polyarginine peptides 
 are known as one of the widely used classes 
 of cell - penetrating peptides ( cpps ) and cellular delivery tools . it has been reported that the presence of the 
 guanidine group in the side chain of arginine plays a key role for 
 improved ability of arginine - rich peptides to cross the cell membrane . various systematic structural investigations have been performed 
 to determine the required number of arginine residues and the length 
 of the peptide for the optimization of cellular uptake . short polyarginine peptides containing less than six arginine residues 
 did not exhibit significant cellular uptake in several previously 
 reported investigations . thus , the presence of more than six arginine residues in the structure 
 of polyarginine peptides is critical for their efficient cell - penetrating 
 functions . however , several investigations were conducted to 
 increase the 
 cellular uptake of polyarginines by attaching the fatty acid to the n - terminal of the peptide . it has been previously reported 
 that the acylation of the n - terminal by fatty acids 
 can facilitate the intracellular uptake of polyarginines . for instance , katayama et al . synthesized acylated octa - arginines 
 and discovered that the introduction of hydrophobic fatty acid enhanced 
 the intracellular uptake of octa - arginine peptide and its conjugated 
 ubiquitin . furthermore , lee et al . designed 
 a class of lipopeptides carrying 715 arginine residues . among 
 them , myristoylated - hendeca - arginine ( c14r11 ) was found to be the most efficient cell - penetrating peptide . however , the fatty acylated polyarginine peptides 
 that contain 715 arginine residues can potentially cause toxicity , 
 and they can be easily degraded by proteases . moreover , linear 
 peptides carrying l - form are not stable 
 in serum and therefore have a limited application for in vivo studies ( figure s2 , supporting information ) . replacing l - form amino acids 
 with d - form to improve the peptide stability leads to high 
 cost production . thus , the synthesis and development of cyclic 
 cpps containing short amino acid sequence with less toxicity is desired . herein , we designed acylated cyclic polyarginine peptides ( acpps ) 
 containing five arginine residues and investigated their ability as 
 cell - penetrating peptides . we compared acpps with a corresponding 
 acylated linear polyarginine peptide ( alpp ) and a nonacylated cyclic 
 polyarginine as controls . we hypothesize that the combination of acylation 
 and cyclization of short polyarginine peptides having less than six 
 arginine residues will increase the intracellular uptake and can generate 
 peptides with molecular transporter properties . for convenience , square 
 brackets [ ] and parentheses ( ) phosphopeptide ptyr - glu - glu - ile ( pyeei ) 
 is an optimal peptide ligand for binding to the src tyrosine kinase 
 sh2 domain . in this study , we used negatively charged fluorescein - labeled 
 phosphopeptide f-gpyeei as a model cell - impermeable compound . all peptides were synthesized by 
 fmoc / tbu solid - phase peptide synthesis strategy either 
 manually or using rainin ps3 synthesizer ( protein technologies inc . ) . manual reactions were carried out in a glass reaction vessel with 
 a sintered glass frit by mixing under nitrogen bubbling at room temperature . fmoc - l - amino acid building blocks , fatty acids , and preloaded 
 h - arg(pbf)-2-chlorotrityl resin were used as starting materials . 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate 
 ( hbtu ) , hydroxybenzotriazole ( hobt ) , and n , n - diisopropylethylamine ( dipea ) in n , n - dimethylformamide 
 ( dmf ) were used as coupling and activating reagents , respectively , 
 for manual synthesis . in the case of using peptide synthesizer , 0.4 
