Patent Application: US-201013263632-A

Abstract:
the embodiment of the invention is a virus - like particle vector , a process for the manufacture thereof , use of the virus - like particle vector and a pharmaceutical composition , which contains the virus - like particle vector . the vector is intended for the delivery of therapeutic agents into specific mammalian tissues , especially low molecular weight agents , in particular low molecular weight anti - cancer drugs into cancer tissues . more specifically , the invention relates to the virus - like particle vector , which constitutes an adenoviral dodecahedron with the therapeutic substance encapsulated or covalently linked .

Description:
fig1 ( a , b ) shows a scheme of the adenovirus , penton and two dodecahedra ( dd ). fig2 shows dd stability analysed using dynamic light scattering ( dls ) technique . dd thermal stability depending on ph and ionic strength . ( a ) dd in 150 mm nacl was tested using dls technique at various ph values at temperature increments of 2 ° c . every 2 min between 12 and 65 ° c . ( b ) electrophoresis analysis of dd and pentameric bases ( pb ) in caps buffer ( ph 9 ) and in carbonate buffer ( ph 10 ). some samples were subjected to temperature changes simulating dls conditions ( marked with dls ). ( c ) dls analysis carried out using dd samples in pbs in various ionic strength conditions . average values from 3 apparatus readings are shown . fig3 shows dd stability during lyophilisation , inside hela cells and in human serum , as well as dd reconstitution from free penton base ( pb ) proteins . ( a ) purified dds were dialysed overnight at 4 ° c . against water or 150 mm aqueous ammonium sulphate . mannitol ( 0 . 4 %) and sucrose ( 0 . 4 %) were added to the samples marked “ cryoprotectant +”. dd samples were frozen at − 80 ° c . or dried with speed - vac or freeze - dried . the dried samples were reconstituted in the initial water volume . the samples were centrifuged for 30 min at 13000 rpm , and the condition of proteins in the supernatant was analysed using agarose gel electrophoresis . ( b ) dd stability in the hela cell culture . purified dd samples ( 2 μg / 100 μl ) were applied onto 2 × 10 4 portions of hela cells . after specified penetration times , cell lysates were isolated and were separated on polyacrylamide gel under denaturing conditions ( left - hand panel ) or on agarose gel under non - denaturing conditions ( two right - hand panels ). in both cases western - blot analysis was carried out using anti - dd antibodies . control dd samples contained 30 ng of protein , and the samples of pentameric bases ( pb ) contained 10 ng of protein . ( c ) dd stability in human serum . dd samples ( 5 μg aliquots ) concentrated by ultrafiltration in microcon ( millipore ) were incubated in human serum ( hs ) at a temperature of 4 ° c . for 2 hours ( lane 4 ) or at 37 ° c . for 15 min or 2 hours ( lanes 5 and 6 , respectively ). the samples were separated on agarose gel under non - denaturing conditions and , subsequently , analysed by western blot using a dd - recognising antibody . the upper part is a coomassie - stained gel with proteins remained after the transfer , and the bottom part is the developed western blot . lanes 1 and 7 correspond to dd samples without serum , non - treated or incubated for 2 hours at 37 ° c ., respectively . lanes 2 and 3 correspond to human serum incubated for 2 hours at 4 or 37 ° c ., respectively . ( d ) purified pbs were dialysed either at 4 ° c . against 50 mm phosphate buffer , ph 6 . 6 , containing 750 mm ammonium sulphate ( left - hand panel ), or at a temperature of 37 ° c . against 100 mm phosphate buffer , ph 7 . 5 ( right - hand panel ). the samples were centrifuged for 30 min at 13000 rpm , and the proteins in the supernatant were analysed using agarose gel electrophoresis under non - denaturing conditions . lane 1 contains the starting pb preparation used for dd reconstitution ; lane 2 corresponds to free pbs with 750 mm ammonium sulphate added before reconstruction ; lane 3 contains free pentameric bases after 2 - hour dialysis ( two dialysis buffer changes ). lanes 4 and 6 correspond to dd reconstructed during 4 - day dialysis at 4 and 37 ° c ., respectively . lanes 7 and 8 correspond to dd , and sample 8 contains 750 mm ammonium sulphate added before reconstruction . fig4 shows the cytotoxicity of bleomycin delivered by the dd . bleomycin has been chemically attached to the dd ( as discussed in example iv ). ( a ) analysis of the dd - blm conjugate using mass spectrometry . ( b ) analysis of the dd - blm conjugate using dynamic light scattering . ( c ) mtt cytotoxicity test . hela cells were treated with : free blm ( 0 . 13 μm ), dd ( 1 μg ) and dd - blm ( 1 μg , which delivers 0 . 