rational utilization 
 of the power of cell - penetrating peptides 
 ( cpps ) has been pursued for decades since hiv s transactivator 
 of transcription ( tat ) protein joined the fascinating family as the 
 first unveiled cpp model . cpps , also known 
 as protein transduction domains ( ptds ) or membrane transduction peptides 
 ( mtps ) , generally consist of 30 or less amino acids and are categorized 
 as amphipathic or cationic depending on their sequences . receptor - independent 
 internalization and noninvasive entry into cells are the two major 
 advantages that draw increasing attention to the field of cpp - based 
 intracellular delivery systems . a variety of cargos , including bioactive 
 molecules , dna / sirna , quantum dots , and nanoparticles , have been delivered 
 using different types of cpps in vitro and in vivo . additionally , some studies have shown that cpps 
 could also promote gastrointestinal absorption of protein drugs as well as direct brain delivery via intranasal administration . however , lack of specificity is the major 
 challenge for cpps to 
 be employed clinically . many approaches have been investigated 
 to confer cpps with the ability of targeting delivery , but no break - through 
 technology has yet been achieved . the nonspecific in vivo biodistribution of cpps is mostly caused by the cationic characteristics 
 of the oligopeptide . therefore , one of 
 the most promising approaches is to reversibly mask the positive charges 
 in a cpp with a polyanionic counterpart . the selective activation 
 of oligoanion - masked cpp can be achieved by specific proteolysis , light activation or differences in the microenvironment at target site . recently , we have designed a recombinant co - oligopeptide 
 containing model amphipathic peptide ( map , klalklalkalkaalkla ) as 
 the cpp sequence , and 10-mer of histidine - glutamic acid repeats map is an amphipathic 
 peptide that shows high cellular uptake and exhibits an -helical 
 structure . , 
 was highly ph - sensitive and could be activated under mildly acidic 
 ph conditions . in cultured hela cells , it exhibited a low surface 
 binding and cellular internalization at ph 7.4 but high surface binding 
 and cellular internalization at ph 6.8 or below . furthermore , the construct showed high accumulation and 
 retention for up to 24 h near the tumor site in a xenograft breast 
 cancer mouse model . in addition to solid 
 tumor tissues , endosomal / lysosomal compartments as well as the infectious / inflammatory sites are also implicated as potential drug delivery 
 targets with acidic bioenvironments . by conjugating with a ligand , 
 he - cpp under an inactive form at physiological ph could be internalized 
 into target cells via a receptor - mediated endocytic pathway , thereby 
 being activated in the endosomal or lysosomal compartments . similar to extracellular ph conditions in tumor 
 tissues , the acidic microenvironments at the sites of infection or 
 inflammation could weaken the masking effect of he repeats , leading 
 to restoration of the membrane - permeability of the cpp . in this 
 study , the systematic design of anionic oligopeptides for 
 neutralizing the cationic charges in oligoarginine cpps is investigated . oligoarginine exhibits many differences from amphipathic cpps like 
 map , such as different intracellular localization , and lacks a secondary structure . therefore , we wanted to determine if the same ph - sensitive masking 
 sequence used on an amphipathic cpp could also be applied to cationic 
 cpps . the efficiency of masking and reactivation of cpps may be influenced 
