Patent Application: US-946708-A

Abstract:
the invention provides a method of making composite particles for efficient delivery of polyelectrolytes to a target . composite particles are made by two methods : 1 ) by first forming disperse polyelectrolyte condensates , by mixing the polyelectrolyte with a condensing agent , and then combining the disperse polyelectrolyte condensates with particles so that the disperse polyelectrolyte condensates bind to the surfaces of the particles or 2 ) combining particles with opposite charge polyelectrolyte to form a polyelectrolyte coated particles followed by a subsequent polyelectrolyte of opposite charge to form a composite particle . the invention includes composite particles , where each composite particle is comprised of a particle with the polyelectrolyte from one or more polyelectrolyte condensates bound to that particle . one advantage of these composite particles is that they permit more efficient and increased amounts of polyelectrolytes to be delivered to a target , in comparison to the prior art . delivery methods include but are not limited to methods whereby the particles are accelerated to a velocity sufficient to penetrate or reach the surface of the target by pneumatic , hydraulic , transferred impulse , macro projectile , centripetal force , explosive , electric discharge , mechanical vibration , magnetic , gravimetric , or electric field .

Description:
in this respect , before explaining at least one embodiment of the invention in detail , it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings . the invention is capable of other embodiments and of being practiced and carried out in various ways . also , it is to be understood that the phraseology and terminology employed herein are for the purpose of the description and should not be regarded as limiting . composite particles formed using the process described in this invention are more efficient in the delivery of the polyelectrolyte to a target due , in part , to increased loading of the polyelectrolyte on the particle surface . an example is shown in table 1 . a dispersed polyelectrolyte condensate was prepared by the mixing of spermidine ( a condensing agent ) and plasmid dna ( polyelectrolyte ) in a low salt aqueous solution with ph between 3 and 11 . 5 . particles are added to the solution and polyelectrolyte condensate ( s ) bind to the particles . naturally occurring polyamines are known to condense dna into dispersed polyelectrolyte condensates of a well characterized size ( vijayanathan , thomas et al . 2001 ; trubetskoy , wolff et al . 2003 ). the size of the dispersed dna condensate depends on the concentration and the chemical structure of the polyamine . non - limiting examples of polyamines that condense dna are spermidine , spermine , and histones . depending on the length , structure ( linear , closed circular ) and concentration of the dna , and the concentration of polyamine , dispersed dna condensates can be formed that have diameters of up to ˜ 1 . 5 micron ( trubetskoy , wolff et al . 2003 ). in contrast to the method described in this invention the standard method , consisting of solutions containing cacl 2 and free base spermidine ( ph 11 ) results in the formation of amorphous aggregates of dna . the amount of dna associated with the particle is determined by the addition of sybr green to the sample and the composite particles are deposited on a microscope slide and imaged using a microscope configured for epifluorescence . quantification of sybr green stained nucleic acid bound to the particles is determined by digital image capture and pixel intensity analysis . the amount of polyelectrolyte condensate bound to the particles using the method described in this invention is compared to the method described in the prior art ( i . e . a process i method ) recommended by the manufacturer and distributor of particle acceleration apparatus , bio - rad , inc and described extensively in the literature . there is more polyelectrolyte bound per particle using the method described in this invention compared to the prior art methodology , at least ten fold as much , and in various aspects , 100 - fold as much , and 1000 - fold as much . in addition , a greater number of the particle population have visualizable polyelectrolyte bound to their surface as 98 % of the composite particles prepared using this novel process are modified with polyelectrolyte ( as determined by visualization of fluorescence that co - localizes with the particles ), compared to only 7 % of the particle population prepared by the method described in the prior art . in one aspect , by this measure , the invention provides populations of composite particles that by sybr green staining are at least 10 percent visualizable ( using conventional fluorescence microscopy as described ), and more preferably , at least 25 percent visualizable , and still more preferably , at least 90 percent visualizable . the novel composite particles described herein also result in an increase in the efficacy of polyelectrolyte delivery ( for example , dna ) using particle acceleration techniques as determined by transfection analysis ( fig1 ). both the total number of cells transfected in a population and the expression levels in those cells is significantly increased using the composite particles described in this invention . ballistic delivery of the novel composite particles results in a greater than 4 - fold increase in the total number of gfp expressing neuro2a cells in the population compared to those transfected using particles prepared by the method described in the prior art ( i . e . employing the method of process i ). for each