08 μm blm ), according to example iv . fig5 shows the effect of dd - blm activity on hela cells . ( a ) cells treated with dd or dd - blm ( 1 μg sample ) were analysed under a confocal microscope using a dd - recognising antibody ( red signal , white / grey on black and white photographs ). cell nuclei were stained with dapi solution . the lowermost row shows cells after 50 - hour treatment , without nuclear staining . the scale bar corresponds to 20 μm . ( b ). cells treated with dd , free bleomycin or dd - blm were analysed after a specified time under a confocal microscope using antibodies : anti - γ - h2ax ( red signal in cell nuclei , grey on black and white photographs ) and anti - tubulin ( green signal in the cytoplasm , white / grey on black and white photographs ). the scale bar corresponds to 10 μm . the applicants initiated own research concerning the adenoviral dodecahedron . the research related to overexpression , purification and characterisation of dodecahedra and also their application for intracellular delivery of low - molecular weight drugs by chemical conjugation with a vector . the research included : preparation of high - quality preparations of dodecahedra purified to achieve a homogeneity . the resulting preparations are devoid of proteins , nucleic acids and proteases from cells , in which dd overexpression occurs . testing vector stability conditions . it was proved that factors such as ph , ionic strength and temperature , affect dd integrity . borderline conditions were developed for the storage , shipment and use of dd preparations in various conditions , suggesting possible use under tropical conditions . testing conditions of dd reconstitution from free penton base proteins . biophysical conditions were developed in order to obtain a dodecahedric vector in vitro from its free constituents , for the possible encapsulation of low - molecular weight therapeutic agents . construction of a dodecahedron ( dd ) conjugate with a low - molecular weight therapeutic agent , especially such as bleomycin ( blm ), by covalent linkage of the therapeutic agent with the dodecahedric vector . use of the dd - blm conjugate in tissue cultures and demonstration of remarkable improvement in conjugate bioavailability with respect to free bleomycin . it appeared that the virus - like particle vector developed according to the invention made it possible to achieve better penetration of hydrophilic anti - proliferation therapeutic agents , especially glycopeptides , such as anti - cancer antibiotics , in particular such as bleomycin , through cell membranes . the use of the dd for the delivery of therapeutic agents most likely means at the same time specific targeting of such agents to newly grown blood vessels , which supply nutrients to neoplastic tumours . it is known that the rgd motif interacts with αv integrins whose levels are elevated only in the endothelial cells , which constitute newly grown vessels , which supply blood to the cancer tissue ( chen , 2006 ). the motif is located in the penton base protein of , which the dd is composed ; therefore , the dd , which contains 60 rgd motifs is a highly specific ligand for αv integrins and , simultaneously , it has strong ability to penetrate cells owing to its endoosmolytic activity and affinity to heparin sulphates . use of dd as a vector for the delivery of bleomycin ( blm ) antibiotic , a low - molecular weight therapeutic agent the biological ( cytotoxic ) effect of a dd - blm preparation , which carried numerous antibiotic copies was tested on human cancer cells in in vitro cultures . it appeared that the chemical cross - linking reaction between the vector and blm did not reduce its cell penetration ability . furthermore , the antibiotic &# 39 ; s cytotoxic activity was retained . namely , dd - blm , when penetrating into human hela cells in in vitro cultures , degrades nuclear dna , similarly to free bleomycin . it was proved that the cytotoxically effective concentration of the antibiotic delivered with the dd was approx . 100 - fold lower than that used with free blm . more than 60 % human cancer cells ( hela ) in in vitro cultures were destroyed after the administration of the dd - blm conjugate , which was proved using the mtt cytotoxicity test ( fig4 c ). the cytotoxic effects were not observed either in the case of dodecahedron or free bleomycin administration in doses equivalent to those carried by the dd - blm conjugate . dd - blm efficiently penetrates through cell membranes using receptors recognised by either dd or blm . most likely , the vector undergoes gradual proteolysis in the cytoplasm of human hela cells , as a result of , which peptides are released with attached bleomycin , wherein the blm - peptides penetrate into the nucleus in , which the antibiotic , bleomycin in this case , is active . the cytotoxic blm activity is known to result from dna damage . phosphorylation of the c - terminal region of the h2ax histone in higher eukaryotic cells is one of chromatin modifications in response to