surface immobilization 
 of poly(ethylene glycol ) ( peg ) , or pegylation , 
 is widely used to modify nanoparticles ( nps ) with the goal to avoid 
 undesired , nonspecific biological interactions . the covalent attachment 
 of peg to nps results in the formation of a relatively inert hydrophilic 
 corona that reduces uptake by the reticuloendothelial system , thereby 
 prolonging the circulation time of nps such as therapeutic proteins , 
 dendrimers , liposomes , and micelles . it has been reported 
 that the length of peg chains plays a major role in determining the 
 cellular interaction of pegylated nps because it affects the particle 
 size , peg conformation ( mushroom vs brush ) , and thickness and surface 
 coverage of the peg corona . the surface charge of nps is another 
 important parameter that can affect cellular interactions of nps . 
 in general , nps with positively charged surfaces ( e.g. , nh2 termini that are protonated under physiological ph ) are known 
 to nonspecifically interact with cells and cause toxicity via membrane 
 destabilization , which can be typically diminished by charge neutralization 
 or carboxylation . in particular , poly(amidoamine ) ( pamam ) dendrimers 
 have been extensively used to study charge - dependent cellular interactions 
 because of their chemically well - defined , dendritic structure that 
 amplifies the effect of surface groups . although the effects of peg corona and surface charges of nps on 
 their cellular interactions have been widely studied independently , 
 it has not been fully understood to date how they collectively affect 
 the nano  bio interactions . our group recently synthesized 
 amphiphilic pegylated dendron - based 
 copolymers ( pdcs ) consisting of hydrophobic poly(-caprolactone ) 
 ( pcl ) , polyester dendron , and multiple hydrophilic peg chains . when the self - assembly characteristics of pdcs 
 were compared to those of similar linear - block copolymers ( lbcs ) , 
 pdcs displayed critical micelle concentrations ( cmc ) ( 1010 m ) that were up to 
 2 orders of magnitude lower than lbcs , indicating their enhanced thermodynamic 
 stability . molecular dynamics ( md ) simulations 
 also revealed that the dendron micelles ( dms ) were almost completely 
 covered by a dense peg layer , whereas lbc - based micelles displayed 
 a considerable degree of pcl core exposure at their surface . this 
 difference was attributed to the high - density peg layers provided 
 by the dendritic architecture of pdcs . interestingly , unlike pamam 
 dendrimers , after the termini of the peg ( 2000 g / mol ) chains of the 
 pdcs were modified to primary amines , the resulting dms ( dm2k - nh2 ) did not exhibit any noticeable cellular interactions 
 or toxicity . this charge - independent 
 end - group effect of the dm2k - nh2 was attributed 
 to a 10-fold higher molecular - weight - to - charge - number of the pdcs 
 than that of pamam dendrimers and back - folding of the terminal amines 
 into the ethylene glycol ( ch2ch2o ) 
 backbone . these results demonstrated that the long peg chain contributes 
 to the sequestration of charge - dependent end - group effects of the 
 dms . our previous findings raised a vital question of whether 
 the end - group 
 charge effects of dms could be tuned through changing the peg chain 
 length . to address this question , we hypothesized that the lack of 
 charge - dependent end - group effects of dm2k - nh2 could turn to be charge - dependent by shortening the peg chain lengths . 
 in this study , we synthesized a series of pdcs with shorter peg chains 
 ( 600 g / mol ) ( pdc600 ) and investigated the relation between 
 the length of peg chains and the end - group charge effect of dms on 
 their cellular interactions . the dm  membrane interactions were 
 further studied in atomistic detail using md simulations . in addition , 
 the effect of polymeric architecture on the encapsulation stability 
 of the resulting micelles was investigated . by comparing dms and lbc - based 
 micelles ( lms ) encapsulating a membrane permeable fluorescent dye , this study presents a systematic investigation of the effect of surface 
 charge , peg chain length , and polymeric architecture on the cellular 
 interactions and stability of polymeric micelles . boc peg amine , hcl salt 
 ( boc - peg600-nh2 ) ( mw 600 g / mol ) , was purchased 
 from jenkem technology 
 usa inc . regenerated cellulose dialysis membrane ( 3.5 kg / mol mwco ) was 
 purchased from spectrum laboratories ( rancho dominguez , ca ) . generation 
 4 ( g4 ) pamam dendrimers and all other reagents were purchased from 
 sigma - aldrich co. ( st . amine - reactive 
 pcl3.5k - g3-pnp ( 4-nitrophenyl ester ) was synthesized according 
 to our published protocol followed by 
 pegylation . briefly , pcl3.5k - g3-pnp ( 150 mg , 0.026 mmol ) 
 was dissolved in dimethylformamide ( dmf ) ( 4 ml ) for 2 h and added 
 into nh2-peg600-boc ( 150 mg , 0.249 mmol ) in 
 dmf ( 3 ml ) in the presence of triethylamine ( tea ) ( 84.17 mg , 0.832 
 mmol ) . the reaction was carried out at room temperature for 24 h. 