 by many factors , such as the number of the positively charged amino 
 acids in cpp , the polyanionic oligopeptide sequences , linker cleavability , 
 and the location of the cpp and the masking sequences . in addition , 
 the cationic charge distribution of the cpp , either as clustered or 
 evenly mixed sequence , may also affect the neutralizing efficiency . using he oligopeptide with various lengths , 
 the masking effect on oligoarginine at a ph range between 6.0 and 
 7.5 was evaluated for the design of activatable cpps with either clustered 
 or mixed positive charges in the oligopeptides . the 
 pgex-4t-1 vectors ( ge healthcare life sciences , piscataway , nj ) were 
 utilized in this study to clone all plasmids . similar to our previous 
 design , the fusion protein contains glutathione 
 s - transferase ( gst ) as a protein cargo fused to an he oligopeptide 
 sequence ( ( he)n , n = 
 8 , 10 , or 12 ) , a short pentaglycine linker ( g5 ) , and a 
 arginine - rich cpp ( yg(rg)6 , ygr6g6 , or tat peptide ( ygrkkrrqrrr ) ) . in order to allow for further characterization 
 of the he - cpp peptide sequences , a tyrosine residue was incorporated 
 in the cpp sequence for quantitation , while a thrombin cleavage recognition 
 sequence ( leu - val - pro - arg - gly - ser , lvprgs ) was 
 reserved between gst and he oligopeptide . the ssdnas of ( he)n ( n = 8 , 10 , and 12 ) , g5-yg(rg)6 and g5-ygr6g6 were synthesized by valuegene inc . after annealing 
 ssdnas into dsdna , the dsdnas coding for ( he)n ( n = 8 , 10 , or 12 ) and g5-yg(rg)6 or g5-ygr6g6 were successively 
 introduced into the pgex-4t-1 vector downstream from the amino - terminal 
 of gst sequence through bamh i , ecor i , and not i restriction enzyme 
 cleavage sites . due to the two - step clones and the ecor i restriction 
 enzyme cleavage site , an extra amino acid residue , phenylalanine ( phe , 
 f ) , was present between the he sequence and g5 linker . after ligation and transformation , colony pcr was performed , and then 
 plasmids were duplicated overnight in escherichia coli ( e. coli ) dh5 competent cells 
 in lysogeny broth ( lb ) media containing 75 g / ml ampicillin 
 and 20 mm l - glucose at 37 c . after sequencing ( genewiz , 
 san diego , ca ) , the plasmids were transformed into e. coli expression strain bl21 . the recombinant protein 
 was expressed and purified using glutathione ( gsh ) agarose beads as 
 the procedure described in our published report . samples collected during purification were analyzed by 
 sds - page followed by coomassie blue staining . the 
 purity was > 90% for all preparations , as determined by band density 
 comparison in sds - page with coomassie blue staining . isoelectric point ( pi ) , calculated 
 using an online software program the purified 
 recombinant gst - fusion 
 proteins were radiolabeled with na  i ( perkinelmer 
 health sciences , chicago , il ) using the chloramine t method as previously 
 described.i - protein was 
 purified by size exclusion chromatography using a gravity - flow column 
 packed with sephadex g50 beads ( ge healthcare life sciences ) . reaction 
 solution was loaded on the column prebalanced with pbs , and liquid 
 flow - through was collected in fractions and monitored by measuring 
 the radioactivity . finally , fraction(s ) containing i - proteins 
 were stored at 20 c until use . human cervical 
 carcinoma ( hela ) cells were originally maintained at 37 c in 
 a 95% humidified atmosphere of 5% co2 in rpmi 1640 medium 
 ( mediatech , manassas , va ) supplemented with 10% fetal bovine serum 
 ( fbs ) , 2 mm l - glutamine , 50 u / ml penicillin and 50 g / ml 
