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WO1992001802A1 - Procedes et appareils servant a introduire sous forme de particules gelees des substances medicamenteuses dans un materiel biologique sans le tuer - Google Patents

Procedes et appareils servant a introduire sous forme de particules gelees des substances medicamenteuses dans un materiel biologique sans le tuer Download PDF

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Publication number
WO1992001802A1
WO1992001802A1 PCT/US1991/005022 US9105022W WO9201802A1 WO 1992001802 A1 WO1992001802 A1 WO 1992001802A1 US 9105022 W US9105022 W US 9105022W WO 9201802 A1 WO9201802 A1 WO 9201802A1
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WO
WIPO (PCT)
Prior art keywords
ice particles
target
pellet
ice
solution
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1991/005022
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English (en)
Inventor
Chris Brinegar
James Gilmore
Michael Johnson
Nigel Walker
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Individual
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Individual
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Filing date
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Publication of WO1992001802A1 publication Critical patent/WO1992001802A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • a variety of techniques are known for the introduction of foreign substances, particularly DNA, into the interiors of living cells without killing those same cells.
  • One class of techniques involves manipulating the cell membrane to make it permeable to DNA molecules. Fcr instance, in bacteria, yeasts and protoplasts cf higher plant cells, treatments with chemicals or heat can be used to make the ceil membranes "leaky,” thereby permitting a desired gene or genes (often integrated in small loops cf DNA called plasmids) to be taken up into the cell.
  • DNA is physically injected intc living cells, particularly ova, of various animal species.
  • DNA ca also be taken up by electrically stimulated cells in a process known as electroporation.
  • the cell is made permeable using a precision laser to burn holes into the cell membrane.
  • Gene transfer is accomplished by infecting certain susceptible dicotyledonous plant species with a particular bacterial species of the genus A ⁇ robacteriu .
  • the bacterium possesses plasmids as a part of its normal complement of DNA.
  • the bacterium is capable of transferring portions of these plasmids to the cells of the plant it infects.
  • the bacterium is capable of transferring portions of these plasmids to the cells of the plant it infects.
  • it is possible to accomplish the introduction of specifically desired genes by engineering Agrobacterium species with recombinant plasmids possessing the desired gene and subsequently infecting a susceptible plant with the engineered bacterium.
  • microprojectile bombardment or “biolistics,” this technique involves coating tiny metal spheres with desired DNA, and then accelerating these projectiles into tissues. Acceleration is generally achieved by shooting the microprojectiles from a gun aimed at the target tissue. Once inside the cell, the foreign DNA detaches from the metal sphere and, in certain of the bombarded cells, is incorporated into the host cell's DNA. See, e.g., Sanford, J.C., “The biolistic process,” Trends in Biotechnology, . 6:299-302 (1988) ; Klein, T. et al. "Stable genetic transformation of intact Nicotiana cells by the particle bombardment process," Proc. Nat.
  • the methods may be taxonomically limited, such as with Agrobacterium- based methods.
  • Membrane permeability methods have limited application with organisms presenting cell walls.
  • Electroporation methods achieve only a very low efficiency of stable transformation.
  • Several methods, such as microinjection and laser-mediated cell membrane manipulation are extremely tedious, ti e consuming and require highly specialized equipment.
  • the design and composition of the microprojectiles presents disadvantages unique to the method.
  • metal microprojectiles can easily clump into larger aggregates that, upon impact, can cause significant cell damage.
  • the type of substances that can be introduced into the cell is limited to negatively- charged molecules such as DNA and RNA.
  • the amount of DNA or RNA carried with each icroprojectile cannot be easily controlled or measured.
  • the long-term chemical and physical effects of the metal microprojectiles inside the cell are unknown.
  • the use of metallic agents requires considerable preparation time, thus affecting the economic efficiency cf the method.
  • a desired substance to be introduced into a cell is added to a desired liquid capable of dissolving, suspending or encapsulating the substance.
  • a desired liquid capable of dissolving, suspending or encapsulating the substance.
  • solution will be defined to include liquids in which the desired substance is either dissolved or suspended or in part both.
  • the solution is then used to make ice particles that contain the desired substance. These ice particles are then accelerated toward a target tissue whereby at least some of the particles impact upon and enter at least some cells in the target tissue without killing the cells. Once inside the cell, these ice particles
  • SUB then melt, leaving behind the desired substance in the protoplasm of the bombarded cell.
