incubation pressure to increase the admission by said cell of said atom, molecule or particle. The method of claim 1, wherein said cell is contained within a mammalian blood vessel that is sealed in two locations to provide said enclosed space.
3. 3. The method of claim 1, wherein said

   ^ P molecule is a charged, therapeutic organic molecule, having a molecular weight of less than 1,000.
4. 4. The method of claim 1, wherein said molecule is a protein or a peptide having a molecular weight
5. 20 of more than 1,000.
6. The method of claim 1, wherein said incubation pressure is between 50 mm Hg and 5 atmospheres above ambient pressure.
7. The method of claim 1, wherein said incubation pressure is between 200 mm Hg and 2.5 atmospheres above ambient pressure.
8. The method of claim 1, wherein said cell is contained in a blood vessel or mammalian organ, and the incubation pressure equilibrates between the outside and inside "of the vessel or organ so that there is no pressure gradient substantially between said outside and inside 8.
9. A system for delivering a substance in a cell, said system comprising: means for enclosing to define at least a part of a boundary of a sealed housing, said sealed housing containing said cell and an extracellular environment of said cell, said extracellular environment containing said substance, and pressurization means for establishing an incubation pressure inside said housing, whereby the establishment of said incubation pressure facilitates the admission of said substance by said cell. of claim 8, further comprising delivery means for delivering said substance to said environment. extracellular 10.
10. The system of claim 8, wherein said means for enclosing comprise a waterproof liner.
11. The system of claim 8, wherein said means for enclosing comprises occlusion means for covering a passage in a tissue.
12. The system of claim 8, wherein said means for enclosing comprise a pressurization chamber.
13. The system of claim 8, wherein said limit is substantially defined by said means for enclosing.
14. The system of claim 8, wherein part of said limit is defined by a fabric.
15. The system of claim 14, further comprising protective means adapted to be placed around said tissue, to prevent trauma to said tissue.
16. 16. The system of claim 15, wherein said protective means comprises an inelastic liner.