 streptomycin . two or three days before cellular uptake and surface 
 binding assay , appropriate number of hela cells was seeded into 6-well 
 plates . after being grown to confluence , cell monolayers were treated 
 with serum - free or serum - containing ( 110% fbs ) medium for 
 10 min followed by incubation in serum - free or serum - containing ( 110% 
 fbs ) medium at ph 6.0 , 6.5 , 7.0 , or 7.5 containing 5 g / ml i - labeled protein and protease inhibitor cocktail . after 1 h of treatment at 37 c , the cell 
 monolayers were washed three times with 1 ml of ice - cold pbs and subsequently 
 treated with trypsin - edta at 37 c for 3 min to detach the cells 
 as well as remove the surface - bound ( noninternalized ) protein from 
 the cell membrane . cells were collected and centrifuged ( pr - j centrifuge , 
 international equipment company , nashville , tn ) at 1500 rpm for 3 
 min to separate intracellular ( cell pellet ) from surface bound ( supernatant ) 
 fractions . the cell pellet was washed twice with ice - cold pbs and 
 then dissolved in 1 ml of 1 n naoh . the i - proteins were 
 detected using a -counter ( packard , downers grove , il ) , and 
 total cell protein was determined by performing the micro - bca assay 
 with a pierce micro - bca assay kit ( thermo fisher scientific , rockford , 
 il ) . in order to study the structure changes of the he - fused cpp peptides 
 under various ph conditions without the interference by gst protein 
 domain , ( he)10-yg(rg)6 , ( he)10-ygr6g6 , and ( he)10-tat were isolated from 
 the respective gst - fusion proteins by on column thrombin cleavage 
 during gst agarose chromatography . after cleavage , the gst - free peptides 
 were purified using hispur ni - nta resins and dialyzed against 10 mm 
 phosphate buffer followed by tricine - sds - page analysis and sterilization 
 by filtration ( 0.22 m micropore membrane ) . the peptide concentrations 
 were determined by their absorption at 280 nm ( uv 160u spectrophotometer , 
 shimadzu , japan ) . for cd analysis , peptides were diluted to 25 mm 
 in pbs at different ph ranging from 6.0 to 7.5 . a j-815 cd spectrometer 
 equipped with a ptc-423s/15 peltier thermostatic cell holder ( jasco , 
 easton , md ) was utilized to measure the spectra . all spectra were recorded from 250 to 160 nm in a 10 mm 
 quartz cuvette at 20 c using a 0.5 nm data interval and a scanning 
 speed of 100 nm / min . the cd spectra were plotted as an average ellipticity 
 ( mdeg ) versus wavelength  ( nm ) . the student s t test was exploited to perform comparison between data 
 sets , where statistically significant differences were assigned for p values of < 0.05 . plasmids 
 encoding gst-(he)n - cpp were 
 successfully constructed in pgex-4t-1 vector ( figure 1a ) according to the positive sequencing results . recombinant 
 gst - fusion proteins were expressed in e. coli bl-21 and purified by gsh affinity chromatography . expression and 
 purification was confirmed by coomassie blue - stained sds - page , where 
 gel of purified gst-(he)n - yg(rg)6 ( figure 1b ) and gst-(he)n - ygr6g6 ( figure 1c ) , n = 8 , 10 and 12 , showed gradient positions 
 with varied he repeats and approximately 90% purity based on the band 
 densities . the yield of each protein obtained from 500 ml lb culture 
 ( table 1 ) indicated that prolonging he - repeats 
 in the oligopeptide sequence decreased expression yield . ( a ) plasmid 
 map of recombinant gst - fusion proteins , where n is equal to 8 , 10 , 
 or 12 and yg(rg)6 or ygr6g6 is used 
 as the cpp . purified gst-(he)n - yg(rg)6 ( b ) and gst-(he)n - ygr6g6 ( c ) were analyzed by sds - page followed by coomassie 