double - strand dna breaks . a specific antibody , which recognises the phosphorylated h2ax histone form was used as the probe for detecting dna damage . dd - blm , when penetrating into human cancer cells in in vitro cultures , degrades nuclear dna , similarly to free bleomycin . in the process of the invention , dd , being a recombinant protein ( rdd ), is obtained with extremely high yield in insect cells in the baculovirus system . the overexpression is 10 mg of rdd per 100 ml of cell suspension . this overexpression yield is comparable to that achieved in the most efficient bacterial systems ( song et al ., 2008 ). rdds have been heretofore purified by saccharose gradient ultracentrifugation . the stage made it possible to eliminate low - and medium - molecular weight cell proteins , but failed to do so with nucleic acids , most likely attached to the rdd surface . due to the planned therapeutic use of the dd , it was needed to prepare a better purified and more homogeneous product , achieved owing to a 2 - stage protein purification process . after initial dd purification in saccharose gradient , ion - exchange chromatography was used . a pure rdd fraction was obtained ( more than 95 % purity ), confirmed in product analysis using electrophoresis technique in polyacrylamide gels and using electron microscopy . the biochemical and biophysical tests conducted ( electron microscopy , agarose gel electrophoresis in agarose gel in non - denaturing conditions and in polyacrylamide gels in denaturing conditions and measurements using dynamic light scattering ( dls )) proved the rdd to be stable up to 40 ° c . in a wide ph range and up to approx . 50 ° c . at a ph of 7 - 8 , at physiological nacl concentration ( 150 mm ). it was shown that high ionic strength conditions largely stabilise its structure , because the rdd is then not denatured up to a temperature of 60 ° c . ( fig2 ). the vector particle retains integrity during dialysis , after freezing and thawing , in speed - vac drying and during freeze - drying in the presence of a cryoprotectant ( fig3 a ). the high vector stability makes rdd handling and storage easier . furthermore , the rdd was found to retain integrity in conditions , which simulate its in vivo use ; namely , it was stable in human serum at a temperature of 37 ° c . for at least 2 hours ( fig3 b ). the results make it possible to use the rdd as a vector for various applications and in various environmental conditions . the analysis carried out using mass spectrometry techniques proved that in the dodecahedron conjugate with the anti - cancer antibiotic prepared by the applicant , one virus - like particle carries 60 drug molecules on average ( fig4 a ), which confirms the multivalency of the vector used . apart from attachment to the vector surface , increased bioavailability of low - molecular weight compounds may be achieved by their encapsulation inside the virus - like particle . the applicants developed conditions in , which dodecahedra associated from their constituents , being pentameric bases ( fig3 d ). owing to dodecahedron in vitro reconstruction in the presence of low - molecular weight compounds , it is possible to obtain a virus - like particle , which contains an encapsulated therapeutic substance . according to the applicant , the properties of the dd discussed above imply the potential of the nanoparticle to be used as a vector for the delivery of therapeutic agents to human tissues . the first example concerns bleomycin , an anti - cancer antibiotic . the applicant found that bioavailability of the antibiotic increased owing to the use of the dd as the vector ; this should enable the use of reduced doses and , in consequence , reduce adverse effects of its activity . after the stage of tests carried out in tissue cultures , studies in the mouse cancer model will be conducted . if the dd - blm preparation used in the model system , such as mice with implanted human brain tumour , proves at least as efficacious as blm delivery by electrochemotherapy used previously , this will make it possible to suggest using the dd - blm conjugate in human anti - cancer treatment . therefore , the use of bleomycin in anti - cancer treatment could be limited to the administration of a dd - blm preparation without any need to use electric shock , which frequently requires complete anaesthesia . due to the planned therapeutic use of the rdd , it was needed to prepare a better purified and more homogeneous product , achieved owing to the addition of the second protein purification stage to the previous protocol ; after initial rdd purification in sucrose gradient , low - pressure ion - exchange chromatography was used , which yielded a pure rdd fraction . for dd expression , a recombinant baculovirus , which comprised the penton base protein gene of the human serotype 3 adenovirus ( ad3 ) was used ( fender et al ., 1997 ). the amplification of the recombinant baculovirus carrying the base protein gene was carried out in a monolayer cell culture of spodoptera frugiperda ( sf21 ). the cells were cultured in tc100 medium containing 5 % foetal bovine serum ( invitrogen ). the recombinant dd was overexpressed in trichoplusia ni cells ( also known as high five , hf ), cultured in suspension in the express five sfm ( invitrogen ) medium in the presence of gentamycin ( 50 mg / l ) and amphotericin b ( 0 . 