 the reaction mixture was then transferred to a dialysis membrane ( 3.5 
 kg / mol mwco ) , dialyzed against distilled water ( dh2o ) for 
 48 h with five water changes , and lyophilized using a freezone 4.5 
 freeze - dryer ( labconco corp . , kanas city , mo ) for 2 days . the resulting 
 pcl3.5k - g3-peg600-boc was obtained as powder . 
 to remove the boc protecting groups in the product , a solution of 
 pcl3.5k - g3-peg600-boc was dissolved in a 1:1 
 mixture of dichloromethane and trifluoroacetic acid ( 2 ml of total 
 volume ) and stirred for 30 min at room temperature . the solvent was 
 then completely removed by rotary evaporation , and the purified product 
 was redissolved in dimethyl sulfoxide ( dmso ) ( 3 ml ) . after dialysis 
 for 24 h and lyophilization for 2 days , briefly , for 
 acetylation , pcl3.5k - g3-peg600-nh2 ( 30 mg , 0.004 mmol ) was dissolved in dmso ( 2 ml ) containing tea 
 ( 38 mg , 0.376 mmol ) . acetic anhydride ( 31.95 mg , 0.313 mmol ) was then 
 added dropwise to the stirring solution . for carboxylation , pcl3.5k - g3-peg600-nh2 ( 30 mg , 0.004 mmol ) 
 was dissolved in dmso ( 2 ml ) containing pyridine ( 12.3 mg , 0.156 mmol ) 
 to which a dmso solution ( 1 ml ) of succinic anhydride ( 15.7 mg , 0.156 
 mmol ) was added dropwise with vigorous stirring . both reactions were 
 carried out at room temperature for 24 h. after dialysis for 24 h 
 and lyophilization for 2 days , pcl3.5k - g3-peg600-ac and pcl3.5k - g3-peg600-cooh were obtained 
 as powder . to prepare rhodamine - labeled pdc600 , pcl3.5k - g3-peg600-nh2 ( 30 mg , 0.004 mmol ) was dissolved 
 in dmso ( 2 ml ) to which an nhs - rhodamine ( 5.16 mg , 0.0098 mmol ) solution 
 in dmso ( 1 ml ) was subsequently added dropwise . the reaction was carried 
 out at room temperature for 24 h. the remaining primary amine groups 
 were then acetylated by addition of acetic anhydride ( 31.95 mg , 0.313 
 mmol ) in the presence of tea ( 38 mg , 0.376 mmol ) , followed by stirring 
 for an additional 24 h at room temperature . the reaction mixture was 
 dialyzed against water for 48 h to remove unreacted rhodamine followed 
 by 2 days of lyophilization , resulting in the final product of pdc600-rhod . the same method was employed to synthesize pdc2k - rhod and linear pcl3.5k - peg2k - rhod 
 ( lbc2k - rhod ) . in addition , g4 pamam dendrimers , used 
 as control in this study , 
 were purified using repetitive dialysis and lyophilization as described 
 earlier , followed by conjugation with 
 rhodamine , using edc / nhs chemistry , resulting in g4-nh2-rhod . to prepare blank 
 micelles , each 
 pdc ( pdc600-nh2 , pdc600-cooh , or 
 pdc600-ac ) ( 5 mg ) or linear block copolymer ( lbc , pcl3.5k - peg2k ) that was synthesized in our laboratory 
 as described earlier was first dissolved 
 in dmf ( 2 ml ) , vortexed vigorously for 5 min , and stirred for 10 min 
 until it was completely dissolved . dh2o ( 1.8 ml ) was then 
 added dropwise into each of the dmf solutions under stirring . each 
 solution was dialyzed against dh2o ( 4 l ) in a dialysis 
 membrane ( 3.5 kg / mol mwco ) at room temperature for 24 h , followed 
 by centrifugation at 9300  g for 5 min to remove 
 large aggregates . the supernatants containing the resulting micelles 
 were collected and immediately used for subsequent experiments . the rhodamine - labeled micelles , dual - dye micelles , and frster 
 resonance energy transfer ( fret ) micelles were prepared using the 
 same method described above except for the inclusion of rhodamine - labeled 
 polymer or hydrophobic dyes in polymer solution . for rhodamine - labeled 
 micelles , pdc - rhod ( 0.5 mg , 10 wt % ) was mixed with each pdc solution 
 in dmf . for dual dye micelles , pdc600-rhod or lbc2k - rhod ( 0.5 mg , 10 wt % ) was incorporated into each polymer solution 
 in dmf , along with 3,3-dioctadecyloxacarbocyanine perchlorate 
 ( dio , 25 g , 0.5 wt % ) . finally , for fret micelles , dio ( 25 
 g , 0.5 wt % ) and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine 
 perchlorate ( dii , 25 g , 0.5 wt % ) were added into pdc600-ac or pcl3.5k - peg2k in dmf . particle size of the 
 various dms ( 1 mg / ml ) was measured using a nicomp 
 380 zeta potential / particle sizer ( particle sizing systems , santa 
 barbara , ca ) . all measurements were performed in triplicate in double 