 m n - methylmorpholine in dmf was used instead of dipea . piperidine in dmf ( 20% ) was employed to deprotect fmoc group at each 
 step . to cleave the linear peptide from the resin , a mixture 
 of trifluoroacetic acid ( tfa)/triisoproylsilane ( tis)/water ( 92.5/5/2.5 , however , in the synthesis of cyclic peptides , the 
 side chain protected linear peptides were first cleaved from the resin 
 by using a cleavage cocktail , containing 2,2,2-trifluoroethanol ( tfe)/acetic 
 acid / dichloromethane ( dcm ) ( 2:1:7 , v / v / v ) . cyclization reaction was 
 performed by employing a mixture of 1-hydroxy-7-azabenzotriazole ( hoat ) 
 and n , n-diisopropylcarbodiimide 
 ( dic ) in anhydrous dmf / dcm for 12 h. after solvent evaporation , the 
 peptide was deprotected and cleaved from the resin by using a cleavage 
 cocktail reagent r  , containing tfa / thioanisole/1,2-ethanedithiol 
 ( edt)/anisole ( 90:5:3:2 , v / v / v / v ) for 23 h. the crude peptides 
 were precipitated and washed with cold diethyl ether . to purify the 
 crude peptides , we used a reversed - phase high pressure liquid chromatography 
 ( rp - hplc ) system using shimadzu lc-8a preparative liquid chromatography 
 on a phenomenex gemini c18 column ( 10 m , 250  21.2 mm ) 
 with a gradient 0100% of acetonitrile ( ch3cn ) containing 
 0.1% tfa ( v / v ) and water containing 0.1% tfa ( v / v ) for 1 h with a 
 flow rate at 15.0 ml / min at the wavelength of 214 nm . as a representative 
 example , the synthesis of dodecanoyl-[r5 ] is described 
 here . h - arg(pbf)-2-chlorotrityl resin ( 660 
 mg , 0.35 mmol , 0.53 mmol / g ) was swelled in dmf for 40 min by n2 . fmoc - arg(pbf)-oh ( 681 mg , 1.05 mmol , 3 equiv ) was coupled 
 to the n - terminal of the resin , using hbtu ( 398 mg , 
 1.05 mmol , 3 equiv ) , hobt ( 142 mg , 1.05 mmol , 3 equiv ) , and dipea 
 ( 366 l , 2.1 mmol , 6 equiv ) in dmf ( 15 ml ) by agitating the 
 resin for 1 h using n2 . after the coupling , the resin was 
 washed with dmf , followed by fmoc - deprotection with piperidine in 
 dmf ( 20% ) . the subsequent three fmoc - arg(pbf)-oh couplings and one 
 dde - lys(fmoc)-oh ( 559 mg , 1.05 mmol , 3 equiv ; dde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl ) 
 coupling was carried out in the same manner , respectively . after removing 
 the fmoc group in side chain of lysine , dodecanoic acid ( 210 mg , 1.05 
 mmol , 3 equiv ) was coupled using hbtu , hobt , and dipea . then dde protection 
 group at n - terminal of peptide was removed by 2% 
 hydrazine in dmf , followed by washing with dmf and dcm . the side chain 
 protected linear peptides were cleaved from the resin by using a cleavage 
 cocktail , tfe / acetic acid / dcm ( 2:1:7 , v / v / v ) , for 1 h. the filtrate 
 was evaporated , and the residue was dried overnight in a vacuum . the 
 cyclization was conducted under a dilute condition with anhydrous 
 dmf / dcm ( 5:3 , v / v , 250 ml ) , using hoat ( 190 mg , 1.4 mmol , 4 equiv ) 
 and dic ( 240 l , 1.54 mmol , 4.4 equiv ) , and stirred for 12 h 
 under nitrogen atmosphere . after cyclization , the solvent was evaporated , 
 and the side chain deprotection was carried out by the addition of 
 reagent  r  for 2 h. the crude dodecanoyl-[r5 ] was precipitated and washed with cold diethyl ether and purified 
 by preparative rp - hplc system as described above . fluorescein - labeled 
 peptides were synthesized with the same protocol 
 before the attachment of fatty acid . we used fmoc-12-aminododecanoic 
 acid instead of dodecanoic acid , and after removing fmoc group , 5(6)-carboxyfluorescein 
 diisobutyrate ( cfdi ) was attached using 7-azabenzotriazol-1-yl - oxytris(pyrrolidino)phosphonium 
 hexafluorophosphate ( pyaop ) , hoat , and dipea . as an example , we started 
 fluorescein - labeled peptide synthesis in smaller scale with h - arg(pbf)-2-chlorotrityl 