transfection the particles were formulated with the same quantity of dna and total number of particles , respectively , and were delivered by the same ballistic method to each cell population . the novel composite particles described herein result in an increased expression level per cell relative to the particles prepared using the prior art method . in one aspect , composite particles of the invention permit at least 4 - fold greater total gene expression levels than particles prepared by process i . in another embodiment of this invention the polyelectrolyte to be delivered can be directly immobilized to the carrier particle surface via interaction with oppositely charged polyelectrolyte bound to the carrier particle . for example , a negatively charged carrier particle can be incubated with a positively charged polyelectrolyte , including but not limited to polyethyleneimine , poly - lysine or poly ( diallyldimethylammonium chloride ) and following washing to remove non - bound polyelectrolyte the carrier particle surface will have a net positive charge at defined ph as determined using zeta potential . this polyelectrolyte coated carrier particle can then be incubated with another negatively charge polyelectrolyte , such as dna or rna , which then immobilizes to the particle surface resulting in a composite particle of defined polyelectrolye layers . the zeta potential measurement of these particle preparations is shown in table 2 . dual or multi layered polyelectrolyte modified particles can be assembled and subsequently delivered to the target . an example of this approach to deliver inhibitory sirna polyelectrolyte is shown in fig2 . when particles modified with sequence specific sirna for gfp are delivered into cells constitutively expressing gfp there is rapid down - regulation of expression as shown by the loss of fluorescence . after 24 hrs there is greater than 95 % inhibition of gfp expression in cells bombarded with polyelectrolyte modified composite particles . in contrast , delivery of control sirna sequence using the composite particles results in maintenance of gfp expression . in addition , there is no detectable delivery of inhibitory sirna using standard methods and non - modified particles since there is no detectable immobilization of the polyelectrolyte to the negatively charged carrier particle surface . when particles are mixed with the dispersed dna condensates , the dna condensates bind to the surface of the particles . at the correct particle to dna ratio , after staining with a fluorescent dna dye , we find that each particle is strongly fluorescent indicating a high degree of dna binding . when these particles are used in particle bombardment applications , a large increase in the transfection efficiency of the particles is obtained as detailed in example 2 . in contrast to all previous protocols , known to us , that specify the addition of cacl 2 and free base spermidine , resulting in an amorphous uncontrolled aggregate , the polyelectrolyte / particle complex ( ie . our composite particle ) are novel compositions and provide increased performance for particle based bombardment delivery applications . the method of the prior art will be referred to as process i : process i : particles + free base spermidine , dna , mixed , and then add cacl 2 . addition of the spermidine and cacl 2 can also be reversed in this process . we will call the preferred procedure for condensate formation process ii : ( note the change in order from process i , and of course , no cacl 2 .) another version of the sequence of addition can be used , which is not quite as desirable as process ii , but also works much better than process i . process iii : polyelectrolye + condensing agent mixed to form dispersed polyelectrolyte condensates , then add particles ; this results in composite particles with improved properties . we will call the preferred process for polyelectrolye layering process iv : process iv : polyelectrolyte coated particle + oppositely charged polyelectrolyte ( s ), mix , spin particles to remove from non - bound polyelectrolyte ( s ), repeat altering charged polyelectrolyte ( s ) until the desired number of layers have been assembled . in one aspect of the invention , particles that consist of disperse polyelectrolyte condensates to be bound to particles are created by polycation - mediated condensation of nucleic acid and their subsequent assembly at the surface of a particle . condensation refers to the condition in which the nucleic acid structure is of finite size and orderly morphology ( bloomfield 1996 ). the condensing agent is added to a solution of nucleic acid to form dispersed polyelectrolyte condensates . particles are mixed with the dispersed polyelectrolyte condensates to form composite particles that can be delivered via a particle gun ( or other acceleration method ). particles may be formed from any material having sufficient density to be efficiently accelerated into the cells or tissues , or other targets . non - limiting examples of materials for making particles include copper , gold , tungsten , nickel , aluminum , silver , iron , steels , cobalt , titanium , glass , silica , polymers , and carbon compounds ( e . g ., graphite , diamond ). metals such as gold are frequently used , as they are inert , nontoxic , and have a high density . the particles should be of a mass sufficiently large to provide the momentum required to penetrate into cells ( or other types of targeted material ), and also have a surface area that can bind a sufficient quantity of the material to be delivered . the particle should be sufficiently small to avoid excessive damage or disruption of biological function once in contact with the targeted material ( e . g . tissue ). particles ranging in diameter from about 0 . 