integrins are a type of cell membrane receptor protein composed of various  and  subunits , among which the v subunit is highly expressed on the surface of hepatic cell carcinoma ( hcc ) . the v subunit is critically important during the infiltration , metastasis , and neovascularization of tumor cells . it is reported that targeting of alpha - v integrins reduces malignancy of bladder carcinoma . currently , research on targeted therapies aiming at integrins include monoclonal antibodies against integrins , integrin antagonists , and targeted modulation of integrin genes , among which integrin antagonists are mainly express in the artificially synthesized polypeptides containing poly ( rgd ) structures , such as cilengitide [ 35 ] . gene therapy studies have reported on the antisense technologies and rna interfering technologies of oligonucleotides [ 611 ] . use of radionuclides in internal radiation therapies has been a research emphasis of tumor therapies , among which some radionuclides decayed through electron capture , like i , emitting many low - energetic auger electrons and coster - kroning ( ck ) electrons during its decay process . however , there is little published data on i marked antisense oligodeoxynucleotide ( asodn ) and small interfering rna ( sirna ) . sirna is easily cleared by the kidneys , it is degraded by enzymes , the cell uptake rate is low , and the markers on it are easy to drop , which limit its wide usage . thus , i was marked on asodn in this study . as a pei derivative , polyethylenimine - arg - gly - asp ( pei - rgd ) is a receptor - dependent nonviral vector . after pei undergoes complexation with nucleic acids and its analogues and rgd binds to the v subunit , pei - rgd together with nucleic acids and its analogues enter cells through pinocytosis . in this study , the v subunit of antisense oligodeoxynucleotide ( asodn ) was marked by radionuclide i. after complexation with pei - rgd , the pei - rgd / i-(v ) asodn compound was introduced into hepg2 cells via receptor - mediated transfected . we analyzed inhibitory effects of the compound on the growth and invasion of hepg2 cells and discussed the integration of antisense technology , rgd inhibitors , and nuclide internal irradiation in hcc treatment . the integrin v subunit asodn ( 5-gccctccttccacaatccca-3 ) sequence was examined using blast and was found to be nonhomologous with other human gene coding sequences . purification through polyacrylamide gel electrophoresis ( page ) and the generation of phosphorothioates at the 5 and 3 ends were performed by shanghai sangon biotech co. , ltd ( shanghai , china ) . pei - rgd was purchased from polyplus - transfection . the innocyte cell invasion assay kit ( qia129 ) approximately 20 g of ( v ) asodn was dissolved in 0.1 m ammonium acetate solution ( ph 5.0 ) and heated at 60c for 30 min . the mixture was then added successively to 10 l of 0.25 mm ki , 10 l of 5 mci nai , and 100 l of 0.03 m tlcl3 , mixed evenly and heated at 60c for 45 min . after cooling at room temperature , 5 l of 0.1 m na2so3 and 20 l of 1 m ammonium acetate solution were added successively ; then , the mixture was adjusted to ph 7.0 and heated at 60c for 60 min . using acetone : acetic ether ( 1:1 , v / v ) as a developer , the marking ratio was computed using paper chromatography . using 0.01 m hydroxyethyl piperazine ethanesulphonic acid ( hepes , ph 7.4 ) as an eluent the radiochemical purity of the purified reactants was separately measured at 24 , 48 , 72 , and 96 h , and the stability was observed in vitro . the hcc strain hepg2 was kindly provided by the model organism open laboratory at the college of life science and technology , tongji university . the strain was cultured in dulbecco s minimum essential medium ( dmem ) with 10% fetal bovine serum in an incubator with 95% humidity and 5% co2 at 37c . cells were grown to logarithmic phase , and drugs were delivered when 5060% of the cells adhered to the walls of the culture flask . the cells were transfected with pei - rgd ( l ) : i-(v ) asodn ( g , 2:1 ) . log - phase hepg2 cells were adjusted with dmem culture solution to 100 000 cells / ml and then inoculated into 96-well plates , with each