Patent Application: US-35933609-A

Abstract:
the present invention provides a fabrication method for the formation of a cell - microparticle hybrid . a biotin - avidin binding system also employs the use of a biodegradable polymer and any cell type that self - assemble to form a hybrid system .

Description:
the inventors now describe a biotin - avidin interaction system that can be utilized to develop a cell - microparticle hybrid . avidin and biotin are broadly used in biological analysis techniques such as immunoassays because they make a highly specific and stable complex . avidin ( mw approximately 68 kda ) is a glycoprotein with 4 subunits that can each bind a biotin ( also known as vitamin h ; mw 244 . 3 ) the affinity constant of the avidin - biotin complex is about 10 15 m − 1 [ 19 ]. in this step of the process , human embryonic kidney 293 ( hek293 , american type culture collection ( atcc )) cells have been biotinylated . this has been achieved by converting native sialic acid residues on the cell surfaces into non - native aldehydes using a mild naio4 solution . sialic acid is a ubiquitous terminal cell surface monosaccharide group with amplified expression in many cancers [ 20 ]. the aldehyde groups have been reacted with biotin - hydrazide to produce biotinylated cells . to achieve this objective , hek293 cells have been grown to 60 - 65 % confluence in 12 - well plates . cell culture media in the wells have been removed and replaced with freshly warmed media and further incubated for 1 h . subsequently , the cells have been washed twice with phosphate buffered saline ( pbs ) and incubated with a 1 mm solution of naio4 in cold pbs for 20 min in the dark at 4 ° c . the hek293 cells have been washed with pbs at ph 6 . 5 supplemented with 0 . 1 % bovine calf serum ( bcs ) at room temperature . next , the cells have been incubated with a 0 . 5 mm solution of biotin hydrazide ( sigma ) in pbs ( ph 6 . 5 ) for 90 min at room temperature . then , the cells have been washed twice with pbs solution at ph 7 . 4 supplemented with 0 . 1 % bcs . the process of cell surface biotinylation is depicted in fig1 . a biotinylated polylactic acid - polyethylene glycol copolymer is synthesized by reacting n - hydroxysuccinimide - biotin with the amine terminus of a bifunctional a - amino - hydroxy - polyethylene glycol that was prepared by reducing a - amine - o - carboxylic acid - polyethylene glycol in a 1m tetrahydrofuran - borane mixture ( sigma ). confirmation of the amide bond between the biotin and the peg was observed by the appearance of a triplet at 7 . 8 ppm in 1h - nmr . lactide was then graft polymerized onto the hydroxyl terminus of the a - biotin - o - hydroxy - polyethylene glycol in the presence of a stannous 2 - ethyl hexanoate initiator as represented in fig2 . [ 21 , 22 ] pla - peg - biotin microparticles have been prepared using a double - emulsion solvent - evaporation approach [ 23 ]. this method utilizes three distinct phases , an inner water phase wherein the relevant proteins or drugs are entrapped , an intermediate organic phase composed of the polymer / methylene chloride solution , and an outer water phase containing an emulsifying agent . these microparticles can be loaded with a wide variety of drugs , proteins , or fluorescent molecules for imaging applications . [ 23 ] the particle size of these microparticles has been determined to be 1 . 4 μm from light scattering measurements using a zetasizer nano zs instrument . the biotinylated cells ( step a ) have been incubated with 1 mg ml − 1 of avidin - saturated biotinylated microparticles ( step b ) for 20 min at 4 ° c . subsequently , the cells have been again washed twice with pbs ( ph 7 . 4 ) prior to imaging by light microscopy ( olympus bx40 ). the degree of biotinylation on the surface of the hek293 cells has been determined using the 2 -( 4 - hydroxyazobenzene ) benzoic acid ( haba )/ avidin assay to be ( 3 . 6 ± 0 . 45 )× 10 9 biotin moieties per cell . the cell viability has been determined using trypan - blue measurements . biotin - functionalized cells demonstrate a viability of 89 . 7 % as compared to 97 . 