 resin ( 208 mg , 0.11 mmol , 0.53 mmol / g ) . fmoc - arg(pbf)-oh ( 214 mg , 
 0.33 mmol , 3 equiv ) , dde - lys(fmoc)-oh ( 176 mg , 0.33 mmol , 3 equiv ) , 
 and fmoc-12-aminododecanoic acid ( 144 mg , 0.33 mmol , 3 equiv ) were 
 used to couple each building block to the resin using hbtu ( 125 mg , 
 0.33 mmol , 3 equiv ) , hobt ( 45 mg , 0.33 mmol , 3 equiv ) , and dipea ( 115 
 l , 0.66 mmol , 6 equiv ) in dmf . cfdi ( 170 mg , 0.33 mmol , 3 equiv ) 
 was coupled with amino group of 12-aminododecaonic chain using pyaop 
 ( 172 mg , 0.33 mmol , 3 equiv ) , hoat ( 45 mg , 0.33 mmol , 3 equiv ) , and 
 dipea . after removal of dde protecting group with 2% hydrazine in dmf 
 and washing with dmf and dcm , side chain protected fluorescein linear 
 peptides were cleaved from resin using tfe / acetic acid / dcm ( 2:1:7 , 
 v / v / v ) . the molecular weights of final products were confirmed by 
 an axima performance matrix - assisted laser desorption / ionization - time - of - flight 
 ( maldi - tof ) mass spectrometer ( shimadzu corporation ) . dodecanoyl-(r6 ) : maldi - tof ( m / z ) c48h96n22o8 calcd . 1108.7781 ; found 1109.7308 [ m + h ] . f-dodecanoyl-(r5 ) : maldi - tof ( m / z ) c69h107n23o14 calcd . 1481.8368 ; 
 found 1482.7586 [ m + h ] . w - dodecanoyl-[r5 ] : maldi - tof ( m / z ) c61h107n25o9 calcd . human ovarian adenocarcinoma 
 ( sk - ov-3 ) , 
 leukemia ( ccrf - cem ) , and embryonic kidney ( hek 293 t ) cells were purchased 
 from american type culture collection . the sk - ov-3 and hek 293 t cells 
 were grown in eagle s minimum essential medium ( emem ) , and 
 rpmi-1640 medium ( atcc , manassas , va ) was used for ccrf - cem cells 
 in a humidified atmosphere of 5% co2 at 37 c . both 
 media were supplemented with fetal bovine serum ( fbs , 10% ) and penicillin streptomycin 
 solution ( 1% , 10,000 units of penicillin and 10 mg of streptomycin 
 in 0.9% nacl ) . cytotoxicity 
 of peptides was examined 
 by mts proliferation assay in two human cancer cell lines ( sk - ov-3 
 and ccrf - cem ) and one human normal cell line ( hek 293 t ) . cells were 
 seeded into 96-well plates ( sk - ov-3 ( 5  10 cells / well ) , 
 ccrf - cem ( 1  10 cells / well ) , and hek 293 t ( 1  
 10 cells / well ) ) . then , the cells were incubated with complete 
 media ( 100 l ) overnight . different concentrations ( 0600 
 m ) of peptide solution ( 10 l ) were added to cells and 
 incubated at 37 c with 5% co2 for 24 h. then , celltiter 
 96 aqueous solution ( 20 l ) was added to each well and incubated 
 for 14 h. the absorbance was measured at 490 nm using microplate 
 reader . sk - ov-3 
 cells were grown in 6-well plates ( 2  10 cells / well ) 
 with complete emem media 24 h prior to the experiment . the fluorescein - labeled 
 peptide stock solution ( 1 mm ) was prepared in water and diluted in 
 gibco opti - mem i reduced serum medium to obtain the final concentration 
 of 5 m . the media were removed , and the mixture containing 
 fluorescein - labeled peptide solution ( 5 m ) was added . after 
 1 h incubation , trypsin  edta solution was added to detach cells 
 from the plate s surface and remove cell surface binding peptides . after 5 min , the complete media ( 2 ml ) were added to neutralize the 
 trypsin . finally , 
 the cells were analyzed by bd facscalibur or facsverse flow cytometer 
 using fitc channel . 5(6)-carboxyfluorescein ( fam ) was used as a negative control . for examination 
 of the cellular uptake mechanism of f-dodecanoyl-[r5 ] at low temperature , the assay was carried out at 4 c 
 to inhibit the energy - dependent cellular uptake pathways . the sk - ov-3 
 cells were preincubated at 4 c for 15 min and incubated with 
 the fluorescein - labeled peptide for 1 h at 4 c . cells were collected 
 and analyzed using flow cytometry with the previously described protocol 
 above . the data collected at 37 c were used as the control . 