 distilled water using unfiltered micelle samples . the morphology of 
 each dm was visualized using transmission electron microscopy ( tem , 
 jem-1220 , jeol ltd . , ( 5 l ) of each micellar 
 suspension ( 0.2 mg / ml ) was placed on a 300 mesh copper grid coated 
 with carbon ( electron microscopy sciences , hatfield , pa ) . all samples 
 were stained with a drop ( 5 l ) of 2% phosphotungstic acid ( ph 
 7 ) and dried in a desiccator at room temperature for 1 day before 
 observed using tem as described earlier . kb cells were seeded 
 in glass bottom culture dishes at a density of 1 or 4  10 cells / well on 8-well millicell ez slides ( millipore corporation , 
 billerica , ma ) or glass - bottomed petri dishes ( mattek corporation , 
 ashland , ma ) and incubated for 24 h. cells were then treated with 
 rhodamine - labeled dm600 ( 8 m ) , rhodamine - labeled 
 dm2k ( 4 m ) , rhodamine - labeled g4 pamam dendrimer 
 ( 1 m ) , or free rhodamine ( 0.2 m ) all in pbs for 2 or 
 4 h. the concentrations of all samples were adjusted to normalize 
 the amounts of rhodamine using fluorescent spectroscopy prior to the 
 experiment . for dio - loaded , rhodamine - labeled ( dual - dye ) micelles , 
 kb cells were seeded in glass bottom culture dishes using the protocol 
 described above , and cells were treated with dual - dye dm600 ( 60 g / ml ) or dual - dye lbc(pcl3.5k - peg2k)-based micelles ( 60 g / ml ) in rpmi without supplements for 
 4 , 8 , 24 , or 48 h. after incubation , the cells were washed twice using 
 pbs and fixed using a 4% paraformaldehyde solution . when the nuclear 
 staining was needed , a mounting agent containing dapi ( vectashield 
 h-1200 , vector laboratories , inc . , burlingame , ca ) was used after 
 fixation . the fixed cells were visualized by a carl zeiss confocal 
 microscope ( lsm 710 , carl zeiss microimaging gmbh , gena , germany ) 
 using a diode laser ( 405 nm ) for dapi , an argon laser ( 488 nm ) for 
 dio , and a dpss laser ( 561 nm ) for rhodamine . a 40 objective 
 ( objective  c - apochromat 40x/1,20 w corr , carl zeiss 
 microimaging gmbh , gena , germany ) was used . all obtained images were 
 processed using zen software ( carl zeiss microimaging gmbh , gena , 
 germany ) . kb cells were seeded in 12-well 
 plates at a density of 2  10 cells / well and incubated 
 for 24 h. cells were then treated with rhodamine - labeled dm600 ( 8 m ) , rhodamine - labeled dm2k ( 4 m ) , rhodamine - labeled 
 g4 pamam dendrimer ( 1 m ) , or free rhodamine ( 0.2 m ) , 
 all in pbs , for 2 h. for dual - dye micelles , cells were treated with 
 dual - dye dm600 ( 60 g / ml ) or dual - dye lbc ( pcl3.5k - peg2k)-based micelles ( 60 g / ml ) in rpmi 
 without supplements for 4 , 8 , 24 , or 48 h. the treated cells were 
 washed twice with pbs and suspended by addition of trypsin / edta . the 
 cell suspensions were centrifuged at 228  g for 5 min , and the supernatants were discarded . the resulting cell 
 pellets were resuspended in a paraformaldehyde solution ( 1% w / v , 500 
 l ) and transferred to flow cytometry sample tubes . the samples 
 were examined using a lsr fortessa flow cytometer ( becton dickinson , 
 franklin lakes , nj ) as described earlier . kb cells were seeded in 96-well plates at 
 a density of 1  10 cells / well and grown in rpmi 
 media for 24 h. cells were then treated with various dms and g4 pamam 
 dendrimers at various concentrations ranging from 1 to 100 m . cell viability was 
 assessed using a celltiter 96 aqueous one solution ( mts ) assay ( promega , 
 madison , wi ) according to the manufacturer s protocol . the 
 uv absorbance was measured at 490 nm using a labsystems multiskan 
 plus microplate reader ( labsystems , finland ) . mean cell viabilities 
 were determined relative to a negative control ( untreated cells ) and 
 a positive control ( cells lysed using 0.1% triton - x ) ( n = 5 ) . fret - dm600-ac micelles 
 ( 60 g / ml ) or fret - lbc ( pcl3.5k - peg2k)-based 
 micelles ( 60 g / ml ) were incubated in pbs at 37 c for 
 up to 48 h. fluorescence intensities were measured using a spectrofluormeter 
 ( rf 1501 , shimadzu , japan ) by scanning the emission spectra from 495 
 to 600 nm at an excitation wavelength of 484 nm . the md 
 simulations were performed with namd and 
 the charmm force field ( charmm27 , c35r revision for ethers and general 
