MXPA99002853A - Gas-driven particle delivery device - Google Patents

Abstract

A gas-driven particle delivery instrument is provided. The device includes various elements which enhance the utility and efficacy of the device, including a rotational flow element, a turbulent flow element, a flow constriction element, and combinations thereof.

Description

DEVICE FOR SUPPLYING GAS-OPERATED PARTICLES FIELD OF THE INVENTION The present invention relates to the field of material supply in cells, more particularly to instruments for the delivery of material in cells using particle-mediated delivery techniques.

BACKGROUND OF THE INVENTION The delivery of materials mediated by particles, particularly nucleic acid molecules, in living cells and tissues has emerged as an important tool in the biotechnology of plants and animals. The expression of the broader and transient term of genetic material supplied via particle-mediated techniques in target cells has been demonstrated in a wide variety of microorganisms, plants, and animals. Successful integration of DNA into embryonic cells has also been demonstrated using these techniques, and particle-mediated gene delivery instruments have been used to deliver other materials into cells, including pharmaceutical and biopharmaceutical compositions such as proteins, peptides and hormones.

REF .: 29876 Since the principles or foundations of particle-mediated delivery technology have been developed, attention has shifted more to the development of devices that offer the operator the ability to effect the delivery of genes mediated by particles in a fast and convenient way. It is also desirable for the operation of the delivery device to be efficient and highly reproducible. A particular device, which uses compressed gas to accelerate the carrier particles carrying or carrying biological materials in target tissue, is described in commonly recognized International Publication No. WO 95/19799. The distribution or extension of carrier particles supplied from a particle-mediated delivery device, such as the device of WO 95/19799, can be decisive in some applications, particularly when the biological material is supplied, for example genetic material. In applications where germline or embryonic line transformation events are desired, the need to control the delivery configuration of carrier particles is substantially more acute than in other applications, such as where only the transient expression of introduced genetic material is necessary. . When a non-frequent embryonic line transformation event is desired, it is generally necessary to uniformly accelerate the particles towards a large target area to increase the probability that one or more target cells will be transformed. Accordingly, even though the device of the patent application WO 95/19799 and other related instruments have been suitable for their intended purposes, these remain a need to provide intensified uniformity and distribution of the particles supplied from such dispositives.

DESCRIPTION OF THE INVENTION The invention is directed to a device for supplying gas-driven particles having elements which modify the flow of gas through the device. In one embodiment of the invention, a particle supply device is provided which comprises a body having an acceleration passage formed therein. A rotating flow element is arranged within the acceleration passage and serves to impart a rotary motion in a gas flow passing through it prior to, or after, this gas flow in an acceleration chamber which forms a descending part of the acceleration passage. In various aspects of the invention, the rotary flow element is used to impart a rotary movement in the gas flow prior to, during, and / or after the gas flow has contacted particles which are to be supplied from Of the device. The rotating flow element can be any feature or structure disposed within an acceleration passage, this characteristic or structure is capable of imparting rotary movement in a gas flow passing through it. A particular rotary flow element comprises a shutter or baffle which is arranged within the acceleration passage in an upward position from a source of particles. Another rotary flow element comprises a structure, such as a plurality of thin, propeller-like vanes or blades, arranged within the acceleration passage in a downward position from a source of particles. In another embodiment, a particle supply device is provided which comprises a body with an extended acceleration chamber formed therein. The device includes a mixing chamber communicating with the inlet of the acceleration chamber, and an upstream gas chamber communicating with the mixing chamber. A rotating flow element is arranged within the countercurrent gas chamber, and imparts a rotary movement in a gas flow passing from the gas chamber countercurrently in the mixing chamber to form a vortex within the mixing chamber. In still a further embodiment, a particulate delivery device is provided which comprises a body with an extended acceleration chamber formed therein. The device includes a source of particles that are arranged adjacent to an inlet for the acceleration chamber. A turbulent flow element is arranged countercurrently or upstream of both the acceleration chamber and the particle source, whereby such an element is used to create turbulence in a gas flow passing through it prior to its contact with the source of particles. In a particular aspect of the invention, the turbulent flow element comprises a gas conduit arranged above the source of particles, wherein the gas conduit has a stepped portion of increased diameter. In another embodiment of the invention, a particle delivery device is provided which comprises a flow constriction element that restricts the flow of compressed gas in the device.

These and other objects, features and advantages of the present invention will become apparent from the following specification, reading in the clarity of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an illustration depicting the general operation of a gas powered particle delivery device. Figures 2A-2C are schematic illustrations of the effect of variations in the geometry of the outlet nozzle in the device of Figure 1. Figure 3 is a pictorial representation of a particle delivery device in which the present invention is embodied . Figure 4 is a sectional view of a portion of the instrument of Figure 3 showing a rotary flow element disposed within a portion of the device. Figure 5 is a side plan view of a rotary flow element in accordance with the present invention.