 to perform the atp - depletion assay , cells were incubated with sodium 
 azide ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) for 1 h before 
 adding the fluorescein - labeled peptide . during the incubation time 
 ( 1 h ) , the fluorescein - labeled bicyclic peptide ( 5 m ) was prepared 
 in the opti - mem i reduced serum medium in the presence of sodium azide 
 ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) . then , the cells were 
 incubated with this solution for 1 h. the following sample preparation 
 and flow cytometry analysis protocol was the same as described above . sk - ov-3 cells 
 were seeded with complete emem on coverslips in 6-well plate ( 1  
 10 cells / well ) and kept until 50% confluency . the media 
 were removed , and cells were incubated with f-dodecanoyl-[r5 ] ( 10 m ) and f-dodecanoyl-(r5 ) ( 10 
 m ) in gibco opti - mem i reduced serum medium ( life technologies , 
 grand island , ny ) for 1 h at 37 c . then cells were washed with 
 1 phosphate buffered saline with calcium and magnesium ( pbs ) for three times . the coverslips were mounted on microscope 
 slides , and images were obtained using carl zeiss lsm 700 system with 
 a 488 nm argon ion laser excitation and a bp 505530 nm band - pass 
 filter . sk - ov-3 and ccrf - cem cells were seeded in 6-well plates ( 2  
 10 cells / well for sk - ov-3 and 1  10cells / well 
 for ccrf - cem ) and grown with complete emem media ( rpmi-1640 for ccrf - cem ) 
 overnight . a mixture of fluorescein - labeled phosphopeptides f-gpyeei 
 ( 5 m ) and peptides ( 10 m ) were prepared in opti - mem 
 i reduced serum medium at room temperature and incubated for 15 min . then the cells were incubated with the premixed solution at 37 c 
 with 5% co2 for 1 h. the sample preparation for facs analysis 
 was carried out by previously mentioned protocol described before . 
 in this assay , the 
 acylated cyclic polyarginine peptides 
 were synthesized by fmoc / tbu solid - phase peptide 
 synthesis method . fmoc - l - arg(pbf)-oh was coupled on h - arg(pbf)-loaded 
 2-chlorotrityl resin in the presence of hbtu , hobt , and dipea in dmf . then dde - lys(fmoc)-oh was attached , and a fatty acid was coupled to 
 the side chain of lysine . dde protecting group was removed by 2% hydrazine 
 in dmf , and a cleavage cocktail containing tfe / acetic acid / dcm ( 2:1:7 
 v / v / v ) was used for 1 h to cleave the side - chain protected linear 
 peptides from the resin . cyclization of linear peptides was carried 
 out in the presence of a mixture of hoat and dic in anhydrous dmf / dcm 
 for 624 h. the side - chain deprotection of cyclic peptide was 
 carried out by a cleavage cocktail reagent r  for 2 
 h. the crude peptides were precipitated and purified with rp - hplc 
 as described above . as a representative example , the synthesis of 
 dodecanoyl-[r5 ] is shown in scheme 1 . a corresponding acylated 
 linear polyarginine peptide ( alpp ) was 
 synthesized for comparative studies with the cyclic peptide ( acpp ) . moreover , a cyclic polyarginine without fatty acid [ r5 ] 
 was also synthesized to investigate the effect of the fatty acid on 
 cyclic peptide and its effect on molecular transporting efficiency . 
 to investigate whether the peptide alone can enter into cells , fluorescein - labeled 
 f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) , where f = fluorescein , were synthesized for facs 
 and microscopy investigations . chemical structures of synthetic peptides 
 used in this study ( f , 
 fluorescein - labeled ; [ ] , cyclic peptide ; ( ) , linear peptide ) . the cytotoxicity 
 of all peptides were tested in two different cancer cell lines , adherent 
 ( sk - ov-3 ) and nonadherent ( ccrf - cem ) cells , and a normal cell line 
 ( hek 293 t ) using mts assay ( figure 2 ) . cyclic 
 polyarginine [ r5 ] without fatty acid was used to explore 
 the effect of n - terminal acylation on cytotoxicity 
 and cellular uptake . alpp ( dodecanoyl-(r5 ) ) and [ r5 ] peptides showed consistently less cytotoxicity in all three 
 cells compared to acpps ( dodecanoyl-[r5 ] and dodecanoyl-[r6 ] ) . after 24 h incubation , acpps showed approximately 20% 
 toxicity in cells at a concentration of 25 m in ccrf - cem cells . however , dodecanoyl - linear ( r5 ) and [ r5 ] did 
 not exhibit significant cytotoxicity at 25 m and showed less 
 than 20% toxicity at the concentration of 100 m . in sk - ov-3 