  • the method of the present invention is superior to existing methods in that it provides a way to introduce not only DNA and RNA, but also non- negatively charged substances, including but not limited to proteins, enzymes, protein/DNA complexes, bacteria, viruses, hormones, etc. into living cells.
  • the ability to introduce enzymes and proteins simultaneously with DNA and RNA may materially improve DNA/RNA viability rates as well as transformation rates of individual cells.
  • the ice projectile ceases to exist after it melts, thereby leaving no residue that may poison or otherwise contaminate the bombarded cell at some future time. Because the ice projectiles are produced from a solution of known concentrations, it is easy to control and predict the amount of DNA or other substance to be delivered to a particular cell.
  • ice particles may be less likely to clump and thus less likely to cause fatal trauma to bombarded cells. Further, it is possible, for a particle of a given diameter, to incorporate more of the desired substance than can be incorporated onto the surface of a metal particle.
  • a further object of the present invention is to provide a method of substance introduction that is efficacious for a wide variety of different species and tissue types.
  • a further object cf the present invention ;s tc provide a simple, self-contained apparatus fcr accelerating ice particles toward a target tissue that is inexpensive to construct and easy to operate.
  • FIG. 1A is a view in cross-section of the ice particle generator of the present invention.
  • FIG. IB is a view in cross-section of an apical portion of the ice particle generator of the present invention.
  • FIG. 2A is a cutaway view in perspective of the pellet of the present invention.
  • FIG. 2B is an exploded cutaway view in perspective of the pellet of the present invention.
  • FIG. 3A is a view in cross-section of the ice particle accelerator of the present invention.
  • FIG. 3B is a view in cross-section of the vacuum chamber portion of the ice particle accelerator of the present invention.
  • FIG. 3C is a schematic view in perspective of the support apparatus within the vacuum chamber of the present invention.
  • the generator 10 is comprised of an inner shell 12 which defines a freezing chamber 14.
  • the freezing chamber 14 is provided with a gas feed line 20 from a regulated nitrogen tank. It is also provided with a nitrogen gas recycling line 16 which leads from coolant chamber 36.
  • the freezing chamber 14 is further provided with a nebulizer 22 for dispersing the solution into a mist of very fine particles and a mist feed line 24 permitting the mist to enter the freezing chamber 14.
  • The-nebulizer 22 is attached externally to a gas feed line 23 that is attached a tank of compressed nitrogen or other substantially inert gas.
  • the nebulizer should be capable of forming mist particles that upon freezing are of an average diameter of 1 micron.
  • One such suitable nebulizer is produced by De Vibliss Company, model no. 644.
  • the freezing chamber 14 is closed above by emplacement of a stopper 26 at the apex and by an end segment 27 at the base of the inner shell 12.
  • the stopper 26 is provided with a thermometer 28 for gauging the temperature of the freezing chamber, and is provided with a vent hole 29. Placed upon the end segment 27, at the base of the freezing chamber,
  • a pellet holder 32 in which are emplaced pellets 50.
  • a funnel 30 supported by a three-legged ring stand 31, for collecting the ice particles formed from the mist produced by nebulizer 22 and for directing the particles into the pellets 50.
  • the generator 10 further comprises an outer shell 34 positioned to surround the inner shell 12 for substantially much of its length.
  • the position of the outer shell 34 with respect to the inner shell 12 defines an coolant chamber 36.
  • the coolant chamber 36 is provided with a coolant feed 38 and a coolant drain 40, which is provided with a flov; valve 42.
  • the inner and outer shells 12 and 34 sit atop a support 44.
  • End segment 27 is supported by a vertically adjustable tripod 48 which allows the insertion, height adjustment, and removal of end segment 27, pellet holder 32, three-legged ring stand " 31, funnel 30 and pellets 50.
  • the inner and outer shells 12 and 34 may be composed of any suitable material resistant to cracking or other degradation that may result from. rapid temperature changes. Suitable materials include galvanized steel, stainless steel, aluminum, sheet metal, copper and other materials such as ceramics and plastics. Metals are preferred because they are inexpensive and easy to form.
  • the stopper 26 is preferably composed of rubber.
  • the collection funnel 30 " is preferably composed of some smooth- surface polymer plastic or glass resistant to rapid temperature changes.
  • the pellet holder 32 is preferably composed of a foamed rubber or polymer plastic. A suitable air rifle pellet is produced by
  • Pellet 50 is comprised of two
  • SUBSTITUTESHEET parts a core 56 comprised of aluminum having a broad rounded base which has been filed flat and a narrower projecting columnar portion, and a skirt 54 comprised of plastic that is configured to be received over the columnar portion of core 56.