Figure 6 is a schematic sectional view of a portion of the particle delivery device comprising a rotary flow element. Figure 7 is a non-schematic view of Figure 6. Figure 8 is a plan view of the countercurrent face of the rotary flow element of the device of Figure 6. Figure 9 is a side plan view of the element of rotary flow of the device of Figure 6. Figure 10 is a sectional view of a turbulent flow element in accordance with the present invention. Figure 11 is a plan view of a flow constriction element in accordance with the present invention. Figure 12 is a graphical representation of the particle delivery study described in Example 1.

DETAILED DESCRIPTION OF THE PREFERRED MODALITY Prior to describing the present invention in detail, it is to be understood that this invention is not limited to particular particle delivery devices or particular carrier particles which may, of course, vary. It is also understood that different embodiments of the described sample delivery modules and related devices can be cut to size for the specific needs in the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the content is clearly dictated otherwise. Thus, for example, the reference to "a particle" includes referring to mixtures of two or more particles, reference to "a therapeutic agent" includes one or more such agents, and the like.

A. Definitions Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention pertains. The following terms are intended to be defined as indicated below.

When used herein, the term "therapeutic agent" is intended to refer to any compound or composition of matter which, when administered to an organism (human or animal) induces a pharmacological, immunogenic, and / or physiological effect. desired by local, regional, and / or systemic action. The term therefore encompasses those compounds or chemicals traditionally referred to as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. More particularly, the term "therapeutic agent" includes compounds or compositions for use in all major therapeutic areas, including, but not limited to, anti-infectives such as antibiotics and antiviral agents, analgesics and analgesic combinations, local anesthetics and general anorexics; antiarthritics, antiasthmatic agents; anticonvulsants; antidepressants; antihistamines; anti-inflammatory agents; antinausea; antineoplastic; antipruritics; antipsychotics; antipyretics; antispasmodics; cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrhythmics); antihypertensive; diuretics; vasodilators; stimulants of the central nervous system; cold and cough preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressive; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides and fragments thereof (if they occur naturally, chemically synthesized or produced recombinantly); and nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) that include both single-stranded and double-stranded molecules, gene constructs, expression vectors, antisense molecules and the like) . The particles of a therapeutic agent, alone or in combination with other drugs or agents, are typically prepared as pharmaceutical compositions which may contain one or more aggregated materials such as vehicles, and / or excipients. "Vehicles" and "excipients" generally refer to substantially inert materials which are non-toxic and do not interact with other components of the composition in a detrimental manner. These materials can be used to increase the amount of solids in particulate pharmaceutical compositions. Examples of suitable carriers include water, silicone, gelatin, waxes, and the like. Examples of "excipients" normally employed include pharmaceutical grades of rose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, ran, starch, cellulose, sodium or calcium phosphates, calcium sulfate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEG), and combinations thereof. In addition, it may be desirable to include a charged lipid and / or detergent in the pharmaceutical compositions. Such materials may be used as stabilizers, antioxidants, or may be used to reduce the possibility of local irritation at the site of administration. The charged, suitable lipid includes, without limitation, phosphatidylcholines (lecithin), and the like. The detergents will typically be a nonionic, anionic, cationic or amphoteric surfactant. Examples of suitable surfactants include, for example, the surfactants Tergitol® and Triton® (Union Carbide Chemicals and Plastics, Danbury, CT), the polyoxyethylene sorbitan, for example, the surfactants TWEEN® (Atlas Chemical Industries, Wilmington, DE), ethers polyoxyethylene, for example, Brij, pharmaceutically acceptable fatty acid esters, for example, lauryl sulfate and salts thereof (SDS), and similar materials. When direct intracellular delivery is desired, the therapeutic agents (or pharmaceutical preparations derived therefrom) can be coated with carrier microparticles using a variety of techniques known in the art. The dense materials are prepared to provide microparticles that can easily accelerate to a target over a short distance, where the microparticles are still sufficiently small in dimension relative to the cells in which they are supplied. It has been found that microparticles having an average diameter of some microns can easily enter living cells without unduly damaging such cells. In particular, microparticles of tungsten, gold, platinum and iridium can be used as carriers for therapeutic agents. Tungsten and gold are preferred. Tungsten microparticles are readily available in particle sizes from 0.5 to 2.0 μm in diameter, and are thus suitable for intracellular delivery. Although such microparticles have optimum densities for use in particular delivery methods, and allow highly efficient coatings with nucleic acids, tungsten may be potentially toxic to certain cell types. Thus, gold is a preferred material for use as carrier microparticles, when gold has a high density, is relatively inert to biological materials and resistant to oxidation, and is readily available in the form of spheres having an average diameter of about 0.2 to 3 μm. Gold spherical microparticles, or pearls, in a range of dimensions of 1-3 microns, have been successfully used in particle delivery technologies, as well as gold is provided in the form of a microcrystalline powder having a range of dimensions measurements of approximately 0.2 to 3 μm.