51 % for untreated cells . a self - assembled cell - microparticle hybrid is represented in fig3 e , 3 j , and 3 k . the inventors now describe another embodiment of biotin - avidin interaction system that can be utilized to develop a cell - microparticle hybrid as an innovative vaccine carrier for the co - delivery of antigen and immunostimulatory adjuvant to the same antigen presenting cell ( apc ). [ e ] transfection of cells prior to biotinylation to express desired protein of interest in this step , 8 × 10 4 hek - 293 cells were seeded on a 24 - well plate 24 hours prior to transfection . polyethyleneimine ( pei ) was used as a non - viral polymeric carrier for the pdna encoding green fluorescent protein ( gfp ). the transfection vehicle was incubated with the cells for 4 hours . at the end of 4 hours the cells were washed and fresh medium was added and cells were incubated at 37 ° c . for 44 hours . success of transfection was assessed using an olympus bx40 fluorescent microscope . cells that were successfully transfected looked as represented in fig4 . the same procedure as described in ( c ) was followed with the additional step of addition of rhodamine 123 in dichloromethane along with the said polymer . the same procedure as described in ( d ) was followed and the hybrid formed is represented in fig4 k . virtually any cell type and size can be attached to surfaces in accordance with the present invention . the cells may be prokaryotic or eukaryotic , plant , fungal , animal or human . the cells may be neuronal , endothelial or epithelial in origin , or may be lymphocytes , fibroblasts or myoblasts . particular cells types include bacteria such as e . coli , staphylococcus , myoblast precursors to skeletal muscle cells , neutrophils , erythroblast , osteoblasts , chondrocyte cartilage cells , basophil , eosinophils , adipocyte fat cells , invertebrate neurons ( helix aspera ), mammalian neurons , adrenomedullary cells , melanocytes , or cancer cells . in addition , naturally occurring or genetically engineered cells , including those form plants , mammals , or invertebrates may be employed as well as mixtures of cells . a particularly useful source of cell lines may be found in atcc cell lines and hybridomas ( 8th ed ., 1994 ), bacteria and bacteriophages ( 19th ed ., 1996 ), yeast ( 1995 ), mycology and botany ( 19th ed ., 1996 ), and protists : algae and protozoa ( 18th ed ., 1993 ), each of which are herein incorporated by reference . biotin - functionalized cells demonstrate a viability of 89 . 7 % as compared to 97 . 51 % for untreated cells . fig1 d shows an image of pla - peg - biotin microparticles that have been incubated with hek293 cells surface functionalized with avidin / biotin . the results of the control experiment in fig1 e shows that the microparticles do not bind as effectively to control hek293 cells that have not been treated with biotin hydrazide . for control hek293 cell samples that have not been treated with biotin hydrazide , it has been observed that the microparticles readily settle in areas where the non - confluent cells have not spread . the ability to specifically bind particles to non - confluent cells using receptor - mediated interactions has significant potential for improving in vitro drug and gene delivery . for example , biotinylated nanoparticles loaded or complexed with plasmid dna could potentially significantly enhance the transfection efficiencies of avidin - biotin surface - engineered cells . to demonstrate that cell - microparticle hybrids can be prepared in solution , the cells have been trypsinized and treated with naio4 and biotin hydrazide as described above . avidinylated pla - peg - biotin microparticles ( 1 mg ml − 1 ) have been self - assembled with the biotinylated cells ( 1 × 10 5 ) by gently pipetting the two solutions into a single vial . control experiments have also been performed wherein the cells are treated in exactly the same manner except for treatment with naio4 . the results of the control experiments show a two - to three - fold reduction in the percentage of cells binding microparticles and a four - fold reduction in the number of particles bound per cell ( fig3 b and c ). control experiments where the cells have not been treated with biotin hydrazide indicate limited or no assembly of microparticle - cell hybrids ( fig1 e and h ). this confirms that the self - assembly process arises from specific biotin - avidin receptor - mediated interactions ( fig3 b , c , e , j , k , and l ). to demonstrate the potential of our cell - microparticle hybrids in dual synthetic - biological drug and protein delivery applications , we