 force field ) . nonbonding interactions were 
 calculated using a cutoff distance of d = 10  , 
 and long - range electrostatic interactions were calculated by the pme 
 method . the systems were simulated in 
 the npt ensemble , using the langevin dynamics with a damping constant 
 of lang = 0.1 p / s , and the time step of 2 fs . the 
 micelles were prepared from nagg = 15 
 individual copolymers ( unimers ) of pdc - nh3 with 
 two different peg chain lengths of molecular weight 600 and 2000 g / mol . initially , the unimers were spherically distributed to form the basic 
 micelle structure and compacted by brief simulations in vacuum . the 
 micelles were then simulated alone in ionic solutions for 10 ns at t = 300 k and p = 1 bar . we also used charmm - gui 
 ( a web - based graphical user interface for charmm ) to prepare a biological membrane consisting of two different types 
 of lipid molecules . the dipalmitoylphosphatidylcholine ( dmpc ) and 
 dipalmitoylphosphatidylglycerol ( dmpg ) lipid molecules were used in 
 a 3:1 ratio . similar to the condition used for the micelles , the membrane 
 ( size 230   230  ) was equilibrated for 2 ns ( lang = 1.0 p / s ) using the charmm36 lipid force field . after 
 equilibrium , both micelles were placed 5  above the membrane , 
 and the whole systems were placed in an ionic solution with a salt 
 concentration of 150 mm ( physiological salt concentration ) . first , 
 the systems were equilibrated for 5 ns while keeping the micelle and 
 membrane atoms fixed . then , all the atoms were released , and the simulations 
 were continued for 75 ns ( lang= 0.1 p / s ) . we continued 
 the micelle simulation without membrane for 30 ns to find out the 
 effect of the negatively charged membrane on the morphology of positively 
 charged micelle . the coulombic potential energy was calculated from 
 eq 11where u(r ) is coulombic potential energy ,  
 is the dielectric constant 
 of water , 0 is the vacuum permittivity , i is the number of positively charged point at the surface 
 of the micelle , j is the number of negatively charged 
 point at the upper layer of the lipid bilayer , and rij is the distance between the positive 
 and negative charge ( qi = qj = 1.6 
  10 c ,  = 80 , and 0 = 8.8542  10 c n m ) . end - group - modified 
 pdcs were synthesized using a modified version of our previously published 
 protocol . briefly , alkyne - core - functionalized 
 polyester g3 dendron ( 870 g / mol ) was conjugated with azido - functionalized 
 pcl ( 3500 g / mol ) using click chemistry , followed by pegylation of 
 the dendritic arms using heterobifunctional nh2-peg600-boc . the end groups of the pdcs were then modified to amine 
 ( pdc600-nh2 ) , carboxylate ( pdc600-cooh ) , and acetamide groups ( pdc600-ac ) , followed by 
 extensive characterization using h nmr and gpc ( table 1 and figure s1 of the supporting 
 information ) briefly , the characteristic 
 peaks of polyester dendron were observed at 4.404.18 and 1.221.15 
 ppm . the repeating unit of pcl appeared at 4.06 , 2.30 , 1.62 , and 1.35 
 ppm , and the repeating unit of peg appeared at 3.62 ppm . also , the 
 characteristic peaks of terminal groups appeared at 3.13 ppm for pdc600-nh2 , 1.96 ppm for pdc600-ac , and 
 2.52 and 2.62 ppm for pdc600-cooh . to employ fluorescence - based 
 analytical techniques , rhodamine - conjugated pdc600 ( pdc600-rhod ) was also prepared and characterized . dms were prepared 
 by self - assembly of the end - group - modified pdcs using the dialysis 
 method . figure 1a depicts the structure of a dm assembled from amine - terminated pdcs 
 as an example . the particle size distributions , zeta - potentials , and 
 cmcs of all three end - group - modified dms , along with dm2k - nh2 included as a control , are listed in table 2 . the hydrodynamic diameters of dm600-nh2 , dm600-ac , and dm600-cooh were 
 measured to be approximately 47 , 23 , and 47 nm , respectively , which 
 was consistent with our previous findings where dm2k - nh2 and dm2k - cooh were larger than dm2k - ac . the zeta - potential measurements 
 confirmed the surface modification , where dm600-cooh and 
 dm600-ac were measured at 21 and 0 mv , respectively , 
 which was significantly different from + 30 mv of dm600-nh2 . the cmcs of the three pdcs were consistent with our previously 