B. General Methods In one embodiment, the invention is directed to a component or topographic feature for use in a particle delivery device, this component or feature provided for an altered gas flow profile within the device. The altered gas flow, in turn, is provided for a dramatic improvement in the distribution of particles in the directions lateral to the main axis of the gas flow. In another embodiment, a component or topographic feature is used in a particle delivery device to provide a turbulent gas flow within the device. Turbulent flow is allowed for a more complete supply of payload from the device. In yet a further embodiment, the invention designs a means for limiting the amount of gas used to deliver particles from a gas powered or activated particle delivery device, wherein this limitation serves to significantly reduce the audible report associated with a gas. particle supply operation without a concomitant reduction in the efficiency of the supply of particles from the device. Various particle delivery devices suitable for delivering a particulate therapeutic agent, or microparticles coated with a therapeutic agent, are known in the art, and all are suitable for use in conjunction with the present invention. Such devices generally use a gaseous discharge to propel particles towards target cells. Optionally, the particles can be releasably attached to a movable carrier sheet, or removably attached to a surface along which a gas stream passes, transfer the particles from the surface and accelerate them to the target. Examples of gas discharge devices are described in US Patent No. ,204,253 and in International Publication No. WO 95/19799. Although the present invention is suitable for use with any particle delivery device, the invention is exemplified herein with reference to the device described in International Publication No. WO 95/19799. However, it is to be understood that any number of methods and devices similar or equivalent to those described herein may also be used in the practice of the present invention. Returning again to the drawings, Figures 1 and 2 provide an illustration of the general method of operation of a particle delivery device as described in International Publication No. WO 95/19799. The components of the device are shown in slightly schematic view in some places for clarity purposes. This particular description is intended to illustrate the basic operating principle of a particulate delivery apparatus, rather than illustrating construction details. Referring now to the device of Figure 1, a particle cartridge 14 is located within the particle delivery instrument. The particulate cartridge 14 is an elongated concave or tubular structure having a concave hollow passage passing through its center. A plurality of particles 16 are disposed inside the cartridge. The particles, as described hereinbefore, may be any particulate therapeutic agent or, preferably, may be comprised of small, dense carrier microparticles that are coated with a therapeutic agent, eg, DNA or RNA, which is intended to be delivered in a target cell or tissue. Such microparticles can be coated alternatively with other types of biological materials such as peptides, cytokines, hormones, or proteins. An actuator 18, for example a gas valve, gate, or rupturable membrane, is located countercurrent to the particle cartridge and is in fluid communication with the interior of the cartridge 14 via an appropriate conduit 17. The actuator is connected, by appropriate pipe generally indicated at 13, with a source of compressed gas 12. The source of compressed gas 12 may be a commercial, conventional compressed gas tank, which preferably contains an inert compressed gas such as helium. A compressed gas reservoir is generally desirable between the gas source 12 and the actuator 18, however, the pipe 13 can function as a reservoir. Adjacent to the particle cartridge 14 is a hole 20 which provides fluid communication with the interior of an acceleration chamber 22 which in turn communicates with a conical outlet nozzle 24. The target, for example a patient, the surface of the tissue, or cell, is designated 19 in the Figure. In general operation, the actuator 18 is used to release an impulse of compressed gas through the device. A particle acceleration passage, disposed between the actuator 18 and the outlet nozzle 24, provides a path through which the released gas creates a gas stream traveling at a significant velocity. The gas stream is accelerated through the particle acceleration passage and, when it passes through the interior of the particle cartridge 14, it dislodges the particles 16. The acceleration gas stream, which contains the particles ejected outside, continues at length of the acceleration passage through the acceleration chamber 22, and in the outlet nozzle 24. A particularly important feature of the device of Figure 1 is the geometry of the outlet nozzle 24. Referring now to Figure 2, three possible different geometries of the outlet nozzle 24 are schematically illustrated as versions A, B, and C. The effect of these different outlet nozzle geometries is also described in the delivery configuration of the particles 16. In Version A, the outlet nozzle 24 does not expand significantly towards the upstream end thereof. Thus, the outgoing gas stream passes substantially linearly from the outlet nozzle 24, and proceeds directly to the target. As a result, the carrier particles continue in a relatively linear path and provide a focused supply configuration that embeds a relatively narrow area of the target.