 cells , dodecanoyl-(r5 ) and [ r5 ] showed more 
 than 80% cell viability at the concentration of 25 m . in normal 
 cells ( hek 293 t ) , all peptides exhibited less than 5% toxicity at 
 25 m . this differential behavior of the peptides in normal , 
 and cancer cells can be possibly rationalized through the interaction 
 between polyarginine peptides and cell membranes . the membrane of 
 cancer cells holds more negative charges compared to that in normal 
 cells because of the presence of anionic lipids such as phosphatidylserine . therefore , polyarginine peptides can be interacted 
 with cancer cells more effectively compared to normal cells . these data indicated 
 that acpps are more toxic than alpp and a nonacylated cyclic peptide 
 [ r5 ] , especially at concentration of 25 m . thus , a noncytotoxic concentration of 510 m was used 
 in cell - based assays . comparison of cytotoxicity between cyclic and linear acylated 
 polyarginine 
 peptides and nonacylated cyclic peptide [ r5 ] at various 
 concentrations against ccrf - cem , sk - ov-3 , and hek 293 t after 24 h. the intracellular uptake studies of fluorescein - labeled 
 acylated cyclic and linear pp , f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) , was carried out in sk - ov-3 
 cells by using flow cytometry and confocal laser scanning microscopy 
 ( clsm ) methods . fluorescein ( fam , f ) alone was selected as 
 a negative control . as it is shown in figure 3 , the f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) showed approximately 13.7- and 10.3-fold higher cellular 
 uptake than that of control 5,6-carboxyfluorescein ( fam ) , respectively , 
 in sk - ov-3 cells . the cellular uptake of f-dodecanoyl-[r5 ] was confirmed by clsm images ( figure 4 ) . f-dodecanoyl-[r5 ] showed higher fluorescence intensity 
 compared to that of f-dodecanoyl-(r5 ) in sk - ov-3 
 cells . therefore , acpp f-dodecanoyl-[r5 ] was found 
 to be a more efficient cell - penetrating peptide compared to its linear 
 counterpart . as shown in figure 4 , the fluorescence 
 signal is extended through the whole cells , suggesting that f-dodecanoyl-[r5 ] can get localized in the nucleus as well as cytoplasm . comparative 
 cellular uptake of f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) ( 5 m ) in sk - ov-3 
 cells ( 1 h ) . confocal laser scanning 
 microscope image of ( a ) f-dodecanoyl-[r5 ] and ( b ) 
 f-dodecanoyl-(r5 ) . the peptides 
 were incubated for 1 h in sk - ov-3 cells at 10 m concentration . the mechanism 
 of the cellular internalization of 
 f-dodecanoyl-[r5 ] was investigated by a temperature 
 control assay at 4 c along with atp depletion assay . these two 
 assays have been widely used to examine the energy - dependent endocytosis . facs results showed that the intracellular uptake 
 of f-dodecanoyl-[r5 ] was significantly reduced 
 at 4 c , indicating that the mechanism of internalization was 
 mainly dependent on the endocytosis pathways ( figure 5 ) . furthermore , atp depletion 
 assay was performed to investigate receptor - mediated endocytosis . to induce atp depletion , sk - ov-3 cells were 
 preincubated with sodium azide ( 10 mm ) and 2-deoxy - d - glucose 
 ( 50 mm ) for 1 h prior to the experiment , and a similar concentration 
 was maintained during the incubation ( 1 h ) with f-dodecanoyl-[r5 ] . the results showed that the cellular uptake of f-acpp 
 was inhibited in the presence of sodium azide and 2-deoxy - d - glucose , suggesting that receptor - mediated endocytosis is involved 
 for the cellular uptake of f-acpp . in atp depletion assay , the basic cellular uptake of fam was higher compared to that in temperature 
 control assay . however , this is evident that the intracellular uptake 
 of f-dodecanoyl-[r5 ] was inhibited , and there was 
 no significant difference between the cells treated with fam compared 
 to those treated with f-dodecanoyl-[r5 ] in temperature 
 control and atp depletion assays , suggesting that endocytosis is the 
 major pathway for the cellular uptake of f-dodecanoyl-[r5 ] . cellular uptake of f-dodecanoyl-[r5 ] ( 5 m ) 
 in sk - ov-3 cells in temperature control assay at 37 and 4 c , 
 and atp depletion assay with nan3 ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) analyzed by flow cytometry . cells with no 
 treatment were used as control . the ability 