have transfected non - adherent el4 cells and adherent hek293 cells with green fluorescent protein ( gfp ). microparticles loaded with rhodamine 123 have been prepared using the double - emulsion solvent - evaporation technique and assembled with gfp - expressing cells using the biotin - avidin interaction . fig3 shows a fluorescent overlay image of rhodamine - labeled microparticles efficiently assembled onto hek293 cells expressing gfp . to demonstrate that this process is compatible with non - adherent cells transfected with antigenic proteins , eg7 cells ( atcc , 1 × 10 5 ) have been engineered with biotin using the same procedure as described above for the hek293 cells . the eg7 cells have been derived from the murine t - cell lymphoma el4 cell line transfected with cdna for a model protein antigen , ovalbumin . fig3 k shows that when the biotinylated eg7 cells are incubated with avidinylated pla - peg - biotin microparticles , cell - microparticle hybrids are readily constructed . cell transplants have been used clinically for bone marrow reconstitution for over 30 years . cell transplants are now being tested for neurological disorders such as parkinson &# 39 ; s [ 24 - 26 ] and huntington &# 39 ; s diseases [ 27 , 28 ]. in addition mimicking the local environment at the site of cell transplant by the presence of signaling molecules , growth factors and proteins can have specific mechanical and biological properties similar to the native extracellular matrix ( ecm ). the interactions between cells and ecm control cellular activities such as migration , proliferation , differentiation , gene expression , and organogenesis [ 29 , 30 ]. hence a hybrid system like the one developed by us can mimic the ecm of the host to the greatest extent and thus serve as an intelligent bio - synthetic building block in tissue engineering . while cpg odn an adjuvant , exhibits potent immunostimulatory effects , the rapid degradation and ineffective delivery into the intracellular compartments of apcs are major bottlenecks to improving its efficacy [ 31 ]. when antigen is administered alone , it elicits strong t h 2 type immune responses . a significant shift in the isotype of antibody response can be achieved by co - administering antigen and cpg odn [ 32 - 35 ]. addition of cpg odn has been reported to result in a significant increase in production of igg2a antibody , increasing the igg2a : igg1 ratio over nine - fold [ 12 ]. this enhanced t h 1 type immune response is essential for counteracting intracellular pathogens including choriomeningitis virus , hepatitis b virus and tetanus toxoid . for example , co - administration of cpg odn and hepatitis b surface antigen ( hbsag ) vaccine to the same site of the muscle significantly enhanced the antibody response [ 36 ]. in contrast , when cpg odn was administered separately following the administration of the vaccine , it did not induce any significant improvement in immunostimulatory effects over the administration of vaccine alone [ 36 ]. these studies highlight the importance of delivery devices that protect cpg odn from enzymatic degradation , improve targeting of cpg odn to the intracellular compartments of apcs , ensure that both cpg odn and antigen are co - delivered to the same apc , and provide long term immunity . with this new hybrid system , cells can be transfected with granulocyte - macrophage colony - stimulating factor ( gm - csf ) and self - assembled with microparticles loaded with immunostimulatory molecules such as cpg oligonucleotides as a new and potent vaccine for cancer delivering both antigen and adjuvant to the same apc . a - hydroxy - x - amine peg ( 1 g ) was dissolved in a mixture of acetonitrile ( 2 ml , aldrich ), methylene chloride ( 1 ml , aldrich ), and et3n ( 80 ll , aldrich ). after the addition of nhs - biotin ( 0 . 250 g , sigma ), the reactants were stirred overnight under argon . subsequently , the reaction was worked - up by the slow addition of diethyl ether ( 40 ml , aldrich ) to precipitate the polymer . the polymer was reprecipitated from hot isopropyl alcohol ( 70 ° c ., aldrich ). the reprecipitated polymer ( 350 mg ) was then dried azeotropically and left under vacuum . lactide ( 2 g , purac biochem bv ) was added to biotin - peg - oh ( 0 . 35 g ) and diluted with 10 ml toluene and sn ( oct ) 2 / toluene ( 0 . 