While the particles 16 diverge a little from their linear path, the divergence is completely small and insignificant. In Version B of Figure 2, the outlet nozzle 24 has an excessively wide tapered taper angle toward the downstream terminal thereof. In this configuration, the gas stream leaves the instrument clearly in a linear fashion, and the particles 16 do not disperse widely. Again, the particles are firmly fixed in a relatively compact portion of the blank. A substantially different supply configuration is obtained, however, when the taper angle of the conical outlet nozzle is less than a critical angle. This phenomenon is illustrated as Version C in Figure 2. In particular, when the accelerated gas stream passes in the outlet nozzle, it itself creates, through a vortex action, a gap between the current passage path of gas and the sides of the outlet nozzle 24. This vacuum causes the gas stream to be attracted externally in all directions perpendicular to the direction of travel of the gas stream. In this way, the gas stream and particles entrained within the gas stream are dispersed in a direction lateral to the main axis of the outlet nozzle (i.e., the direction of travel of the particles). Thus, as can be seen in Version C of Figure 2, the gas stream passing outside the instrument expands laterally over a wider area, whereby the particles 16 are distributed over a wider area and a improved supply profile above the surface area 25 of the target. This avoids overdosing any small area of the target with the supplied or released particles. The exact taper angle of the tapered outlet nozzle 24 can be varied to accommodate the use of different gas pressures and relative dimensions for the acceleration chamber 22. In an instrument which uses a commercial helium tank as the source of force motor, wherein the acceleration chamber 22 has a diameter of approximately 1/16 of an inch, an exit nozzle which tapers from 0.1587 cm (1/16 of an inch) to 1693 cm (2/3 of an inch) over an extension of 8.382 cm (3.3 inches) will provide a satisfactory particle distribution configuration which covers a target surface having a diameter from about 0.1587 cm (1/16 inch) to approximately 1,693 cm (2/3 of an inch). This represents above a 100-fold increase in the particle distribution configuration, with a concomitant 100-fold decrease in particle distribution density. In summary, then, the tapered outlet nozzle 24 of the device of WO 95/19799 can be configured significantly larger along its main axis which is wide in either its countercurrent and downstream limits to obtain a complete distribution wider particle. In addition, it can be adjusted by varying the gas pressure, the force with which the particles are impregnated in target 19. However, to a minimum, the pressure of the gas provided by the source of the moving force may be sufficient to dislodge the particles 16 from the cartridge 14. At the same time, the gas pressure should not be greater than to damage the target 19. When carrier microparticles coated in intact animal skin are supplied using such devices, it has been found that a gas stream discharged will not damage the surface of the skin in white. At high gas pressures, some reddening of the skin may occur, generally at tolerable levels. Regulated gas pressure, such as that available from commercially available compressed helium tanks, has been found to be satisfactory for separating the particles 16 from the cartridge 14, and supplying the same in the epidermal cells of an animal of white without adverse damage to the skin or target cells. Lower pressures or higher or higher pressures can be worked on in particular applications, depending on the density of the particles, the nature of the target surface, and the desired depth of particle penetration. Although the use of the operating parameters and the exit nozzle geometries described above provide a significant distribution of particles delivered above a target surface, the distribution configuration is not as uniform as desired. In particular, although the particle distribution provided by the device of WO 95/19799 is better than that achieved with any other device operated by compressed gas, the configuration is still characterized by a concentration of particles embedded in the center of the target area, with a laterally diminished distribution of particles extending from that centralized area. Accordingly, it is a specific object of the invention to provide an element which serves to increase the uniformity of distribution of particles that are obtained from the particle delivery devices. In a particular embodiment of the invention, there is provided a rotary flow element which can be placed in a particle delivery device within an acceleration passage, for example in an upstream location from a particle source (e.g. , within the fluid conduit 17 of the device of WO 95/19799), or in a downstream location from a source of particles (eg, within the acceleration chamber 22 of the device of WO 95/19799). Accordingly, the element can serve to recanalize all, or a portion, of the gas flow either prior to or after its contact with the particles, thereby imparting a rotational movement in the gas flow prior to, during, and / or after the gas flow has been contacted with the particles. The element can be any characteristic or structure capable of imparting rotary motion in a gas flow. In one aspect of the invention, the element comprises one or more vanes, arranged either inside, or depending on the interior surface of, a gas conduit within an acceleration passage. In one aspect of the invention, the vanes are positioned upstream of a source of particles to be delivered. In another aspect, the vanes are placed downstream of a particle source. The paddle or vanes serve to recanalize at least a portion of the gas flow, forcing it to move or rotate about an axis. In still another aspect, the element comprises a cylindrical stopper or diverter disposed within a gas conduit that resides countercurrently from the particle source. The plug or diverter contains one or more angled channels that allow a flow of gas to expand to pass therethrough so that a rotary flow is initiated in the expansion gas stream prior to, during, and / or after the contact with the particles. These channels can be formed within the shutter or diverter, arranged around the periphery of the shutter or diverter so that a channel wall is provided by the gas conduit, or any combination of internal and peripheral channels can be employed. In any such configuration, the rotary flow element serves to dramatically increase the lateral distribution of the delivered particles, therefore, a more even distribution of particles over the target area or target area is ensured. Without being bound by any particular theory, it is thought that imparting a rotation in the gas flow before, during, and / or after its contact with the particles, aids in a turbulent intermixing of the particles within the gas, which in turn provides a better distribution of the particles within the expansion gas stream. Such rotary flow dynamics can also be performed through the outlet nozzle of the particle delivery device, which assist in the formation of a laterally uniform dispersion of the supplied particles, possibly due to the centrifugal forces. Despite all the mechanism by which the result is achieved, the result is completely clear. The supplied or released particles are distributed laterally from the main axis of the gas stream to their exit from the particle delivery device, providing both an increase that can be measured qualitatively and quantifiably in the uniformity of the particle distribution within the particle. an area of white. Then, in this way, the present invention is broadly applicable for use in any gas powered particle delivery device to provide improved uniformity in a particle distribution. Referring to Figure 3, a particulate delivery device similar to that of Figure 1 is generally indicated at 10. The device 10 comprises a handle 28 through which an inlet conduit 32 passes. The inlet duct 32 it ends at one end with a coupler 31 which allows the connection of the device 10 with an associated source of compressed gas. A trigger 30, located on the handle 28, allows the actuation of the device by releasing a gas flow in the device from an associated source.