 of acpps as a molecular transporter was evaluated and compared by 
 selecting a fluorescein - labeled phosphopeptide , f-gpyeei , 
 as a molecular cargo . phosphopeptide , pyeei ( ptyr - glu - glu - ile ) is 
 an optimal peptide template for the sh2 domain of src tyrosine kinase . several analogues of this peptide have been synthesized as potent 
 ligands for this target . because of the presence of the 
 negatively charged amino acid residues in the structure of the peptide 
 including phosphorylated tyrosine moreover , the internalization of the negatively charged phosphopeptide 
 in cancer cells by diffusion is more difficult because cancer cell 
 membranes are composed of more negatively charged lipids . thus , cellular 
 delivery of cell - impermeable negatively charged phosphopeptides is 
 significantly challenging . we have previously reported different peptide - based 
 carriers for the intracellular delivery of negatively charged phosphopeptides 
 as model cell - impermeable drugs in several cell lines . in this study , the intracellular uptake 
 of f-gpyeei was monitored in the presence and absence of synthetic 
 peptides after 1 h incubation by flow cytometry . as it is exhibited 
 in figure 6 , the acpps ( dodecanoyl-[r5 ] and dodecanoyl-[r6 ] ) delivered the phosphopeptide more 
 efficiently compared to alpps , dodecanoyl-(r5 ) and -[r5 ] . the intracellular uptake of f-gpyeei in the presence 
 of dodecanoyl-[r5 ] and dodecanoyl-[r6 ] was enhanced 
 by 3.4- and 5.5-fold higher than the uptake in the absence of acpps . however , dodecanoyl-(r5 ) and -[r5 ] only improved 
 1.3- and 1.4-fold intracellular uptake , respectively . the results 
 showed that acylated and cyclized polyarginine peptides can deliver 
 the phosphopeptide effectively . however , the intracellular uptake 
 of the phosphopeptide did not improve significantly in the presence 
 of either acylated linear polyarginine peptide or cyclic [ r5 ] . these data suggest that a combination of acylation and cyclization 
 would improve the molecular transporting efficiency of the polyarginine - based 
 peptide ( containing less than six arginines ) for the intracellular 
 delivery of a cell - impermeable phosphopeptide . this has been previously 
 reported that the acylated linear octa - arginine increased the cellular 
 uptake of molecular cargoes by just adding fatty acid to the n - terminal of octa - arginine . however , we discovered that both cyclization and acylation in a 
 short penta - arginine can significantly improve the delivery of a cell - impermeable 
 phosphopeptide in sk - ov-3 cells . cellular uptake of f-gpyeei ( 5 
 m ) in the presence 
 of dodecanoyl-[r5 ] and -[r5 ] , dosecanoyl-(r5 ) , and dodecanoyl-[r6 ] ( 10 m ) in sk - ov-3 
 cell line . phosphopeptide delivery efficiency of dodecanoyl-[r5 ] were compared with known cpps ( r7 , crrrrrrr ; 
 tat , ygrkkrrqrrr ) . the major driving forces for the intracellular delivery are 
 presumed 
 to be structural rigidity through cyclization of the peptide and the 
 interaction of the fatty acid with the cell membrane . it has been 
 previously reported that the cellular uptake of the peptide can be 
 increased due to the structural rigidity by cyclization of arginine - rich 
 peptides . they proposed that the maximal 
 distance between guanidine groups of arginine residue can lead to 
 an efficient transduction of arginine - rich peptides . our investigations 
 showed that dodecanoyl-[r6 ] is able to deliver more efficiently 
 by 1.6-fold higher f-gpyeei uptake compared to that of dodecanoyl-[r5 ] . increasing the number of positively charged arginine residues 
 can enhance the cellular uptake through ionic interactions with the 
 negatively charged phosphopeptide and/or phospholipid in the cell 
 membrane through ionic interactions . however , the higher number of 
 arginine residue is not the only responsible element for the efficient 
 cellular internalization . for example , it has been reported that polyarginine 
 containing 11 amino acids ( r11 ) showed higher cellular 
 uptake compared to the polyarginine containing 13 amino acids ( r13 ) . at the same time , r11 was found to be a more 
 potent transporter compared to r9 in prostate cancer cells . these investigations showed that an optimal 
 number of arginine residues are required for the highest degree of 
 functionality . however , the greater number of amino acid residues 