25 mg / l ). trichoplusia ni cells were infected with the recombinant baculovirus at the moi ( multiplicity of infection ) of 4 infectious units per one cell . 48 hours after the infection , the cells were harvested and lysed by freezing and thawing three times . the supernatant obtained after lysate clarification was centrifuged in 15 - 40 % sucrose gradient ( fender at al ., 1997 ). the vlp product , recovered in 30 - 40 % sucrose , was contaminated with cellular proteins and nucleic acids . final dd purification was achieved by chromatography on an ion - exchange column as a result of , which dodecahedra were prepared as a homogeneous fraction . the oligomeric status of the particles and purity level of the resulting product were analysed in native agarose gels , using electron microscopy and in denaturing polyacrylamide gels . the stability and solubility of purified dd particles was tested . to this end , the purified rdds were dialysed against various buffers ( with 3 changes of each ) and , subsequently , incubated at 30 or 37 ° c . after incubation , the samples were centrifuged and proteins in the supernatant were analysed using agarose gel electrophoresis . the dd remains dissolved at 4 ° c . and ph of 4 . 0 to 10 . 9 , in the presence of 150 mm nacl . without nacl , the dd does not remain in solution and it disappears from the supernatant during centrifugation . therefore , nacl in physiological concentration protects dds against denaturation . in order to test dd resistance to thermal denaturation , dynamic light scattering ( dls ) technique was used owing to , which protein denaturation or aggregation can be monitored . protein samples ( 0 . 2 mg / ml ) were dialysed against suitable buffers and filtered though filters with 0 . 45 μm pore size in order to remove any dust particles . the samples were placed in a cuvette ( 45 μl , greiner , frickenhausen , germany ) and automated particle size measurements were carried out using zs nano zetasizer apparatus ( malvern , worcestershire , gb ). the temperature gradient was 2 ° c . every 2 min between 12 and 65 ° c . the data were evaluated using a cumulative method . in a ph of 4 to 9 , the size of dodecahedric particles was constant up to 40 ° c . ( fig2 a ). above this temperature , particle sizes increased exponentially with increasing temperatures , which indicated denaturation and aggregation . dd denaturation / aggregation starts at a ph of 4 - 5 at a temperature lower by about 10 ° c . than at ph 7 - 8 . at ph 9 ( caps buffer ) and 10 ( carbonate buffer ), small particle size changes occur . protein analysis in native agarose gels proved at a ph of 10 that the dd dissociates into free pentameric bases and at ph 9 ( caps buffer ) the protein disappears , most likely due to aggregation ( fig2 b ). it is noted that caps is an organic buffer , which may cause aggregation by interacting with surface hydrophobic fragments . the addition of 750 mm nacl to pbs leads to increased dd melting temperature ( t m ), which indicates structural stabilisation ( fig2 c ). however , the most significant t m value increase was due to the addition of ammonium sulphate and led to a positive shift of about 12 ° c . ( fig2 c ). the tests completed proved that the vector particle retains integrity during dialysis , after freezing and thawing and in speed - vac drying in the presence of 150 mm ammonium sulphate . a cryoprotectant is required during lyophilisation in order to preserve dd structure ( fig3 a ). vector life in cell cultures was tested in hela cells at various time points after dd addition . purified dd ( 4 μg / 100 μl , 10 . 8 nm ) was applied to hela cells in 24 - well plates ( 2 × 10 4 cells / well ), in the fbs - free medium . the cells were incubated in an incubator at 37 ° c . three hours after dd addition , fbs was added to the medium to a final concentration of 10 %. the cells were harvested at specified time points ( fig3 b ) and lysed in the hypotonic buffer . samples corresponding to half the cells were analysed in polyacrylamide gel in denaturing conditions ( sds - page ) and the other half was analysed in agarose gel in non - denaturing conditions and , subsequently , analysed in both cases by western blot using anti - dd antibodies . the quantity of intracellular dds increased up to 32 hours following transduction . simultaneously , partial dd proteolysis occurred , due to , which only part of the base protein