A countercurrent gas conduit 37 connects the handle 28 to an elongate body 33, this body includes a cartridge chamber 35 capable of housing a particle cartridge. In the particular device of Figure 3, a cartridge holder 36, mounted on the body 33, houses several particle cartridges which are configured as cylindrical tubes coated on their inner surfaces with particles for delivery of the device. In operation, the cartridges of the cartridge holder were individually placed in the position within the chamber of the cartridge 35 so that they are disposed within the path of a gas flow passing through an accelerating passage extending from the countercurrent gas conduit 37 through an acceleration chamber 44. The acceleration chamber 44 terminates in an outlet nozzle 46. The rotary flow element of the present invention is preferably located within the countercurrent gas conduit 37. so that it can impart a rotary motion in the gas stream passing through it prior to the contact of the gas stream with the particles in the cartridge chamber. Referring now to Figures 4 and 5, a particular rotary flow element is shown which comprises a diverter 50 having a countercurrent face 52 and a downstream face 54. The diverter or baffle is configured as a shutter which is it can insert into the countercurrent gas conduit 37 at a location adjacent to the cartridge chamber 35. The diverter 50 includes one or more gas channels 56 arranged in a radial array about its periphery. The diverter or baffle may be comprised of any suitably resilient material which is either machinable or molded, for example, metals, metal alloys and rigid polymeric materials. The gas channels extend along the length of the baffle or deflector in a direction that is substantially in the direction of gas flow through the gas conduit. However, as can be seen with particular reference to Figure 5, each channel can be tilted or angled at an angle? defined relative to the main axis of the deflector. The angle ? particular may vary over a range of about 0-15 °, and is preferably in the range of about 0-11 °. An angle? small of about 0-5 ° provided for the deeper penetration of particles supplied from the particulate delivery device. An angle? medium of about 7-11 ° provides the much wider distribution of particles in a direction lateral to the direction of gas flow through the particle delivery device.

The deflector 50 may comprise external fillets cooperating with the corresponding screwing within the gas conduit 37, or the deflector may have a substantially smooth external surface, for example where the diverter is fixed by compression within the gas conduit. In operation, a gas flow released within the particle supply device passes into the gas conduit 37 where the countercurrent face or side 52 of the diverter or deflector 50 contacts. Then the expansion gas flow is caused to pass. through the gas channels 56, which impart a rotary movement in the gas flow proportional to the angle? This flow of rotating gas then travels in a particle cartridge where it itself removes the particles from the inner surface of the cartridge to supply a target surface. Referring now to Figures 6-9, a deflector of the related rotary flow element is generally indicated at 70. The baffle 70 is arranged within a particle delivery device between a countercurrent chamber 72 which provides the initial chamber at which releases the compressed gas, and a downstream mixing chamber 74. The baffle 70 has a countercurrent face 76, a downstream side or face 78, and an external surface 80. A linear central bore 82 extends between the countercurrent sides or surfaces and of downstream 76 and 78, wherein the central bore is coaxial with the main axis of the baffle 70. An annular seat 84 on the side or countercurrent 76, provides a separate or recessed fixation which accepts and retains a cylindrical particle cartridge 86. The annular seat 84 surrounds and is aligned coaxially with the central bore 82. The particle cartridge 86 has a plurality of particles 88 coated on the inner surface thereof. As can be seen with reference to Figures 6 and 7, the particle cartridges 86 are located within the annular seat 84 and project in the countercurrent chamber 72. Referring now to Figures 8 and 9, the deflector 70 has one or more gas channels 90 on the external surface 80 thereof, wherein the gas channels are arranged in a radial arrangement around the central bore 82. As described here above, the gas channels may be inclined or angled relative to the axis of the baffle 70 to impart a rotary motion in the gas flowing through the particulate delivery device. The relative angle of the gas channels can vary between 0-15 ° depending on the amount of rotary movement desired for the gas flow.