  • the pointed or curved base of core 56 has been filed flat in order to permit the casing 52 to be loaded and fired from gun 110 in a position that is backwards relative to its intended orientation.
  • the skirt 54 may be removed from the core and sealed on one end. Sealing may be accomplished by any appropriate method such as taping, gluing, heat sealing, etc.
  • the desired substance to be introduced into the target cell must be selected. In the case of many substances such as proteins, enzymes, etc., all that is required is the dilution of the substance in an appropriate, liquid to a desired concentration.
  • an appropriate vector capable of cell transformation " is selected.
  • the vector is then manipulated to include the desired gene or genes to be integrated into the genome of the target cells. Once manipulation is complete, the vector can be multiplied and isolated by various techniques that are well known in the art and then dissolved and diluted in a solution to a desired concentration. This solution is then maintained under conditions sufficient to ensure that the biological properties of the selected substance are maintained.
  • the freezing chamber 14 is flushed with compressed nitrogen via feed gas line 20. It is preferred to feed the nitrogen into the freezing chamber 14 at about 5 psi for a period of two minutes. Flushing with nitrogen gas ensures that all water vapor has been evacuated from freezing chamber 14 via vent 29. Failure to rid chamber 14 of water vapor may result in the formation of foreign ice particles that can contaminate the desired ice particles later to be generated.”
  • coolant is added to the coolant chamber 36 through coolant feed 38 in an amount sufficient to fill the chamber. This will cause the temperature within the freezing chamber to drop.
  • the preferred coolant is liquid nitrogen.
  • a sufficient amount of cooling has taken place when the thermometer 28 records a temperature cf 5°C belov.- the freezing point at the desired length. Cooling efficiency is increased when gaseous nitrogen that boils off coolant chamber 36 is transferred into the bottom of freezing chamber 14 via gas recycling line 16.
  • the nebulizer 22 is filled with the sample solution prepared previously. In the De Vibliss nebulizer disclosed above, it is preferred to add approximately 5-7 ml of the desired solution. At this time, nitrogen gas is fed into nebulizer 22 through feed 23 at about 5 psi. This begins mist
  • mist particles fall through freezing chamber 14, they freeze and are collected in collection funnel 30, which directs the particles into the pellets 50. Ice particles collect in pellets 50, largely under the influence of gravity. Material comprised solely of such free frozen granules is referred to as "powder.” A certain proportion of the ice particles generated will collect on the sides of funnel 30. These may be scraped and packed manually. Frozen material collected in this fashion is referred to as "packed.” Generally speaking, nebulization should be carefully regulated to ensure that the biological properties of the desired substance in solution are preserved. Where transformation vectors are employed, excessive pressure or nebulization time can result in shearing of the DNA strand, rendering the vector incapable of transforming the target cell.
  • the pressure and length of time used to nebulize a solution containing such vectors will vary according to the fragility of the vector.
  • Such parameters can easily be determined experimentally using techniques well known in the art, whereby fragility of the vector is generally proportional to vector size and configuration.
  • pellets containing block frozen ice can be fired intact with the particles being generated upon impact with the stopping plate 138.
  • the solution can be block frozen in the pellet and then subjected to sonication which will generate fracture lines within the ice block, thereby causing a more efficient shattering upon impact.
  • One suitable method is to pack the pellets into a suitable container, such as a block of foam plastic polymer with receptacles for receiving the pellets.
  • the plastic block with pellets 50 can then be stored under cold conditions, such as being floated on liquid nitrogen in a covered, insulated container.
  • Accelerator 100 is comprised of three principal parts: a gun 110, a housing 120, an a support structure 130.
  • Gun 110 provides the accelerating force.
  • the make and specifications of gun 110 will depend upon the type of pellet used. However, acceleration using chemical charges or other heat intensive methods are not compatible with ice particle acceleration in the present invention.
  • pellets designed to be shot from air guns Smooth bored guns are preferable over rifles in that frictional heat upon discharge is reduced resulting in a greater amount of the ice remaining frozen.
  • Housing 120 is configured to fit over support structure 130 to form a tight seal so that a partial vacuum can be created and maintained interior to housing 120.
  • Housing 120 is provided with vacuum hose adapter 122 and vacuum gauge 124.