25 microns to about 4 . 0 microns have been used in such particle bombardment applications . particles that are clumped in irregular aggregations are less desirable , as such aggregations will vary widely in their mass and size , thus leading to difficulty in obtaining reproducible results . in one aspect , a particle has a density between 0 . 1 and 23 g / cm 3 most preferably between 5 and 23 g / cm 3 . a particle may be solid , porous , or consists of an assemblage of a number of smaller particles . in one aspect , a particle has a diameter of between 100 nm and 100 microns , where a preferred range is between 300 nm and 3 microns . in one aspect , particles may be composed of one or more of the following materials , gold , silver , platinum , nickel , copper , tungsten , alloys , silica , latex , polystyrene , acrylamide , dextran , and ceramics . in one embodiment of the present invention , the particles are fabricated from metals that include but are not limited to gold , silver , tungsten , platinum , palladium or any alloy thereof . the preferred particle size range is from 50 nm to 5000 nm in diameter and the particle can have a variety of different shapes that include but are not limited to spherical , triangular , elliptical , cylindrical , or platelet . anisotropic particles have a higher surface area for a given volume and can provide for more efficient delivery of biological material to the cells , tissue or target of interest . the particles can be homogenous where the same material is used throughout the particle . alternatively , the particle can be heterogeneous where different areas of the particle have different compositions . one embodiment of a heterogeneous particle is a multi - layer particle where each layer has a different chemical composition . for example , a particle could consist of a solid gold particle that is coated with a layer of silica . alternatively , the particle could consist of an inorganic core with an arbitrarily thick layer of gold . by varying the size of the starting core and the amount of gold deposited , monodisperse gold nanoshelled particles can be fabricated with extremely narrow size distributions (& lt ; 5 % polydispersity ) with a broad range of core sizes ( 0 . 1 - μm in diameter ). another embodiment of a heterogeneous particle is a particle that consists of many smaller particles that have been dried together to form an aggregate . another embodiment of a heterogeneous particle is a particle that is porous . another embodiment is a particle that has an interior that is filled with a liquid . the particles could be composed , in part , of a material that is biodegradable , magnetic , or plays an active role — as a biosensor or as a drug delivery ( synthetic drug , anti - sense dna , rnai , etc ) element . the surface of the particle may be initially coated with a material chosen to aid in having the polyelectrolyte condensate to preferentially be deposited or become associated with such particle . additionally , the surface of the particle may be composed of a material that releases the polyelectrolyte condensate in a preferred form or manner from this surface when the composite particle is inside of a cell , tissue or the targeted material . in one embodiment of the present invention , the particle initially has a negative charge . in one embodiment the zeta potential of the particle is negative in aqueous solution at a specified ph and the particle is not further functionalized . in another embodiment , the particle is coated with one or more layers that impart a negative charge to the surface of the particle . for example , the coating of a gold particle with a positively charged polymer such as poly ( diallyldimethylammonium chloride ) followed by a coating of poly ( sodium 4 - styrene - sulfonate ) will result in a particle with a strong negatively charged surface at a specified ph . in another embodiment , a particle with a positively charged surface is mixed with a negatively charged polymer and then added to polyelectrolyte and a condensing agent . in another embodiment , the particle is functionalized with a polymer that facilitates the release of the bound polyelectrolye after intracellular or intra - tissue delivery . in another embodiment , at a specified ph , a positively charged particle is functionalized with negatively charged polyelectrolyte yielding a negatively charged particle . in another embodiment , the particle is coated with a self assembled monolayer to yield either positive , negative or neutral charge at a specified ph . as discussed above , in a preferred embodiment , ( process ii ), the polyelectrolyte and the particle are first mixed in a solution , and then the condensing agent is added . in another embodiment ( process iii ), the polyelectrolyte and the condensing agent are first added to a solution , mixed , followed by the addition of the particles . in both process ii and process iii the composite particles formed are very effective ballistic delivery vehicles . in a further embodiment ( process iv ), the polyelectrolyte and particle ( s ) of opposite charge are first mixed in a solution , and then removed from non - bound polyelectrolyte , to form a composite particle . an additional number of layers can be assembled by repeating the addition of polyelectrolyte of opposite charge to the composite particle . this process is very effective for delivery of linear , single and double strand polynucleotides including dna and rna . as emphasized , process i is the teaching of the prior art . composite particles produced by such a process are referred to herein as “ process i composite particles .” iv . ratio of nucleic acid ( e . g . dna ) to solid gold particles in one embodiment of this invention a ratio of 0 . 