 blue staining . for ( b ) , lane 1 : mw marker ; lane 2 : gst-(he)8-yg(rg)6 ; lane 3 : gst-(he)10-yg(rg)6 ; lane 4 : gst-(he)12-yg(rg)6 . for ( c ) , lane 
 1 : mw marker ; lane 2 : gst-(he)12-ygr6g6 ; lane 3 : gst-(he)10-ygr6g6 ; lane 
 4 : gst-(he)8-ygr6g6 . to investigate 
 the impact of the length of he repeats on the ph sensitivity of cpp - incorporated 
 recombinant gst - fusion proteins , cellular uptake and surface binding 
 assay confluent hela cell monolayers were grown in 
 6-well plates and treated with i - radiolabeled proteins 
 in serum - free rpmi 1640 media , at various ph ( 6.0 , 6.5 , 7.0 , and 7.5 ) 
 for 1 h at 37 c . the data ( figure 2 ) 
 demonstrated that incorporation of he oligopeptide significantly affected 
 cell uptake and surface binding in a ph - dependent manner . the influence 
 of he oligopeptide on cell uptake and surface binding 
 in hela cells at diverse ph . hela cell monolayers were treated with 
 5 g / ml i - gst-(he)n - cpp for 1 h at 37 c at ph 6.0 , 6.5 , 7.0 , and 7.5 , where n is equal to 8 , 10 , or 12 and cpp is yg(rg)6 or ygr6g6 . cell uptake and surface binding 
 were determined as described in the experimental 
 section . all data were normalized by total cell protein , that 
 is , nanograms per milligram of cell protein , and presented as averages 
  standard deviation from three independent measurements per 
 condition . cell uptake ( a ) and surface binding ( b ) of i - gst-(he)8-yg(rg)6 and i - gst-(he)8-ygr6g6 ; cell uptake ( c ) and surface 
 binding ( d ) of i - gst-(he)10-yg(rg)6 and i - gst-(he)10-ygr6g6 ; cell uptake ( e ) and surface binding ( f ) of i - gst-(he)12-yg(rg)6 and i - gst-(he)12-ygr6g6 . asterisk represents statistically 
 significant differences in cellular uptake or surface binding at diverse 
 ph between cpps in the same group ( * , p < 0.05 ; 
 * * , p < 0.01 ; * * * , p < 0.001 ) . as seen in the cell uptake figures 
 ( figures 2a , c , and e ) , the internalization 
 of the fusion proteins increased 
 with decreasing ph . the fusion protein with mixed arginine configuration , 
 gst-(he)n - yg(rg)6 , showed a 
 5- , 4- , and 3-fold increase for n = 8 , 10 and 12 , 
 respectively , at ph 7.5 vs 6 . with a clustered arginine configuration , gst-(he)n - ygr6g6 was more sensitive to ph changes , where a 5- , 6- , and 4 fold increase 
 for n = 8 , 10 , and 12 , respectively , was observed 
 at ph 7.5 vs 6 . the different lengths of ( he ) repeats also affected 
 the ph - dependent uptake patterns . the fusion proteins with 8 and 10 
 ( he ) repeats showed higher uptake than the 12-(he ) repeat peptide 
 across the entire ph range for both gst-(he)n - yg(rg)6 and gst-(he)n - ygr6g6 . furthermore , the 12-(he ) repeat peptides 
 showed less of a difference in the clustered vs mixed configuration 
 than the 8- and 10-(he ) repeats . the surface binding of the fusion 
 proteins showed a weaker ph - dependency than the cell uptake , where 
 a 2-fold difference was noted at ph 7.5 vs 6 for all fusion proteins 
 tested ( figures 2b , d , and f ) . as a control , 
 the cell uptake and surface binding of gst-(he)10 was also 
 measured . the results shown in figure 3 indicate 
 that both binding and uptake of gst-(he)10 was not dependent 
 on ph , and was low across the ph range tested . comparison of cell uptake 
 ( a ) and surface binding ( b ) between gst-(he)10-ygr6g6 ( open triangle ) and gst-(he)10 ( black / white 
 circle ) in hela cells at diverse ph . hela cell 
 monolayers were treated with 5 g / ml i - gst-(he)10-ygr6g6 or i - gst-(he)10 for 1 h at 37 c at ph 6.0 , 6.5 , 7.0 , and 7.5 . all data were normalized by 