In operation, a gas flow released in the countercurrent chamber 72 travels to the countercurrent side 76 of the baffle 70, with a portion of the gas flow entering the particulate cartridge 86. The reduced diameter of the central bore 82 relative to the diameter of the annular seat 84 and consequently to the particle cartridge 86, restricts the amount of gas that can flow through the particulate cartridge to a fixed percentage of the total gas flow. The volume of the gas flow thus travels around the outer surface of the baffle 70 and through the gas channels 90. This induces the formation of a vortex at a central point within the mixing chamber 74 where the gas flowing to the through the gas channels converge. An axial particle beam 88 which has been dislodged from the inner surface of the particle cartridge 86, travels through the central bore 82 of the baffle in a substantially linear direction, and is therefore supplied at the center of the vortex formed within the mixing chamber. When the gas flow forms a vortex and the particle beam enters the acceleration chamber 92, the flow of gas that is spun is brought into contact with the axial beam of particles which are then accelerated and centrifuged to impart a final path when they pass through the nozzle 94, distributing the particles uniformly over a target zone. Here again the baffle 70 can be comprised of any suitable mouldable or machinable material, which can withstand the force of a flow of compressed gas suitable for supplying particles through the particulate delivery device. The deflector 70 may additionally include external threads or fillets on the external surface 80 thereof to facilitate coupling with the countercurrent chamber 72. The rotary flow elements of the present invention serve to impart a rotating component in the flow of the flow of compressed gas passing through the particulate delivery device. This rotating flow component causes some surprising results in the operation of particle delivery devices. An unexpected result is that a device fixed with a rotary flow element, referred to herein sometimes as a "rotating needle or pin" instrument, is more effective in removing particles from inside the particulate cartridge 14. Without However, the rotary flow elements of the present invention provide their much greater benefit by substantially improving the uniformity in the distribution of particles above a target area.

In another embodiment of the invention, a turbulent flow element is provided, which can be placed in a particle delivery device in a countercurrent location from a particle source (e.g., within the fluid conduit 17 of the WO 95/19799). The element serves to alter all, or a portion, of the gas flow prior to its contact with the particles, thereby improving the reliability of particle release from the particle source. Therefore, the turbulent flow element can be any characteristic or structure capable of interrupting a gas flow in such a way as to impart a flow turbulence. In one aspect of the invention, the element comprises one or more topographic features disposed on the interior surface of a countercurrent gas chamber. Such features may comprise a flange or bridge, shoulder, notch, corrugation, or any combination thereof, the features serve to interrupt or disturb the flow of gas passing through the chamber or conduit. In a particular aspect of the invention, the turbulent flow element comprises a small step on the inner surface of a cylindrical gas chamber, which establishes a chamber area of slightly increased diameter limited by areas of smaller diameters, through these areas passes a flow of expansion gas prior to contact with a source of particles. Referring now to Figure 10, a turbulent flow element is generally indicated at 100. The flow member 100 is adapted by insertion into a particle delivery device, where it itself accepts a flow of gas released through an opening. countercurrent 102 which has a first diameter A. When the gas flow proceeds through the element, it itself enters a stepped portion 104 having a slightly larger diameter B. The stepped portion of the element 100 extends along a substantial portion of the total length of the element and is joined on its countercurrent side with a downstream opening 106 having a diameter A equal to that of the countercurrent opening. The stepped portion 104 of the element is sufficient to introduce a turbulence in the gas flow prior to its contact with a source of arranged particles adjacent to the downstream opening 106. This turbulence improves the reliability of the particle release from the source of particles, improving the efficiency of the particle supply. In a particular embodiment, the diameter A of the top and bottom openings 102 and 106 is approximately 0.635 cm (0.250 inches), and the diameter B of the step portion 104 is approximately 0.711 cm (0.280 inches), providing a step or step of approximately 0.076 cm (0.03 inches). In yet a further embodiment of the invention, a flow constriction element is provided for use in a particle delivery device. Referring to Figure 3, the constriction element is configured by insertion in the particle supply device adjacent to the point of coupling between the device and the associated source of compressed gas. In particular, the constriction element can be inserted into the junction between the inlet tube 32 and the connector 31. The constriction element is generally comprised of a disc of flexible or resilient material sized to restrict the passage of the gas in the device. supply to a small hole. The orifice can be completely small, for example, an orifice of approximately 200 to 250 μm has been found to be sufficient. Referring now to Figure 11, a flow constriction element constructed in accordance with the present invention is generally indicated at 60. The element is configured as a disk having an orifice 62 passing therethrough. The purpose of the restriction provided by the orifice 62 is to isolate an aliquot of compressed gas in the instrument for each particle delivery operation. With a particular reference to the device described in International Publication No. WO 95/19799, the source of compressed gas supplies the additional compressed gas through the instrument at all times, not only during the supply operations. As a result, more gas is supplied to the instrument during the actuation or operation that is effectively required for the effective delivery of the particles to a target surface. Indeed, the excess gas that travels through the instrument only helps to fix the supply operation on the target without providing a corresponding benefit. By providing the constriction element of the flow 60 in the device, the compressed gas is mixed through the orifice in the interior of the gene delivery instrument between supply operations. When the device is operated, then, there is a charge or aliquot of compressed gas already present in the instrument by itself, occupying several chambers and being conducted inside the device. This charge of compressed gas is, when present, sufficient to deliver the particles from the particle source to the target surface. When the compressed gas charge has been released, the restriction of the flow provided by the constriction element of the flow 60 prevents an additional volume of compressed gas flowing through the instrument. After the supply operation, the compressed gas again recharges the instrument until a pressure equilibrium is reached. In other related aspects of the invention, the concept of providing a single aliquot (charge) of gas within the particle delivery device for discharging a particle payload can be accomplished using other mechanisms. For example, instead of using a flow constriction element such as element 60, a combination of valves can be used to achieve the same effect. In a particular arrangement, an inlet valve can be provided which closes during the operation of the instrument, and then opens when the instrument is not being operated to load the instrument for subsequent operation. For example, if a solenoid or electric operated valve is used as the main valve for the instrument, two valves can be operated alternatively where an inlet valve is closed each time the main valve is open, and the valve entrance is open in case the main valve is closed. The benefits of the flow constriction element are several times. For example, the audible report created by the operation of the instrument is dramatically reduced. At 35,181 kg / cm2 (500 psi) of compressed gas, a typical particle delivery device will generate a report of approximately 103 dB during discharge, while the same instrument that has the flow constriction element, present, generates only a report of 88 dB during the download. There is also a discernible decrease in the sensory perception of gas discharge from a particle delivery device when the present constriction element is employed. In addition, there is less damage to sensitive tissues or target cells when the flow constriction element is used. It is specifically intended herein that the various embodiments of the invention can be used alone or in any combination. In this regard, each embodiment is capable of independently providing a unique and advantageous improvement in the operation of a particle delivery device. However, it is particularly advantageous to use both a rotary flow element and a flow constriction element to obtain optimum results.