  • the vacuum gauge 124 is provided with a three way valve 125 through which air may be reintroduced into the interior housing 120 when a vacuum is no longer required.
  • the housing can be comprised of any convenient material that is sufficiently strong to contain a partial vacuum without structural failure. Plexiglass is preferred because it is inexpensive and easy to form.
  • Support structure 130 is designed to receive, position and hold the tissue sample for impact during acceleration of the ice particles.
  • the support structure comprises a base plate 132 and a sandwich comprised of a seal 134 above and a plexiglass sheet 135 which is placed below.
  • Base plate 132 ⁇ seal 134 and sheet 135 are provided with an aperture 146 extending continuously through the two plates when joined.
  • Aperture 146 provides an entry point into the accelerator for gun 110 which is positioned below.
  • Base plate 132 is further provided with a bore hole 147 through which a set screw can be inserted and used to position and secure the barrel of gun 110 in proper alignment within aperture 146.
  • the "base plate 132 should be durable and strong and is comprised of metal, preferably steel.
  • Four rods 136 are secured to the base plate 132.
  • the interior structure is stabilized by the -addition of a structural support plate 144 which is secured to rods 136 opposite the base plate 132.
  • the rods are threaded along their length to accommodate bolts 137 that are used to position various elements of support structure 130 that will presently be described.
  • a stopping plate 138 Just above the base plate 132 is positioned a stopping plate 138. Stopping plate 138 has a hole located at each of the four corners of the plate to allow stopping plate 138 to slide down and over the ends of rods 136. Stopping .plate 138 is positioned at a
  • SUBSTITUTESHEET desired distance from seal 134 by resting the plate on bolts 137 that have been screwed down along rods 136 to a desired position. Once in place, stopping plate 138 is secured into place by a second set of bolts 137.
  • Stopping plate 138 is provided with an aperture 148 positioned on the plate so that aperture 148 is in direct vertical alignment with aperture 146. After discharge, the pellet containing the ice particles will impact at aperture 148. Aperture 148 should be small enough in diameter to stop the pellet 50 but large enough to permit the ice particles to pass and continue toward the target tissue. Stopping plate 138 is comprised of any durable material capable of sustaining repeated bullet impacts without rapid deterioration; steel is preferred.
  • deflector 140 is positioned a metal deflector 140.
  • the tip of deflector 140 should be positioned over aperture 148 of stopping plate 138 so that discharged ice particles that have passed through aperture 148 go on to strike deflector 140 thereby being dispersed into smaller fragments and over a larger area. This helps to minimize the incidence of large frozen fragments that might lethally damage tissue on impact and helps to ensure that a greater area of the tissue will be impacted by the ice particles.
  • Deflector 140 is laterally attached to one the rods 136 and may be positioned at any desired point along a rod in the same way as stopping plate 138.
  • the deflector is a length of sterilizable material (steel is preferred) that can withstand repeated impact. It has a conical tip extended to a sharp point which further aids in dispersing ice projectiles. Use of a deflector is not required in all instances.
  • SUBSTI Above deflector 140 are positioned support wires 142. Bridging the support wires are a pair of adjustable wires 143. The adjustable wires 143 are slidable along support wires 142 so as to accommodate target material of varying size and shape. Target tissue is placed on the adjustable wires 143 and backing plate 144. It should be understood that the positions of each element of the infrastructure of support structure 130 can be adjusted relatively to one another along rods 136.
  • Stopping plate 138 is secured to rods 136 at a distance from gun 110 sufficient to allow an optimum number of ice particles to be accelerated through aperture 148.
  • Deflector 140 if desired, may then be positioned above stopping plate 138 at a sufficient distance to ensure that ice particles are scattered for impact over a large area of the target tissue.
  • the desired target tissue is placed on adjustable wires 14-3 at a desired distance from gun 110 to ensure " that the tissue is impacted with ice particles in a substantially ncn-lethal manner.
  • outer housing 120 is placed over support structure 130 such that a good seal is formed between the support structure 130 and the outer housing 120.
  • a moderate vacuum is then applied to the chamber to reduce heat of friction between the ice particles and the atmosphere.
  • the amount of vacuum varies according to tissue type, although it is generally preferred to reduce atmospheric pressure to between 20 to 30 inches of mercury prior to acceleration.
  • the temperature of the accelerator should be lowered sufficiently to ensure that the ice particles remain frozen prior to tissue impact.