 total cell protein and are presented as averages  standard deviation 
 from three independent measurements per condition . cell - penetrating 
 tat[4757 ] peptide is one of the most extensively studied cpps . the cell internalization and binding of gst-(he)12-tat 
 was compared to the blocked oligoarginine construct at a 1:1.5 
 ratio of cationic amino acids in the cpp sequence ( r , k ) to number 
 of ( he ) repeats ( i.e. , gst-(he)12-tat vs gst-(he)10-ygr6g6 ) . in the cell experiments ( figure 4 ) , gst-(he)12-tat exhibited a stronger 
 ph sensitivity in both cell uptake ( 4.4-fold increase at ph 6.0 versus 
 ph 7.5 ) and surface binding ( 2.0-fold increase at ph 6.0 versus ph 
 7.5 ) . therefore , the ph sensitivity was comparable to the arginine 
 in a clustered configuration , which is consistent with the tat peptide 
 sequence . comparison of cell uptake ( a ) and surface binding ( b ) between gst-(he)10-ygr6g6 ( open circle ) and gst-(he)12-tat ( closed diamond ) in hela cells at diverse ph . upon confluence , 
 hela cell monolayers were incubated with 5 g / ml i - gst-(he)10-ygr6g6 and i - gst-(he)12-tat for 1h at 37 c at ph 6.0 , 6.5 , 
 7.0 , and 7.5 . all data were 
 normalized by total cell protein , and presented as averages  
 standard deviation from three independent measurements per condition . to mimic physiological conditions , fbs was added in the media at 
 different concentrations from 110% , and the cell uptake and 
 surface binding were assayed at ph 6.0 and 7.5 . as expected , the surface 
 binding and uptake of both gst-(he)10-ygr6g6 and gst-(he)10-yg(rg)6 decreased with 
 increasing concentrations of fbs ( figures 5 and 6 ) . however , the ratio of either the 
 surface binding or uptake at ph 6.0 versus 7.5 was similar in the 
 presence of 110% fbs ( table 2 ) . therefore , 
 serum did not affect the ph sensitivity of the constructs . the effect 
 of serum on cell uptake ( a ) and surface biding ( b ) of 
 gst-(he)10-ygr6g6 in hela cells at 
 ph 7.5 ( open bars ) and 6.0 ( closed bars ) . hela cell monolayers were 
 incubated in medium containing 5 g / ml i - gst-(he)10-ygr6g6 in the presence of 0% , 1% , 
 5% , or 10% fbs for 1 h at 37 c . cell uptake and surface binding 
 were determined as described in the experimental 
 section . all data were normalized by total cell protein and 
 presented as averages  standard deviation from three independent 
 measurements per condition . the effect of serum on cell uptake ( a ) and surface biding ( b ) of 
 gst-(he)10-yg(rg)6 in hela cells at ph 7.5 ( open 
 bars ) and 6.0 ( closed bars ) . hela cell monolayers were incubated in 
 medium containing 5 g / ml i - gst-(he)10-yg(rg)6 in the presence of 0% , 1% , 5% , or 10% fbs for 
 1 h at 37 c . cell uptake and surface binding were determined 
 as described in the experimental section . all data were normalized by total cell protein and presented as averages 
  standard deviation from three independent measurements per 
 condition . ratios were calculated based on 
 the results shown in figures 5 and 6 . indicates 
 a statistically significant 
 difference ( p = 0.03 ) compared to the ratio in the 
 respective 0% fbs control . all other differences compared to the respective 
 0% fbs controls were nonsignificant ( p > 0.05 ) . as shown in figures 7b and 8 , ( he)10-ygr6g6 and ( he)10-tat exhibited 
 a random - coiled structure with a minimum at 198 nm . the ( he)10-yg(rg)6 peptide , on the other hand , exhibited a -sheet 
 conformation with a minimum at 218 nm and maximum at 195 nm ( figure 7a ) . the content of -sheet conformation ( he)10-yg(rg)6 was evaluated at various ph , and the 
 -sheet content increased with increasing ph . the cd spectra 