 in cyclic peptides can decrease the structural rigidity , which lowers 
 the ability of the peptide to get into cells . dodecanoyl-[r5 ] was also compared with several commonly 
 cpps , such as cr7 and tat ( ygrkkrrqrrr ) peptides . the acpp 
 improved the cellular uptake of the phosphopeptide by 1.4- and 1.8-fold 
 higher than those of cr7 and tat , respectively ( figure 6 ) . these results revealed that although other peptides 
 containing arginine can deliver the molecular cargo , acpp dodecanoyl-[r5 ] with a shorter peptide sequence than cr7 and 
 tat can work as a molecular transporter with higher efficiency . chemical structures 
 of acpps with different length of fatty acid 
 chains ( c8 , c12 , and c16 ) . to investigate the effect 
 of the chain length on the cell penetration 
 potency , we synthesized octanoyl-[r5 ] , dodecanoyl-[r5 ] , and hexadecanoyl-[r5 ] ( figure 7 ) . acpps showed less than 20% toxicity in cells at the concentration 
 of 25 m ( figure 8a ) . the in 
 vitro toxicity results showed that increasing the fatty acid 
 chain length caused enhanced toxicity in cells as hexadecanoyl-[r5 ] was more cytotoxic than dodecanoyl-[r5 ] and octanoyl-[r5 ] . these data indicate that the fatty acid chain length could 
 alter the interaction with the cell membrane and disturb the membrane 
 integrity . on the basis of the cytotoxicity data , ( a ) cytotoxicity 
 assay of cyclic polyarginine peptide - fatty acid 
 conjugates against sk - ov-3 cells ( 24 h incubation ) . ( b ) cellular uptake 
 of a phosphopeptide , f-gpyeei ( 5 m ) , in the presence 
 of peptide - fatty acid conjugates ( 10 m ) in sk - ov-3 cells . it has been previously reported that there was a relationship 
 between 
 the length of fatty acid in polyarginines and their cellular uptake , meaning that the optimal length of fatty acid 
 is required for optimal functionality based on the peptide sequence 
 and cell type . the results exhibited that the cellular uptake of f-gpyeei 
 was improved in the order of octanoyl-[r5 ] < dodecanoyl-[r5 ] < hexadecanoyl-[r5 ] ( figure 8b ) . the cellular uptake of f-gpyeei in the presence 
 of hexadecanoyl-[r5 ] was 9.3- and 6.0-fold higher than 
 that in the presence of octanoyl-[r5 ] and dodecanoyl-[r5 ] , respectively . these data suggest that the length of the 
 attached fatty acid chain in the structure significantly influences 
 the efficiency of the peptide as a molecular transporter . sixteen - carbon 
 chain length ( c16 ) was found to be an optimized length 
 for the intracellular delivery of f-gpyeei in sk - ov-3 cells . after acpps were found to act as cpp and molecular 
 transporter , a systematic investigation was performed to modify the 
 fatty acid to another hydrophobic moiety . w4-[r5 ] , 
 the whole fatty acid chain was replaced with four tryptophan residues . the second conjugate w - dodecanoyl-[r5 ] had one more tryptophan 
 at the end of the dodecanoyl fatty acid chain . w4-[r5 ] enhanced intracellular delivery of f-gpyeei by 4.1-fold 
 higher compared to that of dodecanoyl-[r5 ] in sk - ov-3 . 
 however , w - dodecanoyl-[r5 ] improved the uptake of f-gpyeei 
 by 1.3-fold higher compared to that of w4-[r5 ] in ccrf - cem cells ( figure 9 ) . thus , it is not straightforward 
 to compare the cellular uptakes in sk - ov-3 and ccrf - cem . however , 
 the results could be assessed indirectly by relative comparison with 
 the cellular uptake by w4-[r5 ] . these data suggest 
 that the presence of hydrophobic tryptophan moieties could enhance 
 the molecular transporting efficiency . in other words , appropriate 
 modification of hydrophobic moiety in cpps can increase the drug delivery 
 ability of acpp . thus , cyclic nature , short length of polyarginine , 
 and hydrophobic segments were found to be critical elements to generate 
 a peptide with an optimized molecular transporter efficiency . cellular uptake 
 assay of a phosphopeptide , f-gpyeei ( 5 
 m ) , in the presence of w4-[r5 ] and w - dodecanoyl-[r5 ] ( 10 m ) against sk - ov-3 and ccrf - cem cell lines ( 1 
 h incubation ) . cells with no treatment 
 were used as control . to evaluate 
 whether the presence of peptides can enhance the pharmacological effect 
 of a molecular cargo , the antiproliferative activity of doxorubicin 
 ( dox ) was examined in the presence of dodecanoy-[r6 ] ( [ r6]-c12 ) in mcf-7 cells in a time - dependent assay . 