  • a pellet 50 containing nebulized or block frozen solution containing the desired substance is retrieved from storage and loaded into the gun 110 so that the open end of pellet 50 which contains the core 56 rests at the bottom of the gun chamber. This ensures that the open end of pellet 50 which contains the frozen solution will be facing toward aperture 148.
  • the gun is discharged, thereby accelerating pellet 50 toward stopping plate 138.
  • Pellet 50 is stopped upon impact with stepping plate 138, but the frozen solution within pellet £0 continues through aperture 148.
  • impact with the stopping plate fragments the frozen solution, if block frozen, or mostly disperses the frozen solution, if nebulized, into ice particles as the pellet strikes the stopping plate.
  • the ice particles are further dispersed as they strike deflector 140.
  • the deflector acts to enlarge the impact zone of the ice particles on the target tissue by making the trajectories of the ice particles more oblique relative to their original paths. After passing the deflector, the ice particles travel on tc and imbed in the target material. After impact, the target material may be left in place and reimpacted as desired.
  • air is bled into the housing 120 via the three way valve 125 until pressure inside the accelerator 100 is equal to atmospheric pressure. The housing is then separated from the rubber seal 134 and the target material removed.
  • GUS beta-glucuronidase
  • NPT II neomycin phosphotransferase II
  • GUS beta-glucuronidase
  • NPT II neomycin phosphotransferase II
  • Replication of the plasmid to produce a sufficient quantity of plasmids for experimentation was accomplished by transforming competent cells of Escherichia coli strain JM109 (obtained form Promeg , Inc.) using standard, well known methods.
  • the transformed E. coli were grown to a high density in a rich liquid medium of a standard composition well known in the art for effective E. coli culture.
  • the pBI121 plasmids were isolated from the E. coli cells by alkaline lysis, followed by a cesium chloride density gradient centrifugation according to procedures well known in the art.
  • the plasmids were precipitated by ethanol and dried according to standard techniques in the art.
  • the plasmids were then redissolved in a sterile 10 mM aqueous solution of sodium chloride to a concentration of 0.1 mg/ml. Standard Tris-EDTA buffer was not used to redissolve the plasmids in order to avoid the introduction of potentially toxic compounds into the target tissue.
  • the plasmid containing solution was then frozen according to the techniques set forth and described above. In instances where the ice particles are formed according to the nebulization technique of the present invention, care was taken to avoid treatment that would destroy the integrity of the plasmid and thus its ability to achieve transformation of the target tissue.
  • nebulization pressures in excess of 5 psi and nebulization times in excess of ten minutes should be avoided when using plasmid pBI121 as the desired gene vector. Pressures and times in excess of these figures will shear substantially all the plasmid intc segments toe small to effect transformation.
  • the prepared pellets were loaded then accelerated toward the target material positioned within the ice particle accelerator according to the method described above. After this treatment, the material was incubated in a sterile container at 25 e C under a relative humidity approaching 100% to permit transformation and expression.
  • the treated tissues were histochemically stained for GUS activity using "X- gluc” substrate according to the methods set forth in the manufacturer's directions.
  • the GUS enzyme acts on the "X-gluc” substrate, resulting in an insoluble blue dye that forms crystalline deposits in transformed cells.
  • the target tissue was allowed to incubate overnight at 37°C in the "X-gluc” substrate, and was examined thereafter to ascertain the number of transformed cells.
  • Tobacco Leaf 6.0 0.5 a) 10 ul. of plasmid (0.1 mg/ml) b) Pellets filled with nebulized plasmid powder (0.1 mg/ml)
  • Example 2 In this example, the effect of varying the volumetric amount of frozen plasmid solution having a standard plasmid concentration of 0.1 mg/ml was investigated. The results are set forth in Table II. Table II

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Abstract

Selon la présente invention, une substance désirée devant être introduite dans une cellule est ajoutée à un liquide désiré capable d'assurer la dissolution, la suspension ou l'encapsulation de la substance. Conformément aux pièces descriptives et aux revendications, cette solution est définie comme une solution contenant des liquides dans lesquels la susbtance désirée se trouve soit sous forme dissoute soit en suspension soit encore en partie sous ces deux formes. La solution est ensuite utilisée pour produire des particules gelées contenant la substance désirée. Ces particules gelées sont ensuite accélérées pour être dirigées vers un tissu cible contre lequel une partie au moins de ces particules se heurte, pour pénétrer ensuite dans certaines au moins des cellules du tissu cible sans tuer les cellules. Une fois à l'intérieur de la cellule, ces particules gelées fondent, libérant ainsi la substance désirée dans le protoplasme de la cellule bombardée.