 showed an isosbestic point at 205 nm , consistent with a two - state 
 transition from random coil to -sheet . for both for ( he)10-ygr6g6 and ( he)10-tat , 
 ph had limited influence on the secondary structure ( figure 8) . circular dichroism ( cd ) spectra of ( he)10-yg(rg)6 ( a ) and ( he)10-ygr6g6 ( b ) 
 at ph 6.0 ( gray line ) , 6.5 ( long dashed black line ) , 7.0 ( regular 
 black line ) , and 7.5 ( bold black line ) . ( he)10-yg(rg)6 and ( he)10-ygr6g6 were isolated 
 from their corresponding recombinant gst - fusion proteins , gst-(he)10-yg(rg)6 and gst-(he)10-ygr6g6 , after overnight thrombin treatment . after buffer exchange 
 and desalting , isolated peptides were diluted to 25 mm in 10 mm phosphate 
 buffer saline ( pbs ) . data were plotted as ellipticity 
 ( mdeg ) versus wavelength ( nm ) after deduction of background . circular dichroism ( cd ) spectra of ( he)10-yg(rg)6 ( bold line ) , ( he)10-ygr6g6 ( regular line ) and ( he)10-tat ( long 
 dashed line ) at ph 
 6.0 ( a ) and 7.5 ( b ) . gst-(he)10-yg(rg)6 , gst-(he)10-ygr6g6 , and gst-(he)10-tat 
 were treated with thrombin for 18 h. next morning , ( he)10-yg(rg)6 , ( he)10-ygr6g6 , and ( he)10-tat could be eluted from corresponding gsh 
 columns with pbs with or without 250 mm acetate . buffer exchange and 
 desalting were then performed . afterward , isolated peptides were diluted 
 in 10 mm pbs at a final concentration of 25 mm . data were plotted as ellipticity ( mdeg ) versus wavelength ( nm ) after 
 deduction of background . it is well established that positively charged 
 amino acid residues , 
 that is , arginine and lysine , are essential for both the binding and 
 uptake of cpps in mammalian cells . however , the polycationic charge 
 is also responsible for the nonspecific cellular interaction and , 
 consequently , the cytotoxicity of cpps that limits the in 
 vivo application in drug delivery . therefore , the reversible 
 masking of the positive charge has been a promising approach to avoid 
 the nonspecific binding of cpps to cell surface . in this report , we 
 used oligoarginine peptides , with arginyl residues in a clustered 
 ( i.e. , ygr6g6 ) or a mixed ( i.e. , yg(rg)6 ) configuration , as the cpp models to investigate the influence 
 of the negative charges generated from a ph - dependent anionic he oligopeptide 
 on the binding and uptake of cpp in cultured hela cells . due 
 to the number of glutamic acid residues , a longer he oligopeptide 
 should provide a higher masking efficiency , which should yield a lower 
 cellular binding of the cpp . in our preliminary studies , we found 
 that oligopeptides with a 1:1 ratio of he - to - cationic amino 
 acid ( r / k ) showed a lack of masking effect at ph 7.5 , with lower ph 
 sensitivity . therefore , in this 
 study , an excess of negative charges in he repeats was designed to 
 ensure a complete neutralization of the six positive charges in yg(rg)6 or ygr6g6 at ph 7.5 . as shown in figure 2e , both yg(rg)6- and ygr6g6-fused proteins showed a decrease in cell uptake and surface 
 binding with an increase of the length of he oligopeptide . when linked 
 to a 12-mer he oligopeptide , the cell absorption capacity of yg(rg)6 and ygr6g6 was immensely restrained , 
 leading to a minimal cell uptake and a poor ph sensitivity in the 
 ph range from 7.5 to 6.5 ( figure 2e ) . although 
 gst-(he)8-ygr6g6 showed the highest 
 uptake , gst-(he)10-ygr6g6 actually 
 had the greatest ph sensitivity among the three proteins ( 5- , 6- , 
 and 4-fold increase for he n = 8 , 10 , and 12 at ph 
 7.5 vs 6.0 ) . moreover , nonspecific uptake and binding of gst-(he)10-ygr6g6 were lower than gst-(he)8-ygr6g6 at ph 7.5 . thus , the 10-mer 