mesh containing 500 l . we established 5 study groups ( 2/1 , 4/2 , 8/4 , 16/8 , and 32/16 l/g pei - rgd / i-[v ] asodn ) and used a i-(v ) asodn group and a i group as the controls . the cells were cultured in a co2 incubator for 48 h , and when 5060% of the cells adhered to the bottom of the plates , the drugs were delivered . the cell suspensions were collected into centrifuge tubes to measure the total counts per minute cpm(a0 ) . the dose - effect tests included 5 pei - rgd / i-(v)asodn groups : 2/1 , 4/2 , 8/4 , 16/8 , and 32/16 l/g . seven groups were used for the control tests : a pei - rgd / i-(v)asodn group ( 2 l/1 g ) , a i-(v)asodn group , a ( v)asodn group , a pei - rgd/(v)asodn group , a pei - rgd group , a i group , and a blank group . after transfection ( in the same manner described above ) , the cell inhibition rate was computed using the methyl thiazolyl tetrazolium ( mtt ) method . using the cytotoxicity test as a comparison , cell invasion was tested using the innocyte cell invasion assay kit ( qia129 ) . approximately 300400 l of serum - free culture solution was added to the upper chamber . after incubation for 3060 min , approximately 500 l of 10% foetal bovine serum medium was added to the lower chamber . then , 300350 l of the transfected cell suspension was added to the upper chamber and placed into a co2 incubator for 48 h. five hundred microliters of cell staining solution was added to the lower chamber . the cells that adhered to the bottom side of the upper chamber were knocked into the lower chamber and cultured for another 30 min . approximately 200 l of the cells in the staining solution was used for fluorescence detection , photographing , and calculation of the inhibition rates . all data were processed with spss , version 19.0 ( ibm corporation , armonk , ny , usa ) using analysis of variance ( anova ) and multi - sample paired - comparisons . the results are expressed as means  standard deviation . the integrin v subunit asodn ( 5-gccctccttccacaatccca-3 ) sequence was examined using blast and was found to be nonhomologous with other human gene coding sequences . purification through polyacrylamide gel electrophoresis ( page ) and the generation of phosphorothioates at the 5 and 3 ends were performed by shanghai sangon biotech co. , ltd ( shanghai , china ) . pei - rgd was purchased from polyplus - transfection . the innocyte cell invasion assay kit ( qia129 ) approximately 20 g of ( v ) asodn was dissolved in 0.1 m ammonium acetate solution ( ph 5.0 ) and heated at 60c for 30 min . the mixture was then added successively to 10 l of 0.25 mm ki , 10 l of 5 mci nai , and 100 l of 0.03 m tlcl3 , mixed evenly and heated at 60c for 45 min . after cooling at room temperature , 5 l of 0.1 m na2so3 and 20 l of 1 m ammonium acetate solution were added successively ; then , the mixture was adjusted to ph 7.0 and heated at 60c for 60 min . using acetone : acetic ether ( 1:1 , v / v ) as a developer , the marking ratio was computed using paper chromatography . using 0.01 m hydroxyethyl piperazine ethanesulphonic acid ( hepes , ph 7.4 ) as an eluent the radiochemical purity of the purified reactants was separately measured at 24 , 48 , 72 , and 96 h , and the stability was observed in vitro . the hcc strain hepg2 was kindly provided by the model organism open laboratory at the college of life science and technology , tongji university . the strain was cultured in dulbecco s minimum essential medium ( dmem ) with 10% fetal bovine serum in an incubator with 95% humidity and 5% co2 at 37c . cells were grown to logarithmic phase , and drugs were delivered when 5060% of the cells adhered to the walls of the culture flask . the cells were transfected with pei - rgd ( l ) : i-(v ) asodn ( g , 2:1 ) . log - phase hepg2 cells were adjusted with dmem culture solution to 100 000 cells / ml and then inoculated into 96-well plates , with each mesh containing 500 l . we established 5 study groups ( 2/1 , 4/2 , 8/4 , 16/8 , and 32/16 l/g pei - rgd / i-[v ] asodn ) and used a i-(v ) asodn group and a i group as the controls . the cells were cultured in a co2 incubator for 48 h , and when 5060% of the cells adhered to the bottom of the plates , the