 measured values ( on the order of 10 m ) of pdcs 
 with 2k peg ( pdc2k ) as well as that of lms ( 2.4  
 10 m ) . transmission electron 
 microscopy ( tem ) images ( figure s2 ) revealed 
 that all dm600 were spherical with sizes similar to those 
 of dm2k . interestingly , the 
 core size of the both dm600 and dm2k were similar , 
 even though the pdc600 has a lower hlb ( 16.56 for pdc2k and 12.37 for pdc600 ) , which suggested an increased 
 copolymer aggregation number ( nagg ) for 
 dm600 . collectively , these data demonstrate 
 that the end - group - modified pdcs were successfully prepared and formed 
 dms with various surface groups . number - averaged 
 molecular weight , mn , estimated by h nmr . measured 
 by gpc using dn / dc values . calculated 
 by weight - average molecular 
 weights divided by number - average molecular weights using the data 
 obtained by gpc . not measured 
 due to the limited 
 solubility of amine - terminated pdc in thf . cellular interactions of end - group - modified 
 dms and g4 pamam dendrimers . ( b ) flow cytometry results of the cell association of end - group - modified 
 dms and g4 pamam dendrimers after 2 h of incubation . confocal images 
 of kb cells treated with ( c ) rhodamine - labeled dm2k - nh2 , ( d ) dm600-nh2 , and ( e ) g4 pamam at 
 37 c for 2 h. nuclei were stained using dapi ( blue color ) . note that both amine - terminated dm600-nh2 ( d ) and pamam dendrimers ( e ) exhibit significant 
 cell interactions as opposed to dm2k - nh2 ( c ) , 
 dm600-ac , and dm600-cooh , indicating that the 
 role of the surface charge and peg chain length on cell interactions 
 of dms . the end - group effect of dms 
 with shorter peg chains on their cell 
 interactions was first evaluated in terms of cellular uptake and cytotoxicity . after incubation with kb cells , the in vitro behaviors 
 of dm600 with nh2 , cooh , and 
 ac terminal groups , dm2k - nh2 , and g4 
 pamam dendrimers ( g4-nh2 ) were observed using confocal 
 microscopy and flow cytometry , as shown in figure 1b  e . pdc600-rhod was incorporated at 10% 
 ( w / w ) of each dm600 for fluorescence - based detection . no 
 significant cellular interactions were observed for charge neutral 
 dm600-ac and negatively charged dm600-cooh . the positive control g4-nh2 exhibited a strong , charge - dependent 
 interaction as expected . as we reported earlier , dm2k - nh2 , despite their positively 
 charged surfaces , did not show any noticeable cellular interactions . markedly , dm600-nh2 displayed a significant 
 cellular interaction , which proved our hypothesis that shortening 
 the peg chain length would enhance the end - group effect of dms , converting 
 previously observed charge - independent interactions to be charge - dependent . we verified this result using the mcf-7 cell line as well ( figure s3 ) , which confirmed that the observed 
 charge - dependent cell interactions of dm600 were not cell 
 specific . the charge - dependent cytotoxicity profiles of dm600 were also evaluated using an mts assay ( figure 2 ) . unlike dm2k - nh2 , dm600-ac , and dm600-cooh that all exhibited no apparent 
 cytotoxicity , dm600-nh2 displayed moderate cytotoxicity 
 at 100 m . however , the cytotoxicity of dm600-nh2 appeared to be significantly smaller than that of g4-nh2 , probably due to its 5-fold higher molecular - weight - to - surface - charge 
 ratio than g4-nh2 . these cell uptake and cytotoxicity results 
 indicate that the length of the peg corona modulates the charge - dependent 
 cellular interactions of dms . cell viability of kb cells after treated with 
 end - group - modified 
 dms and g4 pamam dendrimers at concentrations of 1100 m 
 for 24 h , as measured using mts assay . amine - terminated pamam dendrimers 
 induce significant cytotoxicity , and dm600-nh2 induced a modest level of cytotoxicity only at a high concentration . all other micelles that do not show cell interactions do not cause 
 any noticeable cell death . * p < 0.05 compared 
 to the negative control . to better understand 
 these observations , we conducted atomistic molecular dynamics ( md ) 
 simulations of the experimental systems . figures 3a and 3d show the conformation of dm600-nh3 and dm2k - nh3 in 150 mm nacl solution after 30 ns equilibration . both micelles were stable , as determined by 