1 μg - 10 μg dna per 0 . 95 cm 2 surface area of particles is preferred . in general , the preferred ratio may depend on the size , surface area , porosity , and density of the particles . condensing agents coordinate the assembly of dispersed polyelectrolyte condensates assembled in process ii and process iii , and are involved in the subsequent interaction of the polyelectrolyte condensate with the particle to make a composite particle . in one aspect of the present invention , the condensing agents are polycationic molecules selected from the group that consists of spermidine , spermine , basic histones , high mobility group polypeptides , transition protein tp2 , non - naturally occurring spermidine and spermine derivatives , cobalt hexamine , poly ( ethylenimine ), poly - l - lysine , and poly - l - ornithine ( bloomfield , va ., 1996 , curr . opin . struc . biol ., 6 , 334 - 341 ). in a preferred embodiment the condensing reagent concentration is between 0 . 5 - 100 mm . there are a number of chemicals that can disrupt the polyelectrolyte layering and condensation processes . it is an objective of the present invention that these substances be excluded from the composite particle fabrication process . for example , concentrations of mono or divalent cations such as ca , na , or mg in their ion or salt form in combination reduce the amount of polyelectrolyte that can be loaded onto a particle . in one aspect , the concentration of such ions should not exceed a molar ratio of 0 . 1 / 1 with the dna condensing agent or polyelectrolyte . example of the first positive charged polyelectrolyte coating layer used in process iv include but is not limited to polyethyleneimine , poly - lysine or poly ( diallyldimethylammonium chloride ). the negative charged polyelectrolyte layer includes but is not limited to polynucleotides such as dna or rna . examples of the polyelectrolyte to be delivered using this present invention include polynucleotides such as dna or rna or another biologically active molecule that can be condensed using condensing agents . in any of the referred embodiments , the composite particles prepared in this present invention are suitable for particle gun mediated delivery into cells and tissues from animal , plants , eukaryotes , prokaryotes , fungi , other living organisms , and other non - living materials . the composite particles formed using the methods described herein have other non - ballistic applications where a layer of polyelectrolyte / high polyelectrolyte binding capacity is useful . one application is the use of the composite particles for delivery of polyelectrolyte to the lungs . another application is the use of the composite particles for microinjection , or as a nucleic acid transport vehicle in conjunction with other known methods that lead to composite particle internalization by a cell or tissue ( electroporation , endocytosis , pinocytosis , magnetofection , lipofectamine 2000 , and the like ). the following examples are intended to provide illustration of the application of the present method . the following examples are not intended to completely define or otherwise limit the scope of the invention . in this example , the benefit of formulating the particles using a nucleic acid condensing agent is demonstrated by the increased association of fluorescently labeled dna at the surface of the gold particles . in the improved method described in this invention gold particles ( 1 . 6 μm average diameter ), typically 2 . 5 mg from a stock solution of 200 mg / ml in water , are resuspended in 100 μl of 20 mm sodium acetate ph 4 . 5 in a microfuge tube . the genetic material to be transferred to the cells by bombardment , in this example gwiz plasmid dna containing the gene for gfp ( green fluorescent protein ) ( aldevron , fargo , s . d .) is added to a concentration of 2 . 0 μg per mg of particles from a stock resuspended at a concentration of 1 mg / ml in water . the solution is vortexed and the nucleic acid condensing reagent , in this example spermidine - hcl ( 50 mm in water ), is added to yield a final concentration of 25 mm . the solution is incubated at room temperature for 10 minutes , 2 μl sybr green dna stain ( molecular probes , inc ., eugene , oreg .) is added , 6 μl of the sample is placed under a coverslip and the sample is visualized with a combination of darkfield and bright field microscopy . a parallel sample is created using the “ standard method ” as described both in the bio - rad helios gene gun system instruction manual and extensively in the literature . the gold particles ( 2 . 5 mg ) are placed in a microfuge tube and suspended in 100 μl of a 50 mm free base spermidine solution ( ph 11 ). a 100 μl aqueous solution containing gwiz plasmid dna is added to yield a ratio of 2 . 