drugs were delivered . the cell suspensions were collected into centrifuge tubes to measure the total counts per minute cpm(a0 ) . the dose - effect tests included 5 pei - rgd / i-(v)asodn groups : 2/1 , 4/2 , 8/4 , 16/8 , and 32/16 l/g . seven groups were used for the control tests : a pei - rgd / i-(v)asodn group ( 2 l/1 g ) , a i-(v)asodn group , a ( v)asodn group , a pei - rgd/(v)asodn group , a pei - rgd group , a i group , and a blank group . after transfection ( in the same manner described above ) , the cell inhibition rate was computed using the methyl thiazolyl tetrazolium ( mtt ) method . using the cytotoxicity test as a comparison , cell invasion was tested using the innocyte cell invasion assay kit ( qia129 ) . approximately 300400 l of serum - free culture solution was added to the upper chamber . after incubation for 3060 min , approximately 500 l of 10% foetal bovine serum medium was added to the lower chamber . then , 300350 l of the transfected cell suspension was added to the upper chamber and placed into a co2 incubator for 48 h. five hundred microliters of cell staining solution was added to the lower chamber . the cells that adhered to the bottom side of the upper chamber were knocked into the lower chamber and cultured for another 30 min . approximately 200 l of the cells in the staining solution was used for fluorescence detection , photographing , and calculation of the inhibition rates . all data were processed with spss , version 19.0 ( ibm corporation , armonk , ny , usa ) using analysis of variance ( anova ) and multi - sample paired - comparisons . i-(v)asodn was purified and placed at 37c , and the radiochemical purity at 0 , 24 , 48 , 72 , and 96 h was 96.681.38% , 95.611.56% , 90.571.95% , 87.381.67% , and 77.791.86% , respectively . the radiochemical purity after 48 h was still greater than 90% , but it obviously decreased after 96 h. thus , we suggest that further experiments with this marker should be finished within 48 h after marking . the cell intake rates of the pei - rgd / i-(v)asodn groups were 10.921.20% ( 2 l/1 g ) , 12.770.85% ( 4 l/2 g ) , 7.781.37% ( 8 l/4 g ) , 7.111.76% ( 16 l/8 g ) , and 1.421.03% ( 32 l/16 g ) , 2.191.35% ( i-[v]asodn ) and 0.310.28% ( i ) . for the dose - effect tests , the inhibition rates of the pei - rgd / i-(v)asodn doses were 9.633.15% ( 2 l/1 g ) , 38.837.64% ( 4 l/2 g ) , 60.297.65% ( 8 l/4 g ) , 69.406.47% ( 16 l/8 g ) , and 92.644.41% ( 32 l/16 g ) ( figure 1 ) , with a correlation coefficient of r=0.879 . for the control tests , the inhibition rates are shown in table 1 and figure 2 . the invasion test fluorescence fields for all groups are shown in figure 3 , and the inhibition rates are listed in table 2 and figure 4 . the inhibition rate in the pei - rgd / i-(v ) asodn group was significantly greater than those in the other groups ( p<0.01 ) , indicating the greater inhibitory effect of pei - rgd / i-(v ) asodn on hepg2 cell invasion . i-(v)asodn was purified and placed at 37c , and the radiochemical purity at 0 , 24 , 48 , 72 , and 96 h was 96.681.38% , 95.611.56% , 90.571.95% , 87.381.67% , and 77.791.86% , respectively . the radiochemical purity after 48 h was still greater than 90% , but it obviously decreased after 96 h. thus , we suggest that further experiments with this marker should be finished within 48 h after marking . the cell intake rates of the pei - rgd / i-(v)asodn groups were 10.921.20% ( 2 l/1 g ) , 12.770.85% ( 4 l/2 g ) , 7.781.37% ( 8 l/4 g ) , 7.111.76% ( 16 l/8 g ) , and 1.421.03% ( 32 l/16 g ) , 2.191.35% ( i-[v]asodn ) and 0.310.28% ( i ) . for the dose - effect tests , the inhibition rates of the pei - rgd / i-(v)asodn doses were 9.633.15% ( 2 l/1 g ) , 38.837.64% ( 4 l/2 g ) , 60.297.65% ( 8 l/4 g ) , 69.406.47% ( 16 l/8 g ) , and 92.644.41% ( 32 l/16 g ) ( figure 1 ) , with a correlation coefficient of r=0.879 . for the control tests , the inhibition rates are shown in table 1 and figure 2 . the invasion test fluorescence fields for all groups are shown in figure 3 , and the inhibition rates are listed in table 2 and figure 4 . the inhibition rate in the pei - rgd / i-(v ) asodn group was significantly greater than those in the other groups ( p<0.01 ) , indicating the greater inhibitory effect of pei - rgd / i-(v ) asodn on hepg2 cell invasion . the integrin v subunit is highly expressed in several types of malignant tumour cells ( e.g. , hcc and melanoma ) [ 1416 ] , and it is involved in the recognition of ligands , mainly through the rgd in its homologous repeated sequence identification ligands . the integrin v subunit interacts with several ingredients in the extracellular matrix ( ecm ) , such as vitronectin , fibronectin , and fibrinogen . it is excited by extracellular growth hormone and/or ecm signals , and it alters the proliferation , differentiation , and migrating abilities of tumor cells by activating ras / map and other signal pathways . the integrin v subunit promotes the growth of tumor cells , inhibits their apoptosis , and controls their invasion and metastasis . reported a human monoclonal antibody 264rad targeting v6 integrin reduced tumor growth and metastasis , and modulated key biomarkers in vivo . existing research concerning the treatment of hcc by the integrin v subunit focuses on immunotherapy and gene therapy ; these methods have partially entered the stage of clinical drug trials . as previously reported , lm609   and vitaxin , the monoclonal antibodies of integrin v3 , both inhibited the invasion of tumors in vivo . in gene therapy , tumor - specific asodn or nuclear enzymes are primarily used to reduce or alter the expression configuration of the integrin v subunit and interfere with v - subunit - mediated adhesion , movement , and transfer . because most of the selected transfecting vectors are viruses or liposomes , problems will likely emerge , such as insecurity and poor specificity of target cells in vivo , as well as the use of time - consuming and complex steps ( e.g. , vector cloning , sequencing , and validation ) . thus , these vectors have not been shown to be favorable for use in vivo experiments and clinical applications . therefore , in the present study , we used receptor - mediated gene transfer technology ; namely , the asodn was introduced into cells through selective combination between the vector pei - rgd and hcc membrane surface . 1 ) pei is a cationic compound that can bind via ionic bonds with asodn ; it carries asodn and absorbs h in the lysosome , thus inactivating nucleic acid enzymes in the lysosome and protecting asodn from degradation . 2 ) the pei surface is occupied by many short rgd polypeptides , such as the integrin v subunit , that are natural ligands . these polypeptides specifically bind with v and enter target cells through endocytosis , thus realizing the targeted transfection . reported cyclic rgd - poly(ethylene glycol)-polyethylenimine was more suitable for glioblastoma targeting gene transfer in vivo . in the present study , the cell intake rate was maximized at 4 l/2 g pei - rgd / i-(v ) asodn , and it decreased with increased dosage , likely because the receptor v was saturated on the hepg2 cell surface or the compound was toxic to the cells . therefore , the results showed that , with the increased dosage , the compound inhibited hepg2 cells in a concentration - dependent manner . the latter results also correlated with the studied dosage range ( r=0.879 ) . because of the high cytotoxicity under high dosage and to reduce the effects during paired comparison , we selected the smallest dosage of 2 l/1 g for the control group . 1 ) the inhibition rates were not significantly different between the pei - rgd / i-(v)asodn group and the pei - rgd/(v ) asodn group ( p>0.05 ) , but both groups were differed significantly from the other groups ( p<0.001 ) . additionally , at a low dosage ( 2 l/1 g ) , the toxic effects of the pei - rgd / i-(v)asodn group and the pei - rgd/(v)asodn group were not significantly different , but they were higher than in the other groups , indicating that the radiation bioeffect of i was not obvious inside the cells . 