remained in the cells after 4 days ( fig3 b , left - hand panel ). analysis in native agarose gel proved that 96 hours after penetration most of the intracellular vector migrated between dd and pentameric bases ( pb ), which indicated removal of external dd loops with retained molecule integrity ( fig3 b , right - hand panel ). dd samples ( 5 μg liquots ) concentrated by ultrafiltration in microcon ( millipore ), were incubated in human serum ( sl ) at a temperature of 4 ° c . for 2 hours and at 37 ° c . for 15 min or 2 hours . the dd retains integrity in conditions , which simulate its potential in vivo use ; namely , it is stable in freshly prepared human serum at a temperature of 37 ° c . for at least two hours ( fig3 c ). a homogeneous fraction of free pentameric bases ( pb ) was obtained during purification on an ion - exchange column . the purified rpbs were dialysed against 50 mm ph 6 . 6 or ph 7 . 5 phosphate buffers containing 750 mm ammonium sulphate with several buffer changes . after the end of dialysis , the samples were centrifuged and the oligomeric status of proteins in the supernatant was analysed using agarose gel electrophoresis . in high ionic strength conditions during 4 - day dialysis at a temperature of 4 ° c . or 37 ° c ., association of dodecahedra from pentameric bases occurs . owing to dodecahedron in vitro reconstruction from their constituent parts in the presence of low - molecular weight compounds , it is possible to obtain a vector , which contains a therapeutic substance encapsulated in the virus - like particle . the results provided indicate that the dd can be conveniently stored and transported and reconstructed in vitro from its constituents ; this proves that it can be used for various therapeutic purposes , in various configurations and in various environmental conditions . a process for the preparation of the vadenoviral dodecahedron particle with bleomycin bleomycin a 5 hydrochloride ( hangzhou xiangyuan co ., ltd ., china ) was chemically attached to previously purified rdd particles during a two - stage conjugation procedure using carbodiimide ( edc ) and succinic acid ester ( s - nhs ) ( pierce , rockford ill ., usa ). dodecahedra at a concentration of 27 nm were activated in the 0 . 1 m ph 6 . 0 mes buffer containing 0 . 5 m nacl , in the presence of 0 . 31 mm edc and 5 mm s - nhs . conjugation with bleomycin ( 23 mm ) was carried out for two hours at room temperature upon gentle stirring . the reaction was terminated by adding hydroxylamine to a final concentration of 10 mm . the reagents used and unbound bleomycin were eliminated during 24 - hour dialysis with four changes of 20 mm ph 7 . 5 tris buffer containing 150 mm nacl and 5 % glycerol . bleomycin quantity attached to the dd was determined using mass spectrometry technique . the analysis was carried out using a perseptive biosystems mass spectrometer ( framingham , mass . ), by way of a pulse nitrogen laser at a wavelength of 337 nm . the samples were concentrated in ziptipc4 ( millipore ) and extracted with saturated sinapinic acid solution prepared in 80 % mixture of aqueous acetonitrile ( vol ./ vol .) comprising 0 . 3 % trifluoroacetic acid according to the manufacturer &# 39 ; s instructions . the eluent mixture was transferred onto a steel plate and dried on air . the apparatus was calibrated using bovine albumin ( biosystems ) with a molecular weight of 66431 da . in the dd - blm conjugate , the penton base protein monomer ( of , which dd comprises ) carries between 0 and two blm particles ( the blm molecular weight is 1400 ) with significant majority of monomers containing one blm molecule ( fig4 a ). the data indicate that one dd molecule , which contains 60 base protein monomers carries 60 blm residues on average . tests using dynamic light scattering technique ( dls ) proved that the melting temperature of the dd - blm conjugate is very similar to that of the initial dodecahedron , which indicates that the cross - linking reaction does not change the biophysical properties of the vector . the results of the studies underscore vector polyvalency , wherein one dd particle is able to provide multiple copies of the therapeutic substance . hela human cancer cells were treated with the dd - blm conjugate prepared according to the invention . similarly to free bleomycin , the dd - blm conjugate led to the inhibition of cancer cell proliferation . what was the most important , the cytotoxically effective blm concentration delivered with the dd was 100 times as low as in the case of free bleomycin . the cytotoxic dd - blm activity was quantitatively evaluated in vitro using the mtt test ( mtt , 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide ). in the test , the ability of live cells to reduce the soluble yellow tetrazolium salt ( mtt ) to blue formazan crystals is used . hela cells cultured in 96 - well plates ( 10 4 cells / well ) were incubated for 3 hours at 37 ° c . in 100 μl of the emem medium containing a ) various quantities of dd ( 1 μg corresponds to 2 . 