Patent Application: US-59721505-A

Abstract:
materials and methods are provided for making nanoparticles having a core including metal and / or semiconductor atoms , which core is covalently linked to a plurality of ligands comprising a rna ligand . the rna ligands may include sirna or mirna . also provided are uses of these nanoparticles in therapy and diagnosis .

Description:
the her - 2 / neu oncogene and its encoded product p185her - 2 / neu belong to the epidermal growth factor receptor tyrosine kinases ( bargmann et al , 1986 ). the her receptor family consists of four transmembrane tyrosine kinases : egfr ( also known as her - 1 or erbb - 1 ), erbb - 2 ( her - 2 ), erbb - 3 ( her - 3 ), and erbb - 4 ( her - 4 ). her - 2 / neu signalling pathways are known to play critical roles in cell growth and differentiation , malignant transformation , and resistance to chemotherapeutic agents ( yarden & amp ; sliwkowski , 2001 ). her - 2 / neu is over - expressed in about one third of cases of human breast or ovarian cancers , and its over - expression is associated with poor prognosis ( berchuck et al , 1990 ). numerous attempts have been made to inhibit her - 2 / neu expression in cancer cells as a potential therapeutic approach . a humanized monoclonal antibody against her - 2 / neu ( trastuzumab or herceptin ) has been effective in her - 2 / neu - overexpressing metastatic cancer ( mendelsohn & amp ; baselga , 2000 ; baselga et al , 1996 ) but was found to up - regulate her - 3 expression . an antisense oligonucleotide against her - 2 / neu has been shown to induce apoptosis in human breast cancer cell lines that overexpress her - 2 / neu ( roh et al , 2000 ). gene therapy with e1a , delivered by liposomes or by adenoviral vectors , can reduce mortality among tumour - bearing mice in a model of her - 2 / neu - overexpressing ovarian cancer and can reduce the incidence of distant metastases in a model of breast cancer ( chang et al , 1996 ). the down regulation of her - 2 / neu expression was found to lead to decreases in pi3k , akt , and phosphorylated akt which resulted in decreased expression of cyclin d1 , a cyclin involved in the regulation of g0 / g1 cell arrest and oncogenic transformation ( sherr & amp ; roberts , 1999 ). a recent study comparing the efficacy of antisense oligonucleotides and sirna demonstrated that sirnas are at least 10 times more efficient on a nm basis at silencing a reporter gene ( miyagishi et al , 2003 ). several previous studies have demonstrated that her - 2 / neu stimulates the transcription of vegf , a potent proangiogenic factor ( kumar & amp ; yarmand - bagheri , 2001 ) the level of which was markedly decreased after silencing of her - 2 / neu expression . down regulation of her - 2 / neu by retroviral sirna increased thrombospondin - 1 levels , a powerful inhibitor of angiogenesis ( izumi et al , 2002 ). in vitro data demonstrated that her2 sirna treatment also significantly up - regulates hla class i surface expression in human tumours ( choudhury et al , 2004 ). sk - br - 3 human mammary adenocarcinoma from atcc ( cat .# htb - 30 ). ovcar - 3 human ascites adenocarcinoma from nci - frederick cancer dctd tumor / cell line repository ( vial 0502296 ). the sense sirna had a sequence r ( gccucacagagaucuugaa ) d ( tt ) 3thss . ( mw of k - salt 7416 . 25 ) and the antisense sequence r ( uucaagaucucugugaggc ) d ( tt ) 3thss ( mw of k - salt 7409 . 57 ) were obtained from qiagen . the control ( non - silencing ) sirna duplex sequences from qiagen ( cat # 1022076 ) where sense r ( uuc ucc gaa cgu guc acg u ) d ( tt ) and antisense r ( acg uga cac guu cgg aga a ) d ( tt ) and the mw of the annealed k - salt was 14839 . 5 . dissolve contents ( 296 . 65 μg ) of one sense sirna tube in 1 ml sterile buffer ( 100 mm potassium acetate , 30 mm hepes - koh , 2 mm magnesium acetate ph 7 . 4 ) to make a 40 μm stock . each μl will contain 0 . 297 μg sirna . the contents ( 296 . 38 μg ) of one antisense sirna tube were dissolved in 1 ml sterile buffer ( 100 mm potassium acetate , 30 mm hepes - koh , 2 mm magnesium acetate ph 7 . 4 ) to make a 40 μm stock . each μl contained 0 . 296 μg sirna . to anneal , 30 μl of each rna oligo solution was combined with 15 μl of 5 × annealing buffer . the final buffer concentration was 50 mm tris , ph 7 . 5 - 8 . 0 , 100 mm nacl in depc - treated water . the final volume was 75 μl and the final concentration of sirna duplex was 16 μm . the solution was incubated for 1 minute in a water bath at 90 - 95 ° c ., and allowed to cool to room temperature ( i . e . below 30 ° c .). the tube was centrifuged briefly to collect all liquid at the bottom of the tube . slow cooling to room temperature took 45 - 60 minutes . the resultant solution was stored at − 20 ° c . until ready to use and was resistant to repeated freezing and thawing . haucl 4 ( 99 . 999 %) and nabh 4 were purchased from aldrich chemical company . 2 - thioethyl - β - d - glucopyranoside was synthesized in our laboratory using standard procedures . for all experiments and solutions , nanopure water ( 18 . 1 mω ) treated with depc ( diethylpirocarbonate ) was used . all eppendorfs , spatulas and vials were rnase free . annealed double - stranded sirna was purchased from qiagen - xeragon inc . the specifications were : to a solution of 2 - thioethyl - β - d - glucopyranoside ( 0 . 