2 ) the pei - rgd group , i-(v)asodn group , ( v)asodn group , and i group did not differ significantly from the control group ( p>0.05 ) , indicating that the 4 substances at low dosage were not cytotoxic . the likely causes for this result include the following : low - dosage pei - rgd was not cytotoxic ; without the operation of a vector , the asodn transfection rate was low , leading to a low targeting capability ; and the marked i and free i could not enter the nucleus and , thus , could not demonstrate radiation bioeffects against dna . to reduce the effects of compound cytotoxicity on hepg2 cell invasion , we selected the lowest dosage ( 2 l/1 g ) for the cell invasion tests . the results showed that the inhibition rate of the pei - rgd / i-(v)asodn group was significantly different from those of the other control groups , indicating that the inhibitory effect on hepg2 cell invasion was greater than those in the other groups . 1 ) the combination of pei - rgd with v on the surface of hepg2 cells reduced the probability of v binding with other natural ligands ( e.g. , other cells , ecm , and basal membranes ) , indicating the competitive inhibitory effect . 2 ) the combination of pei - rgd with v induced endocytosis into the cytoplasm , thereby degrading v and reducing the density of v on the hepg2 cell surface . 3 ) under the protection of pei - rgd , ( v)asodn entering cells avoided the degradation by nucleases and bound with v mrna , thereby interfering with the expression of v . the marked i produced auger electrons and ck electrons via disintegration , forming local high - energy deposition within 10 nm ( 10-bp dna chains ) around the disintegration locus , which would induce non - repairable injuries , such as double - strand breaks ( dsbs ) . compared with the pei - rgd/(v)asodn group , the cytotoxicity of the pei - rgd / i-(v ) asodn group was not significantly different , but the inhibitory effect on hepg2 cell invasion was significantly different , which indicated that although the ionizing radiation of i did not significantly inhibit the growth of hepg2 cells , it weakened the invasion ability of the hepg2 cells . moreover , the inhibition effects on cell invasion were significantly different between pei - rgd/(v)asodn and pei - rgd , but the differences were not very large , indicating that the antisense technology did not satisfactorily inhibit the invasion of the hepg2 cells . therefore , rna interference technology should be used in future works , which is characterized by a higher inhibitory effect , longer working time , and greater stability . this study showed that pei - rgd / i-(v)asodn can efficiently inhibit the growth and proliferation of hepg2 cells and can weaken their invasive ability . although the radiation bioeffect of i was not obvious inside the cells , it weakened the invasion ability of the hepg2 cells . we also found that the usefulness of antisense technology for inhibiting the invasion of hepg2 cells was limited .
backgroundto investigate the in vitro inhibitory effects of pei - rgd/125i-(v)asodn ( pei , polyethylenimine ; rgd , arg - gly - asp ; asodn , antisense oligodeoxynucleotide ) on the growth and invasion of hepg2 cells.material/methodsasodn of the integrin v - subunit was marked with 125i and underwent complexation with pei - rgd , a pei derivative . 
 next , pei - rgd/125i-(v ) asodn was introduced into hepg2 cells via receptor - mediated transfection , and its inhibition rate on hepg2 cell growth was tested using the methyl thiazolyl tetrazolium ( mtt ) method . 
 the effects of pei - rgd/125i-(v ) asodn on hepg2 cell invasion ability were evaluated using the boyden chamber assay.results1 ) the 125i marking rate of ( v ) asodn was 73.784.09% , and the radiochemical purity was 96.681.38% ( greater than 90% even after a 48-h incubation period at 37c ) , indicating high stability . 
 2 ) the cytotoxicity assays showed that the cell inhibition rates did not differ significantly between the pei - rgd/125i-(v)asodn group and the pei - rgd/(v ) asodn group , but they were both significantly higher than in the other groups and were positively correlated ( r=0.879 ) with the dosage within a certain range . 
 3 ) the invasion assays showed that the inhibition rate was significantly greater in the pei - rgd/125i-(v ) asodn group compared to the other groups.conclusionspei-rgd/125i-(v ) asodn can efficiently inhibit the growth and proliferation of hepg2 cells and can also weaken their invasive ability .