 the maintenance of their integrity and spherical shape . we also modeled 
 a biological membrane in the same solution , using dipalmitoylphosphatidylcholine 
 ( neutral ) and dipalmitoylphosphatidylglycerol ( negatively 
 charged ) lipid molecules in a 3:1 ratio . then we placed both dms within 
 5  of the membrane and simulated the whole system . over the course of 75 ns simulations , significant 
 differences in the behavior of the two dms were observed . after 30 
 ns of equilibration , individual pdcs were pulled away from the core 
 of dm600-nh3 and drawn toward the 
 membrane surface , due to strong coulombic attraction , as shown in 
 figure 3b . after 60 ns , dm600-nh3 began to flatten and spread over the membrane 
 surface , and at 75 ns it formed a pancake - like shape ( figure 3c ) . coherent  binding of the terminal amine groups 
 of pdcs to the negatively charged membrane . the short peg chains provide 
 direct links between the nh3 groups 
 and the dendron holding them , which means that several nh3 groups attached to the same dendron can act in 
 a concerted way ( like a polyion ) in the presence of charged membrane 
 pulling them by coulombic coupling to the membrane . on the other hand , 
 dm2k - nh3 stably settled on the membrane 
 but did not display any noticeable conformational changes ( figure 3e , f ) . the coulombic binding energies between dms 
 and the membrane were calculated with our code ( no ions considered ) 
 and found to be 17% higher for dm600-nh3 than dm2k - nh3 ( 0.224  
 10 j vs 0.189  10 j ) . these results were in agreement with our in vitro observations of the dm600-nh3 cell 
 insertion ( stronger interactions with the membrane ) . atomistic md simulations 
 of ( a  c ) dm600-nh3 and ( d  f ) 
 dm2k - nh3 in an ionic solution ( 150 
 mm nacl ) with and without 
 lipid membranes . the functionalized peg chains are shown in atomistic details 
 with the amine termini highlighted in red . ( a , d ) show the micelles 
 in solution ; ( b , c , e , f ) show the micelles placed on lipid membranes 
 for 30 ns ( b , e ) and 75 ns ( c , f ) . arrows indicate the stretching 
 and deformation of dm600-nh3 upon 
 interaction with the lipid membrane . next , we analyzed further the mechanism of peg 
 chain - length - dependent 
 cellular interactions of dms . our previous study suggested that the 
 lack of cellular interactions of dm2k - nh2 was 
 in part due to the back - folding of the amine termini into the peg 
 layers , impeding the cellular interactions . we calculated the number of oxygen atoms in the peg chains present 
 within a distance of 2.5  from the terminal amine hydrogens 
 of dm600-nh3 by averaging the results 
 over 300 frames ( 1.2 ns of equilibration ) to quantitate the number 
 of intramolecular hydrogen bonds that can be formed . the average numbers 
 of oxygen atoms were 165  8.9 and 212  8.6 per frame for 
 dm600-nh3 and dm2k - nh3 , respectively . a lower number of oxygen atoms 
 that are available for hydrogen bond formation would indicate less 
 sequestration of the amine termini by peg chains , thus increasing 
 the exposure of amine groups at the dm600-nh3 surface . this phenomenon was also supported by the zeta - potential 
 measurements of dm600-nh2 and dm2k - nh2 , as summarized in table 2 . the higher surface exposure of the amine termini on dm600-nh2 resulted in a higher zeta - potential value ( 30 
 mv ) compared to that ( 21 mv ) of dm2k - nh2 . taken together , these results revealed that charge - dependent end - group 
 effect of dms could be pronounced or sequestered by the number of 
 hydrogen bonds formed between the peg backbone and the amine termini . the increased cellular interactions of dm600-nh2 can be also explained by the intrinsic properties of the 
 corresponding pdcs . the charge - number - to - molecular - weight ratio is 
 approximately 2-fold higher for pdc600 than pdc2k , which accentuates the end - group effect of the dms . furthermore , 
 shorter peg chains lead to a decreased hlb of the copolymer , resulting 
 in a higher nagg of micelles . a dm with a higher nagg contains more pdcs , and thus more terminal groups are expected to 
 be present on its surface . the lower the number of intramolecular 
 hydrogen bonds , the higher the charge - number - to - molecular - weight ratio 