0 μg dna per mg particles and the sample vortexed . while continuing to vortex the sample 100 μl of a 1 m cacl 2 solution is added dropwise to the sample . the sample is allowed to stand for 10 minutes at room temperature to induce the aggregation and precipitation of dna . after this incubation period , 2 μl of sybr green dna stain is added and 5 μl of this solution is placed under a coverslip . the sample is visualized using a combination of darkfield and bright field microscopy . to determine the amount of dna associated with the particles for each of the two preparation methods digital images were acquired using a macrofire ccd . equivalent exposure times were used for each sample . image - pro image analysis software was used to determine the relative amount of fluorescence per field and the number of particles that had detectable associated dna fluorescence . the data are presented in table 1 . in this example of the invention the benefit is illustrated by an increase in the number of transfected cells obtained when using particles formulated with the invention , “ process ii method ”, as compared to that using the standard “ prior art ” method . the particles prepared as described in example i were suspended in an ethanol solution . particle - dna samples where coated onto tefzel tubing , dried with nitrogen gas and used for ballistic transformation . these particles can be used to biolistically introduce any plasmid containing a gene of interest and we have used the reporter plasmid containing a coding region for the green fluorescent protein ( gfp ; commercially available aldevron , fargo , n . d .). an optimum plasmid to particle ratio is in the range of 0 . 1 g to 10 μg plasmid per cm 2 of particle surface area . neuro2a mouse neuroblastoma cells were used for analysis of biolistic transfection efficiency . cells were maintained at 37 ° c . in dulbecco &# 39 ; s modified eagles medium ( invitrogell , carlsbad , calif .) supplemented with 10 % fetal bovine serum and penicillin / streptomycin . the cells were routinely passaged after they had reached confluence . for biolistic transfection analysis the neuro2a cells were plated into 35 mm dishes the day prior to biolistic delivery . the medium was removed from the cells and the biolistic delivery device ( any commercially available pds - 1000 or helios gene gun ; bio - rad , hercules , calif .) was positioned above the cells and particle - dna complexes were delivered by gas pressure mediated acceleration through a 5 μm pore size polycarbonate membrane . the cells were placed at 37 ° c . for an additional 4 - 24 hours and expression levels were determined using fluorescence microscopy . the total number of cells expressing gfp and the intensity output per cell were determined using image - pro image analysis software . the data obtained for each of the two particle sample preparation methods is shown in fig1 a - 1c . preparation of polyelectrolyte coated particles . in the method described in this invention gold particles ( 1 . 0 μm average diameter ), typically 100 mg were suspended in 1 ml of appropriately buffered solution to yield the desired zeta potential for immobilization . a positive charge polyelectrolyte , such as poly - lysine , polyethyleneimine or poly ( diallyldimethylammonium chloride ) ( pdadmac ), was added to a final concentration of between 0 . 1 - 1 % weight volume and the samples were incubated . the non - bound polyelectrolyte was removed and a second layer is assembled onto the positive charge particles by addition of a negative charge polyelectrolyte , for example sirna or oligonucleotide . the measured zeta potential of these different particles after each assembly step is shown in table 2 . the optimal ratio of negative charged polyelectrolyte to positive charged particle is between 50 - 400 pmole per milligram of gold particle . the samples were incubated and non - bound polyelectrolyte was removed from the suspension . samples with this dual layer can subsequently be processed for ballistic delivery or used for further layers of polyelectrolyte addition . particles were prepared as described using a negatively charged polyelectrolyte second layer compromised of a 21 base long synthetic rna molecule ( qiagen , valencia , calif .) of sequence composition complementary to green fluorescent protein ( gfp ) gene sequence , or alternatively , for use as a negative control , a synthetic rna whose sequence was non - complementary to gfp . the composite particles were resuspended in an ethanol solution , coated onto tefzel tubing , dried with nitrogen gas and used for ballistic transformation . the medium was removed from the cells , the biolistic delivery device ( a commercially available pds - 1000 or helios gene gun ; bio - rad , hercules , calif .) was positioned above the cells , and particle - polyelectrolyte complexes were delivered by gas pressure mediated acceleration through a 5 μm pore size polycarbonate membrane . the cells were placed at 37 ° c . and expression levels were monitored at varying times using fluorescence microscopy for an additional 144 hours . digital images were obtained using a macrofire ccd , and image intensity per unit area was determined using image - pro image analysis software . the data obtained for each of the two particle populations ( rna complementary and non - complementary to the sequence of gfp ) are shown in fig2 a - 2c . with respect to the above description then , it is to be realized that the optimum dimensional relationships for the parts of the invention , to include variations in size , materials , shape , form , function , assembly and use , are deemed readily apparent and obvious to one skilled in the art , and all equivalent relationships to those illustrated in the drawings and described in the specification are intended to be encompassed by the present invention . therefore , the foregoing is considered as illustrative only of the principles of the invention . further , since numerous modifications and changes will readily occur to those skilled in the art , it is not desired to limit the invention to the exact construction and operation shown and described , and accordingly , all suitable modifications and equivalents may be resorted to , falling within the scope of the invention . antipov , a . a ., g . b . sukhorukov , et al . 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