1 g in 1 ml ). the reaction mixture was then brought to reflux at 110 ° c . for 4 h under argon . the product was precipitated from a dichloromethane ( dcm ) solution into a cold stirring solution of diethyl ether and isolated by vacuum filtration . the final product was characterized by gpc and 1h nmr spectroscopy . pla - peg - biotin ( 50 mg ) was dissolved in 5 ml of dcm . for rhodamine - loaded microparticles , 1 mg rhodamine 123 ( sigma ) was also dissolved in 5 ml dcm . the polymer solution was then added to 500 ll of a 1 % ( w / v ) pva ( mw : 250 000 , 88 % hydrolyzed , sigma ) solution and ultrasonicated for 30 s . the primary emulsion was then added to 50 ml of 1 % ( w / v ) pva solution and homogenized further for 3 min at 13 500 rpm . the emulsion was then left stirring overnight over a magnetic stirrer to allow dcm to evaporate and to enable the formation of microparticles . the average diameter of the microparticles was 1 . 4 lm , as determined by measurements made using a zetasizer nanozs ( malvern instruments ) instrument . hek293 cells and eg7 / el4 ( crl - 2113 / tib - 39 ) cells were obtained from atcc ( manassas , va .). the cells were cultured in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) obtained from gibco brl ( grand island , n . y .) supplemented with 10 % fetal bovine serum ( fbs ), streptomycin at 100 lg ml - 1 , penicillin at 100 u ml - 1 , and 4 mm l - glutamine at 37 ° c . in a humidified 5 % co2 - containing atmosphere . the hek293 cells were passaged at pre - confluence every 4 days in a 1 : 4 ratio using 0 . 25 % trypsin . fresh dmem medium was replenished every 2 days during cell culture . the eg7 / el4 cells were passaged every alternate day in a 1 : 3 ratio by aspirating two - thirds of the medium and replacing with fresh medium . gfp - plasmids ( clontech ) were transformed to escherichia coli dh5a and amplified in terrific broth media at 37 ° c . overnight at a shaking speed of 300 rpm . the plasmid was purified using an endotoxin - free qiagen giga plasmid purification kit ( qiagen , valencia , calif .) according to the protocol provided by the manufacturer . purified pdna was dissolved in saline , and its purity and concentration were determined by uv absorbance at 260 and 280 nm . hek293 and el4 cells were seeded into 24 - well plates at a density of 8 × 104 cells per well 24 h before starting transfection . each well of the 24 - well plate was transfected with 0 . 5 ml reduced - serum opti - mem media ( gibco ). polyethyleneimine ( pei , 25 000 branched , sigma )/ pdna complexes in a ratio of 5 : 1 comprising 5 lg pei in 40 ll opti - mem and 1 lg dna in 40 ll opti - mem were added to each well . after 4 h , the transfection media was removed and the cells were washed . after 2 days of further incubation in serum - containing media , the wells were washed with pbs and imaged live . the cells were then ready for use in microparticle - cell assembly experiments . this analysis was carried out using a spectrophotometer ( spectramax 384 plus , molecular devices , ca ) at a fixed wavelength of 500 nm . 180 ll of haba / avidin reagent was added to a 96 - well plate and three readings of absorbance were recorded with an average read time of 0 . 5 s . after the initial recording , 120 ll of the supernatant collected following cell treatment with biotin hydrazide was added to each of the 96 - well plates . three sets of absorbance readings were recorded again after allowing 3 min for reaction . the absorbance decreased proportionately depending on the amount of biotin present on the cell surface because the biotin displaces haba owing to its higher affinity for avidin . the changes in absorbance were used in conjunction with a calibration curve to calculate the extent of biotinylation of the cell surface . cell - microparticle hybrids were seeded onto a poly ( l - lysine ) coated ( 1 lg cm - 2 ) coverslip and fixed with 2 . 5 % glutaraldehyde solution . after 1 h , the hybrids were washed twice with a 0 . 2 % sodium cacodylate buffer solution and dehydrated with 25 , 50 , 75 , 95 % ( 4 min each ), and 100 % ethanol ( 10 min each ) solutions . the cell - microparticle hybrids were then treated with hexadimethylsilazane ( hdms ) for 10 min and dried for 3 h . the samples were then sputter coated and visualized using sem ( hitachi s4800 ). a . k . boal , f . ilhan , j . e . derouchey , t . thurn - 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