9 mg , 3 . 75 μmol ) and sirna ( 0 . 148 mg , 0 . 01 μmol ) in tris buffer 100 mm , ph 7 . 7 ( 250 μl ), an aqueous haucl 4 solution ( 22 μl , 0 . 025m ) was added . then , 1n aqueous solution of nabh 4 ( 30 μl ) was added in several portions with rapid shaking . the brown suspension formed was shaken for an additional 1 h at 4 ° c . the suspension was purified by centrifugal filtering ( amicon mw 10000 , 30 min , 4 ° c ., 14000 rpm ). the process was repeated twice , washing with 125 μl of tris buffer . the residue in the amicon filter was dissolved in 250 μl of tris buffer and lyophilised to afford 4 mg of rna - au - glc nanoparticles ( resuspension of the solid in 1 ml of water should give a 6 ± 1 μm solution of rna in 20 mm tris buffer ). the filtrate was desalted using amicon ( mw 3000 , 4 ° c ., 14000 rpm ) and lyophilised . the weight of the residue was & lt ; 50 μg . the transmission electron micrograph ( tem ) shown in fig1 shows that the average size of the particles was 2 . 8 nm , with an average of 807 gold atoms / particle , sirna and 100 molecules of the glucose derivative as represented in fig4 and has an approximate mw & gt ; 160 , 000 . rna - au - glc nanoparticles , glc - au nanoparticles and the residue of washing the rna - au - glc nanoparticles which presumably containing rna oligonucleotide and glucose derivative were each dissolved in 30 μl of water . aliquots of these solutions ( 1 μl ) were mixed with aqueous solution of ethidium bromide ( etbr ) ( 1 μl , 0 . 1 % v / v ). fluorescence was observed under a uv lamp ( see fig2 ) demonstrating that the so - prepared nanoparticles have incorporated sirna ( fig2 b , tube 1 ), while the nanoparticles containing only glucose do not show any fluorescence ( fig2 b , tube 2 ). 4 mg of the sirna / nanogold complex generated from 148 μg sirna after were dissolved in 1 ml water to obtain a 6 ± 1 μm stock solution in 20 mm tris . each μl of solution contained the equivalent of 0 . 078 μg sirna . 1 . 24 h before transfection , 6 × 10 4 cells were pipetted into a 24 - well plate and the volume made up to 0 . 5 ml with appropriate culture medium . 2 . the cells were allowed to reach 50 - 80 % confluency , taking approximately 24 h . 3 . the culture medium was removed and replaced with 300 μl fresh medium / well . 1 . 3 . 3 μl ( or 12 . 8 μl of the nanogold complex ) of the appropriate duplex sirna stock was dispensed into a 24 - well plate corresponding to that with cells . 2 . to each well 96 . 7 μl ( or 87 . 2 μl for the nanogold complex ) of the appropriate culture medium was added and mixed well by pipetting up and down 5 times . 3 . to each well ( other than the nanogold complex wells ) 6 μl of rnaifect was added and mixed well by pipetting up and down 5 times . 4 . the solutions were incubated at room temperature for 10 - 15 minutes to allow complex formation . 5 . the cells in 300 μl culture medium were overlayed with the 100 μl of the appropriate transfection complex . 7 . the plate was incubated at 37 ° c . in a co 2 incubator for 48 - 72 h . 8 . the medium was removed and the cells washed three times with ice cold pbs . 9 . the cells were lysed and the protein content of the lysates determined . 10 . the proteins were separated by sds - page followed by western blotting using a her2 / erbb2 polyclonal rabbit antibody from cell signalling technology ( cat .# 2242 ). 11 . the blots were treated with an anti - rabbit igg - hrp conjugate followed by ecl development . preliminary observations using 1 μg sirna / well are shown in fig3 a and 3 b . sirna - gold nanoparticles were added to cells without rnaifectamine . the skbr3 cells were slower to reach 80 % confluency than the ovcar cells . the skbr3 results are from lysates 48 h after transfection while the ovcar results were from lysates 72 h after transfection . a schematic of the nanoparticles are shown in fig4 . cells were transfected with sirna alone and with sirna conjugated to gold glyconanoparticles . fig5 shows that the nanoparticle - conjugated sirna was effective and had no toxic effects . a dose - dependent effect on cell number was seen indicating that the sirna nanoparticles increased cell proliferation . ovcar cells were transfected with sirna - nanoparticles with and without the transfection reagent usually required for transfection of cells with sirna . fig6 shows that the transfection reagent was not required for entry of sirna - nanoparticles into the cells . the results show that the sirna nanoparticles were effectively delivered into the cells even in the absence of transfection reagent ; indeed , delivery appeared to be more efficient without transfection reagent . the dose - dependency of the effect on cell number indicates a genuine response to the sirna - nanoparticles . pal - bhadra et al . rnai - mediated targeting of heterochromatin by the rits complex . science ( 2004 ) vol . 303 , 669 . verdel et al . heterochromatic silencing and hp1 localization in drosphila are dependent on the rnai machinery . science ( 2004 ) vol . 303 , 672 . elbashir et al . duplexes of 21 - nucleotide rnas mediate rna interference in cultured mammalian cells . nature 2001 ; 411 : 494 - 8 . bargmann et al . the neu oncogene encodes an epidermal growth factor receptor - related protein . 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