C. Experimental The following are examples of specific embodiments for carrying out the present invention. The examples are given for purposes of illustration only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (eg, quantities, temperatures, etc.), but some deviation and experimental error, of course, should be calculated.

Example 1 Distribution of Particle Supply To test the effect of a rotating flow element on the operation of a particle delivery device, the following study was carried out. A particle delivery device, such as the "gene gun" described in International Publication No. WO 95/19799, is used to deliver gold microparticles in a Parafilm® block covered by a Mylar® film. This arrangement approximates a target surface of typical skin tissue, and is sufficiently opaque to allow optical and / or visual testing of the distribution of particles in a target area. In the present study, a Model GS300 optical reader (Hoefer Scientific) was used to generate a graphical representation of the distribution of gold microparticles in a cross section through the center of the expanded particles. Six different particles supplied were conducted, using payloads of identical particles which is supplied from an ACCELL® particle delivery device (PowderJect Vaccines, Madison, Wl) operated at 28,124 kg / cm 2 (400 psi) of helium. Each supply was conducted using a rotating flow element that provides a different degree of turns (0 °, 2 °, 3.5 °, 5 °, 7 °, and 11 °). The results of the study are presented in the graph of Figure 12. In the graph, the ordered value represents the density of particles, while the value of the abscissa represents expanded particles. As can be seen in Figure 12, curve A (obtained from the supply of particles using a rotating flow element that provides 0 ° turns) is both high and narrow, demonstrating that the particles do not expand laterally and are concentrated in the center of the target area. In contrast, the curve C (obtained from the supply of particles using a rotary flow element that provides a turn of 3.5 °) is much wider in relation to the curve A, indicating a larger particle expansion above the target area . In addition, curve C is lower, indicating that there is not a particularly high concentration of particles in the center of the target area. Curve F (obtained from the supply of particles using a rotating flow element that provides a 11 ° turn) shows that this higher degree of rotation significantly disperses the particles over the entire target area. Subsequent gene delivery experiments in animal skin confirm that the rotary flow element improves the operation of particle delivery devices. The use of the element was shown to provide comparable levels of reporter gene expression in test animals, when compared to parallel experiments using devices without the flow elements. Furthermore, it is observed that the occurrence of erythremia in the skin of test animals was significantly reduced when the rotary flow element was used in the particle delivery devices. Accordingly, new gas flow modifying elements have been described for use with particle delivery devices. Although the preferred embodiments of the disclosed invention have been described in some detail, it is to be understood that obvious variations can be made without departing from the spirit and scope of the invention as defined by the appended claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description. Having described the present invention as above, the content of the following is claimed as property

Claims (19)

1. A device for supplying particles in a tissue or target cell, the device is characterized in that it comprises: a main body or part having an acceleration passage formed therein, the passage having an inlet and an outlet; and a rotary flow element arranged within the acceleration passage, wherein the rotating flow element imparts a rotary motion in a gas flow passing therethrough.