 and the greater nagg , which all contribute 
 to the increased cellular interactions of dm600-nh2 and a more pronounced end - group effect compared to dm2k - nh2 . low - generation pamam dendrimers 
 ( generations 25 ) 
 ( 26 nm ) have been reported to internalize into cells 
 via energy - dependent endocytosis . on the other hand , in addition 
 to energy - dependent mechanisms , energy - independent mechanisms such 
 as nanoscale hole formation have been proposed for larger g7 pamam 
 ( 10 nm ) dendrimers . the nanoscale 
 hole formation causes cytoplasm leakage and is usually accompanied 
 by higher cytotoxicity . since the end groups of dms are coordinated 
 by g3 dendrons and the self - assembled structures are much larger than 
 g7 dendrimers , we sought to determine which dendrimer internalization 
 mechanism governs cell entry of dm600-nh2 . the cellular uptake studies were carried out at 4 c to investigate 
 whether the nonspecific charge - dependent cell interactions of dm600-nh2 were energy - dependent . figure 4a shows that dm600-nh2 was mostly observed 
 on the cell surface without significant cell entry , indicating that 
 their uptake was primarily energy - dependent . additionally , flow cytometry 
 analysis ( figure 4b ) revealed that the cell 
 association of dm600-nh2 at 4 c was only 
 25% of that at 37 c . we then concluded that the cellular internalization 
 mechanism of dm600-nh2 was similar to that of 
 low - generation pamam dendrimers , which was in good agreement with 
 their moderate cytotoxicity shown earlier . however , further investigation 
 is obviously required to fully understand the uptake mechanism of 
 dm600-nh2 . the md simulation results demonstrated that the 
 nonspecific membrane interactions may likely result in a partial pulling 
 of individual pdcs from dm600-nh2 , followed 
 by its large - scale deformations , causing the destabilization of the 
 micelle structure following the cell membrane interaction . since this 
 may deteriorate the therapeutic effect when dms are used as drug carriers , 
 we examined the integrity of dm600-nh2 upon 
 cell internalization . to investigate this , we prepared a dual - dye 
 dm system by encapsulating dio into rhodamine - labeled dm600-nh2 , which enabled colocalization studies using confocal 
 microscopy . as shown in figure 4c  e , 
 red fluorescence ( rhodamine ) and green signals ( dio ) were overlapped 
 inside the kb cells after treatment with dm600-nh2 at 37 c for 4 h. a colocalization analysis of the red and 
 green signals was then performed using imagej . the pearson s coefficient of these two colors was 
 0.939 , which indicated almost complete colocalization of dm600-nh2 with dio . to exclude the possibility that dio was 
 released extracellularly and then cointernalized into cells along 
 with rhodamine - labeled dms , kb cells were also treated with a mixture 
 of dio and rhodamine - labeled dm600-nh2 . the 
 poor correlation ( pearson s coefficient : 0.317 ) between the 
 two signals as shown in figure 4f h 
 confirmed that extracellularly released dio was not cointernalized 
 into cells with dm600-nh2 . these results demonstrated 
 that dm600-nh2 maintained its structural integrity 
 during its nonspecific interaction with the cell membrane and carried 
 the hydrophobic cargo into the cells . ( a ) kb cells treated with rhodamine - labeled 
 dm600-nh2 at 4 c for 2 h. ( b ) flow cytometry 
 histograms representing 
 the cell association of dm600-nh2 at and 37 
 c . the lack of internalization and the significantly reduced 
 cell association at 4 c indicate that dm600-nh2 internalizes into kb cells in an energy - dependent manner . ( c  e ) kb cells treated with dual - dye dm600-nh2 at 37 c for 4 h. ( f  h ) kb cells treated with 
 the mixture of free dio and rhodamine - labeled dm600-nh2 37 c for 4 h. ( c , f : red channel for rhodamine ; d , 
 g : green channel for dio ; e , h : merged images ) the correlation ( pearson s ) 
 coefficients between red and green signals are 0.939 and 0.317 in 
 ( e ) and ( h ) , respectively . the colocalization of the two signals shown 
 in ( e ) confirms that dm600-nh2 internalizes 
 into the cells together with the encapsulated dye . one of the major rationales behind 
 our dm design was to take advantage 
 of the dendritic structure to achieve enhanced structural stability 