 as it is shown in figure 10 , the antiproliferative 
 potency of dox ( 5 m ) was enhanced by 7% , 11% , and 14% in the 
 presence of [ r6]-c12 ( 5 m ) when compared 
 with that of the drug alone after 24 , 48 , and 72 h incubation , respectively . these results showed that the cell proliferation of mcf-7 cells was 
 inhibited in a time - dependent manner suggesting a sustained drug release 
 by peptide leading to a higher inhibitory effect . the presence of 
 the peptide possibly improved the cellular uptake of dox and enhanced 
 the antiproliferative activity . time - dependent antiproliferative activity 
 of dox ( 5 m ) in 
 the presence and absence of [ r6]-c12 ( 5 m ) . in 
 conclusion , acylated cyclic polyarginine peptides were synthesized 
 and examined as cpps and potential molecular transporters . acpps showed 
 higher potency as molecular transporter compared to the corresponding 
 linear counterpart and cyclic polyarginine without fatty acid . the 
 mechanism of the peptide internalization was found to be energy - dependent 
 endocytosis . cyclization and acylation reactions on the structure 
 of the peptide enhanced the intracellular uptake of polyarginine peptides 
 although they carry a short length of sequence . this intracellular 
 delivery property of acpps can be optimized by modifying the length 
 of fatty acid chain . to the best of our knowledge , this is the first 
 report of cyclic fatty acylated polyarginine peptide as molecular 
 transporter of a cell - impermeable phosphopeptide . this study provided 
 insights about how a combination of the cyclic nature and acylation 
 can improve the cell internalization of polyarginines . further investigations 
 are undergoing to determine whether conjugation of cell - impermeable 
 hydrophobic drugs to acylated cyclic polyarginines can be an efficient 
 method for designing novel drug delivery systems .
many 
 of the reported arginine - rich cell - penetrating peptides ( cpps ) 
 for the enhanced delivery of drugs are linear peptides composed of 
 more than seven arginine residues to retain the cell penetration properties . 
 herein , we synthesized a class of nine polyarginine peptides containing 
 5 and 6 arginines , namely , r5 and r6 . 
 we further 
 explored the effect of acylation with long chain fatty acids ( i.e. , 
 octanoic acid , dodecanoic acid , and hexadecanoic acid ) and cyclization 
 on the cell penetrating properties of the peptides . 
 the fluorescence - labeled 
 acylated cyclic peptide dodecanoyl-[r5 ] and linear peptide 
 dodecanoyl-(r5 ) showed approximately 13.7- and 10.2-fold 
 higher cellular uptake than that of control 5,6-carboxyfluorescein , 
 respectively . 
 the mechanism of the peptide internalization into cells 
 was found to be energy - dependent endocytosis . 
 dodecanoyl-[r5 ] and dodecanoyl-[r6 ] enhanced the intracellular uptake 
 of a fluorescence - labeled cell - impermeable negatively charged phosphopeptide 
 ( f-gpyeei ) in human ovarian cancer cells ( sk - ov-3 ) by 3.4-fold 
 and 5.5-fold , respectively , as shown by flow cytometry 
 . the cellular 
 uptake of f-gpyeei in the presence of hexadecanoyl-[r5 ] was 9.3- and 6.0-fold higher than that in the presence of 
 octanoyl-[r5 ] and dodecanoyl-[r5 ] , respectively . 
 dodecanoyl-[r5 ] enhanced the cellular uptake of the phosphopeptide 
 by 1.42.5-fold higher than the corresponding linear peptide 
 dodecanoyl-(r5 ) and those of representative cpps , such 
 as hepta - arginine ( cr7 ) and tat peptide . 
 these results 
 showed that a combination of acylation by long chain fatty acids and 
 cyclization on short arginine - containing peptides can improve their 
 cell - penetrating property , possibly through efficient interaction 
 of rigid positively charged r and hydrophobic dodecanoyl moiety with 
 the corresponding residues in the cell membrane phospholipids .