7 nm ), b ) dd - blm ( 1 μg corresponds to 2 . 7 nm dd and 0 . 08 μm blm ) or c ) free bleomycin ( 0 . 13 , 1 and 8 μm , respectively ). after 3 hours , foetal bovine serum was added to a final concentration of 10 %. after various incubation times at 37 ° c ., the incubation medium was removed and 100 μl emem containing 0 . 5 mg / ml mtt ( sigma ) was added . the plates were incubated according to the manufacturer &# 39 ; s instructions ; optical density measurements were carried out using an hti reader ( biotek , vt winooski , usa ). the number of live cells was calculated according to the protocol ( mosmann , 1983 ). more than 60 % of human cells in in vitro cultures are destroyed after dd - blm treatment . the cytotoxic effect was not observed when free bleomycin was applied in doses equivalent to the antibiotic quantity contained in the preparation used . the dd - blm preparations containing about 0 . 08 μm blm were proved to be highly cytotoxic , whereas free blm added in the same quantity had no cytotoxic effect ( fig4 ). similar cell mortality was observed only when 8 μm of free blm solution ( results not shown ) was added , that is , 100 times more than in the case of bleomycin delivered by dds . the subsequent stage included microscopic examination of human cancer cells subjected to dd - blm treatment . in order to prepare preparations for the confocal microscope , hela cells ( 5 × 10 4 ) were plated onto special coverslips . on the next day , various amounts of pure dd , dd - blm conjugate or free bleomycin were applied onto the cells ; all samples were suspended in the serum - free emem medium . after 3 - hour incubation , foetal bovine serum was added to a final concentration of 10 %. after the end of incubation , the cells were washed with cold pbs and subsequently fixed and permeabilised for 10 min in 100 % cold methyl alcohol . preparations obtained in this way were incubated for 1 hour with antibodies ( ab ): polyclonal dd - recognising ab , monoclonal tubulin - recognising ab ( sigma , st louis mo ., usa ) and polyclonal anti - γ - h2ax ab ( calbiochem , darmstadt , germany ). after washing the cells using pbs , secondary antibodies were applied , conjugated with dyes : texas red ( jackson , immunoresearch laboratories , west grove pa ., usa ) or green , fitc ( santa cruz biotechnology , santa cruz calif ., usa ). dapi solution was used for the labelling of cell nuclei ( applichem ). because no bleomycin - recognising antibody is available , which could be used in confocal microscopy , anti - dd abs were used for the detection of the dd - blm conjugate . the dd and also dd preparation with covalently bound blm were found to penetrate into 100 % cells in in vitro cultures , which is proved by the red signal from the anti - dd antibody in the cytoplasm of cells observed 1 hour after the application of the preparations ( fig5 a , 1 hour , dd and dd - blm ). fifty hours after the application of the free dd , the vector quantity ( visible in the cytoplasm only ) decreased significantly in comparison with shorter incubation times ( red signal , fig5 a ), which indicates proteolysis and elimination of the vector from cells . the dd - blm conjugate induces the occurrence of enlarged cells , which is visible 30 hours after conjugate application , being even more pronounced at a later time . fifty hours after dd - blm application , the dd signal is present throughout the cell , which indicates that nuclear membrane integrity has been destroyed ( one of cell death symptoms ). the cytotoxic , blm activity is known to result from dna damage ( mir et al ., 1996 ). phosphorylation of the c - terminal region of the h2ax histone in higher eukaryotic cells is one of chromatin modifications in response to double - strand dna breaks ( kinner et al ., 2008 ). a specific antibody , which recognises the phosphorylated histone form ( anti - γ - h2ax ; calbiochem , darmstadt , germany ) was used as the probe for detecting dna damage . in control hela cells and in cells treated with a pure dodecahedron , no dna damage was found , indicated by the lack of the red signal from the anti - γ - h2ax antibody ( fig5 b , rows hela and dd ). conversely , the dd - blm conjugate , when penetrating into cells , degrades nuclear dna , which is indicated by the presence of the signal from the specific antibody . application of free bleomycin has a similar effect ( fig5 b , row : blm ). the effect of the action of 0 . 08 μm blm - containing conjugate is stronger than damage induced by free blm at a concentration of 8 μm and , therefore 100 times as high ( fig5 b , rows : dd - blm and blm ). carter b . j ., de vroom e ., long e . c ., van der marel g . a ., van boom j . h ., hecht s . m . 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