 ( he ) repeat showed the optimal ph sensitivity and low binding at neutral 
 ph . from the cellular uptake assay ( figures 2a , c , and e ) , the internalization of gst-(he)n - yg(rg)6 showed only a moderate increase 
 with the 
 decrease in ph from 7.0 to 6.0 . on the other hand , a much sharper 
 increase of cell uptake was observed in gst-(he)n - ygr6g6 profiles , with a nearly linear 
 increase from ph 7.0 to 6.0 for gst-(he)8-ygr6g6 and gst-(he)10-ygr6g6 and a shift of the ph sensitivity of cellular uptake to lower ph 
 for gst-(he)12-ygr6g6 . this result 
 in ph sensitivity is consistent with the estimated pi of ( he)n - ygr6g6 fused oligopeptides 
 ( table 1 ) . although ( he)n - yg(rg)6 and ( he)n - ygr6g6 had a similar pi with n = 8 , 
 10 , or 12 , they exhibited a totally different ph - dependent profile 
 in cellular internalization . the different profile is possibly due 
 to the different interaction patterns between he and cpp with either 
 clustered or mixed positive charges . however , regardless of the length 
 of he oligopeptide moiety , all three fusion peptides with ygr6g6 showed a significantly higher cell uptake at 
 ph 6 than that of their yg(rg)6 counterpart . at ph 6.5 
 ( i.e. , ph = pka for histidine ) , half of 
 the anionic charges in he peptide will have been neutralized by the 
 protonated histidyl residues , which , according to our data , is sufficient 
 to begin to recover the binding and uptake of the cpp sequence . the 
 significantly higher cellular uptake of clustered arg - peptide than 
 that of mixed arg - peptide at ph 6.5 or lower is possibly due to the 
 fact that the insertion of glycine residues between the arginine residues 
 will result in a decreased cell uptake of the oligoarginine . however , the difference in cell uptake between 
 these two he fusion peptides , especially at ph 6.5 , appeared much 
 greater than that what has been observed between ygr6g6 and yg(rg)6 . it is likely that the difference 
 is also due to the presence of ( he)n sequence 
 in the fusion oligopeptides which may influence the overall conformation 
 of the fusion peptides . therefore , the difference in conformation 
 between ( he)10-ygr6g6 and ( he)10-yg(rg)6 was further investigated by using cd 
 spectroscopy . in order to further evaluate the oligopeptides 
 for further use 
 in in vivo studies , the ph sensitivity was evaluated in vitro in the presence of fbs . as shown in figures 5 and 6 , increasing amounts 
 of fbs led to decreased cell uptake and binding . this result was expected 
 because the addition of fbs has been shown to decrease the nonspecific 
 binding and the electrostatic adsorption of cpps . however , the presence of up to 10% fbs did not affect the ph sensitivity 
 of the constructs because the ratio of cell uptake and of surface 
 binding at ph 6.0 versus ph 7.5 was similar across the 010% 
 fbs range ( table 2 ) . cd spectra have 
 been studied on some arginine - rich peptides with 
 cluster of arginine residues , that is , hiv-1 tat , r7 , and r9 , to assess their secondary structures . however , no evidence showed 
 that they have an ordered structure regardless of solvent / buffer systems 
 used in those studies . as shown in figure 7b , ( he)10-ygr6g6 tends to have an 
 unstructured conformation in the range from ph 6.0 to 7.5 , with a 
 minimum absorption around 198 nm and no positive peak . however , the spectra of ( he)10-yg(rg)6 , as 
 shown in figure 7a , indicated an antiparallel 
 -sheet conformation , whereas its 
 secondary structural content varied in different ph environments . 