2. A device for supplying particles in a cell or target tissue, the device is characterized in that it comprises: a main body or part having an acceleration passage, the passage having an entrance and an exit; an actuator or actuator for admitting a gauging flow through the entrance in the passage, the gaseous flow that accelerates through the passage and transports particles out of the outlet; and an inductor element of the rotary flow located in the passage to impart a rotational movement to the gas flow.

3. A device for supplying particles in a cell or target tissue, the device is characterized in that it comprises: a main body or part having an elongated or extended acceleration chamber formed therein, the chamber having an inlet and an outlet; and a rotary flow element arranged countercurrently from the inlet of the acceleration chamber, wherein the rotary flow element imparts a rotational movement in a gas flow passing therethrough, prior to the entry of the gas flow in the acceleration chamber.

4. The device according to claim 3, characterized in that the rotating flow element comprises a baffle placed inside a gas chamber or duct arranged countercurrent to the acceleration chamber, the baffle having a countercurrent side or surface and a side or surface to downstream.

5. The device according to claim 4, characterized in that the baffle is comprised of a substantially cylindrical obturator having an external surface with a gas channel formed therein, the gas channel allows the passage of a gas flow from the side or surface countercurrent to the side or descending surface of the deflector.

6. The device according to claim 5, characterized in that the gas channel is arranged in angular relation to the main axis of the baffle.

7. The device according to claim 5, characterized in that it further comprises a plurality of gas channels arranged in a radial shape around the external surface of the baffle, wherein the gas channels are each arranged in an angular relationship with the main axis deflector and allows the passage of a gas flow through it.

8. The device according to claim 5, characterized in that the deflector also comprises a substantially linear central bore passing between the upward and downward sides of the deflector, the central bore or bolt allows the passage of a gas flow therethrough.

9. The device according to claim 8, characterized in that the deflector further comprises an annular seat coaxially aligned with the central bolt or hole and disposed within the countercurrent face or side of the deflector.

10. The device according to claim 9, characterized in that the annular seat is configured to accept and retain a cylindrical cartridge containing particles to be supplied from the device.

11. A device for supplying particles in a cell or target tissue, the device is characterized in that it comprises: a main body or part having an elongated acceleration chamber formed therein, the chamber having an inlet and an outlet which terminates in a outlet nozzle; a mixing chamber having an inlet, and an outlet communicating with the inlet of the acceleration chamber; a countercurrent gas chamber having an outlet communicating with the inlet of the mixing chamber, wherein a rotary flow element is arranged at the outlet of the countercurrent gas chamber and imparts a rotational movement in a flow of gas that passes from the gas chamber to countercurrent in the mixing chamber.

12. The device according to claim 11, characterized in that the rotary flow element comprises a substantially cylindrical deflector having an external surface, a countercurrent side and a downward side, the deflector additionally has a plurality of gas channels formed in the surface external thereof, which allows the passage of a gas flow from the face or side to countercurrent to the downward side of the baffle.

13. The device according to claim 12, characterized in that the gas flow through the plurality of gas channels in the baffle creates a vortex within the mixing chamber.

14. The device according to claim 13, characterized in that the deflector also comprises a substantially linear central bore passing between the upward or downward sides of the baffle, the central auger allows a flow of gas containing particles passing therethrough.

15. A device for supplying particles in a cell or target tissue, the device is characterized in that it comprises: a main body or part having an elongated acceleration chamber formed therein, the chamber having an outlet and an inlet; a source of particles that are supplied from the device, wherein the source is adjacent to the entrance of the acceleration chamber; and a turbulent flow element arranged countercurrently from the inlet of the acceleration chamber and the particle source, whereby the turbulent flow element creates turbulence in a gas flow passing through it prior to the flow contact gas with the particle source.

16. The device according to claim 15, characterized in that the turbulent flow element comprises a gas conduit having a stepped portion of increased diameter.

17. A device for supplying particles in a cell or target tissue, the device is characterized in that it comprises: a main body or part having an elongated acceleration chamber formed therein, the chamber having an outlet and an inlet; a source of compressed gas coupled to the main body or part for the supply of a gas flow at the inlet of the acceleration chamber; and a flow constriction element that limits the flow of gas from the source to the main body or part.

18. The device according to claim 17, characterized in that the flow constriction element comprises a shutter having a hole passing through it.

19. The device according to claim 17, further characterized by comprising a rotary flow element arranged countercurrently from the entrance of the acceleration chamber, wherein the rotary flow element imparts a rotary movement in a flow of gas passing through. of the same prior to the entry of the gas flow in the acceleration chamber.