PCT/US1991/005022 1990-07-19 1991-07-16 Procedes et appareils servant a introduire sous forme de particules gelees des substances medicamenteuses dans un materiel biologique sans le tuer Ceased WO1992001802A1 (fr)

Applications Claiming Priority (2)

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US55572790A 1990-07-19 1990-07-19
US555,727 1990-07-19

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WO1992001802A1 true WO1992001802A1 (fr) 1992-02-06

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000005339A1 (fr) * 1998-07-22 2000-02-03 The Secretary Of State For Defence Transfert de matieres dans des cellules utilisant du silicium poreux
US8409376B2 (en) 2008-10-31 2013-04-02 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US8545855B2 (en) 2008-10-31 2013-10-01 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US8551505B2 (en) 2008-10-31 2013-10-08 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US8722068B2 (en) 2008-10-31 2014-05-13 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US8725420B2 (en) 2008-10-31 2014-05-13 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US8721583B2 (en) 2008-10-31 2014-05-13 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US8731841B2 (en) 2008-10-31 2014-05-20 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US8731840B2 (en) 2008-10-31 2014-05-20 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US8762067B2 (en) 2008-10-31 2014-06-24 The Invention Science Fund I, Llc Methods and systems for ablation or abrasion with frozen particles and comparing tissue surface ablation or abrasion data to clinical outcome data
US8788211B2 (en) 2008-10-31 2014-07-22 The Invention Science Fund I, Llc Method and system for comparing tissue ablation or abrasion data to data related to administration of a frozen particle composition
US8793075B2 (en) 2008-10-31 2014-07-29 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US9050317B2 (en) 2008-10-31 2015-06-09 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US9050070B2 (en) 2008-10-31 2015-06-09 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US9060934B2 (en) 2008-10-31 2015-06-23 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US9060926B2 (en) 2008-10-31 2015-06-23 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US9072799B2 (en) 2008-10-31 2015-07-07 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US9072688B2 (en) 2008-10-31 2015-07-07 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles

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WO2000005339A1 (fr) * 1998-07-22 2000-02-03 The Secretary Of State For Defence Transfert de matieres dans des cellules utilisant du silicium poreux
EP1231259A3 (fr) * 1998-07-22 2003-11-19 Qinetiq Limited Transfert de matières dans des cellules utilisant du silicium poreux
US6770480B1 (en) 1998-07-22 2004-08-03 Psimedica Limited Transferring materials into cells using porous silicon
EP1522578A1 (fr) * 1998-07-22 2005-04-13 PSIMEDICA Limited Transfert de matières dans des cellules utilisant du silicium poreux
US7332339B2 (en) 1998-07-22 2008-02-19 Psimedica Limited Transferring materials into cells using porous silicon
US8409376B2 (en) 2008-10-31 2013-04-02 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US8545855B2 (en) 2008-10-31 2013-10-01 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US8551505B2 (en) 2008-10-31 2013-10-08 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US8722068B2 (en) 2008-10-31 2014-05-13 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US8725420B2 (en) 2008-10-31 2014-05-13 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US8721583B2 (en) 2008-10-31 2014-05-13 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US8731841B2 (en) 2008-10-31 2014-05-20 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US8731840B2 (en) 2008-10-31 2014-05-20 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US8762067B2 (en) 2008-10-31 2014-06-24 The Invention Science Fund I, Llc Methods and systems for ablation or abrasion with frozen particles and comparing tissue surface ablation or abrasion data to clinical outcome data
US8788211B2 (en) 2008-10-31 2014-07-22 The Invention Science Fund I, Llc Method and system for comparing tissue ablation or abrasion data to data related to administration of a frozen particle composition
US8793075B2 (en) 2008-10-31 2014-07-29 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US9050317B2 (en) 2008-10-31 2015-06-09 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US9050070B2 (en) 2008-10-31 2015-06-09 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US9060934B2 (en) 2008-10-31 2015-06-23 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US9060926B2 (en) 2008-10-31 2015-06-23 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles
US9072799B2 (en) 2008-10-31 2015-07-07 The Invention Science Fund I, Llc Compositions and methods for surface abrasion with frozen particles
US9072688B2 (en) 2008-10-31 2015-07-07 The Invention Science Fund I, Llc Compositions and methods for therapeutic delivery with frozen particles

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