 because the cd measurements were obtained from isolated he - cpp peptides 
 without gst , the drastic difference in the spectra between ( he)10-ygr6g6 and ( he)10-yg(rg)6 as shown in figure 7a clearly suggests 
 a difference in the structure of these 2 peptides . glycine is the 
 most conformationally unrestrained amino acid , and it has been utilized 
 between arg to form a -hairpin conformation by other groups . in our study , the insertion of glycine as a 
 spacer between arginine residues to create an alternating rg sequence , 
 that is , ( rg)6 , will most likely facilitate the electrostatic 
 interaction between the guanidine group of arginine residues in the 
 cpp - sequence and the carboxylic group of glutamic acid residue in 
 the he - sequence . in addition , the stability of the ionic pairing may 
 be also responsible for the adoption and maintenance of an antiparallel 
 -sheet conformation of ( he)n - yg(rg)6 fusion peptides . figure 7a shows that the majority of ( he)10-yg(rg)6 still adopts the same conformation even at ph 6.0 , that is , 
 when the net charge of the ( he)10 moiety in the fusion 
 peptide is only slightly anionic ( net charge = 2 ) . consequently , 
 the maintenance of the antiparallel -sheet conformation at 
 mildly acidic ph condition may stabilize the he - oligoarginine complex 
 resulting in the poor ph sensitivity of ( he)n - yg(rg)6 . the conformational dependence of ph sensitivity 
 of ( he)n - fused cpps is consistent with 
 our finding with he - tat[4757 ] oligopeptide . tat[4757 ] 
 consists of clusters of 6 arg and 2 lys residues and is one of the 
 most well - studied cpps . when fused with ( he)12 , the fusion 
 peptide showed a high ph sensitivity between ph 7.0 to ph 6.0 in cell 
 uptake and binding assay ( figures 4a and 4b ) . in conclusion , our results indicate that 
 he oligopeptide can be 
 conjugated to cpps with both clustered and mixed positive charges 
 to form cpp derivatives with a reactivation in mildly acidic environment . however , a higher ph sensitivity can be expected in clustered than 
 in mixed positive charge cpps . in general , the increase of the length 
 of the he - repeat can decrease the nonspecific absorption of cpp at 
 neutral ph , but it will also decrease the reactivation at acidic ph . 
 therefore , when designing a reversible masking polyanion to decrease 
 the nonspecific binding of cpps in drug delivery across biological 
 barriers , both the total charges of the anions and the molecular conformation 
 of the ionic complex should be considered .
overcoming the nonspecific cellular 
 uptake of cell - penetrating 
 peptides ( cpps ) is a major hurdle in their clinical application . using 
 ph as the activation switch 
 , histidine - glutamic acid ( he ) dipeptide 
 repeats were fused to cpps to trigger the membrane - penetrating activity 
 at mildly acidic ph environments ( i.e. , ph 6.5 or below ) while masking 
 the internalization at neutral ph ( i.e. , ph 7.0 or above ) . in this 
 study , a series of recombinant gst - fusion proteins containing an he 
 oligopeptide sequence ( i.e. , ( he)n with n = 8 , 10 , or 12 ) and a cationic cpp ( i.e. , yg(rg)6 , ygr6g6 , or tat ) were engineered for a ph - sensitive 
 study comparing their cellular uptake and surface binding in cultured 
 hela cells . 
 circular dichroism ( cd ) spectroscopy was performed to 
 correlate differences between cpps in secondary structure with the 
 ph sensitivity . 
 ygr6g6 with clustered arginine 
 residues exhibited greater ph sensitivity in cellular uptake than 
 yg(rg)6 with separated arginine residues . increasing the 
 stretch of he repeats decreased cellular uptake and surface binding 
 for both yg(rg)6 and ygr6g6 . 
 the 
 ratio of cellular internalization at ph 7.5 vs 6.0 was not changed 
 by the presence of serum . 
 cd spectral data revealed that both ( he)10-tat and ( he)10-ygr6g6 exhibited 
 an unordered secondary structure , whereas ( he)10-yg(rg)6 adopted an antiparallel -sheet conformation . 
 this 
 -sheet conformation presumably stabilized the association of 
 ( he)10 with yg(rg)6 , leading to weakened ph 
 sensitivity of ( he)10-yg(rg)6 . on the other 
 hand , the random - coiled structures , that is , ( he)10-ygr6g6 and ( he)10-tat , both showed higher 
 ph sensitivity as determined in cell experiments . 
 the data presented 
 in this study provide a basis for the future design of ph - sensitive 
 he - cpp carrier for targeted drug delivery .