 over conventional lbc - based systems owing to its preorganized conical 
 molecular architecture and ability to accommodate a significantly 
 denser outer peg layer . to test this hypothesis , we compared dual - dye 
 dms with lms in terms of their ability to prevent premature release 
 of hydrophobic cargos . kb cells were incubated and monitored after 
 treatment with dual - dye dm600-ac and lms made from pcl3.5k - peg2k - ome . note that we used charge neutral 
 micelles to inhibit their nonspecific cell entry based on electrostatic 
 interactions . as shown in figure 5a , b and figure s4 , the lack of a rhodamine signal indicated 
 that neither dm600-ac nor lms were internalized into the 
 cells even after 48 h of incubation . interestingly , a negligible uptake 
 of dio was observed from kb cells treated with dm600-ac , 
 whereas a significantly stronger dio signal was observed for cells 
 treated with lms . flow cytometry analysis shown in figure 5c further supported that the cell - associated dio 
 signal was higher for the cells incubated with lms than those with 
 dm600-ac . these results are in agreement with a previous 
 report where hydrophobic probes such as dio that were encapsulated 
 into lms composed of pcl  peg or polylactide  peg were 
 released from the micelles extracellularly . because the both micelles showed a great degree of thermodynamic 
 stability ( low cmcs ) and maintained structural integrity at the testing 
 concentration ( 60 g / ml ) as measured by fret ( figure s5 ) , this uncontrolled 
 release was attributed to the hydrophobic cell membranes extracting 
 the hydrophobic cargos from the micelle cores . our results herein 
 indicate the superior stability of dms over lms for hydrophobic drug 
 delivery . despite the similar cmc values of dms and lms , the dendritic 
 architecture , in combination of the dense peg layers of dms , likely 
 allowed enhanced protection of the encapsulated hydrophobic cargo 
 molecules compared to lms . this enhanced stability is particularly 
 important for the future development of targeted nps to minimize premature 
 drug release and ultimately improve therapeutic efficacy . confocal images 
 of kb cells treated with dual - dye ( a ) dm600-ac and ( b ) 
 lbc ( pcl3.5k - peg2k)-based micelles 
 at 37 c for 4 h. images a and b are merged images of red ( rhodamine ) 
 and green ( dio ) channels along with differential interference contrast 
 ( dic ) images . ( c ) flow cytometry data of the cellular association 
 of dm600-ac , lbc - based micelles , and free dio at various 
 time points . scale bar : 10 m . a significantly lower extracellular 
 release of the encapsulated dio was observed in dm600-ac 
 compared to lbc - based micelles , indicating the enhanced stability 
 of the encapsulated dye in dms . here we report that the length of hydrophilic 
 peg chains can control 
 the charged end - group effect of dms . with the short peg chains ( 600 
 g / mol ) , positively charged dm600-nh2 showed 
 charge - dependent nonspecific cellular interactions and modest cytotoxicity , 
 whereas the dms prepared from longer peg chains ( 2000 g / mol ) , dm2k - nh2 , did not . this result was further supported 
 by the md data , where dm600-nh2 exhibited less 
 hydrogen bonding formation than dm2k - nh2 , thereby 
 pronouncing the surface charge effect . surface modification of dm600 to charge neutral or negatively charged groups substantially 
 decreased the charge - based cell interactions . in addition , we found 
 that dm600-nh2 maintained its structural integrity 
 after binding to kb cell membranes and internalized through an energy - dependent 
 pathway . no dms display any noticeable leakage of an encapsulated 
 dye , whereas the lm allowed a degree of dye leakage , indicating that 
 the dense , dendritic peg corona enhanced the encapsulation stability 
 of dms . our results integrating both in vitro and 
 md simulation data support the potential to use dms as a drug delivery 
 platform of which cellular interactions can be facilely modulated 
 by surface functionalization and the peg chain length . this study 
 helps to understand the role of surface charge , peg corona , and polymeric 
 architecture on tuning the biological properties of nps , providing 
 a critical design cue for dms as well as other types of polymeric 
 micelles .