the biocompatibility 
 and ease of programming of peptides have long 
 inspired their development as therapeutics , signaling agents , and sensors . however , significant 
 problems limit their use in vivo , including short durations of activity 
 resulting from rapid digestion by endogenous proteases and efficient 
 renal clearance due to their generally low molecular weights . proteolytic digestion of circulating peptides can be rapid , occurring 
 with half - lives of less than a few minutes , owing to the abundance 
 of active proteases in both serum and tissues . the greatest threats to peptide integrity are found in the lumen 
 of the small intestine , which contains gram quantities of proteases 
 secreted by the pancreas ( i.e. , -chymotrypsin , trypsin , and 
 carboxypeptidases ) , as well as in the brush border membrane of epithelial 
 cells , which houses some 15 peptidases that together cleave amide 
 bonds in peptides and proteins with little specificity . in practice , unmodified therapeutic peptides are typically directly 
 injected at the site of interest to minimize proteolytic degradation , 
 and many are used only as last - resort , salvage treatments in patients 
 with multidrug resistant afflictions . harnessing 
 the inherent specificity , affinity , and low immunogenicity of peptides 
 in therapeutic and diagnostic applications will require the development 
 of simple , widely applicable , and easy - to - access methods that protect 
 active peptides from proteolysis , but do not hinder their function . traditional strategies for limiting enzymatic degradation involve 
 chemical modification of the peptide , including the incorporation 
 of unnatural amino acids ( e.g. , d - amino acids ) , terminal capping via acetylation of the n - terminus 
 or amidation of the c - terminus , introduction of backbone modifications such as n - methylation , use of stabilizing linkers , cyclization , and conjugation to polyethylene glycol ( peg ) . hence , chemistries are chosen such that peptides are no longer recognized 
 by , or become inaccessible to , the active site of a proteolytic enzyme . however , because these strategies modify the connectivity , or amino 
 acid identity of the peptide , they can reduce its bioactivity , often 
 necessitating multiple rounds of structure  function studies 
 to restore the activity of the material . strategies that do not require direct modification of the peptide 
 chemical structure typically involve manipulation of their three - dimensional 
 spatial arrangement via chemical conjugation of the peptide to a higher 
 molecular weight structure . architectures of this type include peptide  polymer 
 conjugates or systems involving the display 
 of multiple copies of the peptide on a small molecule scaffold . however , in practice syntheses of these materials often require 
 multiple conjugation and purification steps , or the preparation of 
 complicated scaffolds that are not generalizable or conveniently deployed . we present a new methodology for protecting active peptides from 
 proteolysis by packaging them into high - density brush polymers via 
 ring opening metathesis polymerization ( romp ) , using an easily prepared 
 initiator . this strategy is inspired by observations we have made 
 in our laboratory over several years , involving the behavior of brush 
 polymers that result from the graft - through polymerization of norbornyl - peptide 
 monomers via romp . specifically , we have observed that polymerization 
 can result in structures that resist proteolysis relative to their 
 monomeric analogues . given these observations , we 
 hypothesized that polymerized peptides , while protected from proteolysis , 
 would maintain their intended biological function and that this phenomenon 
 might be a general feature of peptides arranged in this manner . if 
 so , such an approach could provide a general , accessible route to 
 the development of proteolytically resistant peptide displays capable 
 of performing the functions inherent to the peptide , such as binding 
 a receptor or ligand , initiating a signaling pathway , penetrating 
 a cell , or inducing a therapeutic effect . we envision that such a 
 strategy will provide a feasible route for the preparation and delivery 
 of peptides in future therapeutic applications , where the peptide 
 is active on the polymer or is released at a given place and time 
 via a cleavable linkage . to test the core concept , we prepared 
 two canonical cell - penetrating 
 peptides ( cpps ) , tat ( ygrkkrrqrrr ) and arg8 ( rrrrrrrr ) , as modified norbornene monomers and polymerized them to generate 
 brush polymers via romp . there is a long history of appending peptides 
 with interesting functional properties , such as an ability to penetrate 
 cells or a propensity for localizing inside a cellular 
 nucleus , directly to a material of interest in order to bestow 
 that same property on the new material . indeed , cpps have been used 
 in this regard and have been shown to effectively penetrate cells 
 when chemically conjugated to small molecule drugs , therapeutic peptides , 
 and other structures . while effective for model studies , the downside to 
 this strategy is that cpps ( and all other targeting peptides composed 
 of naturally occurring amino acids ) are generally still susceptible 
 to proteolytic degradation , when conjugated as individual , linear 
 peptide sequences . we reasoned that a 
 cpp organized as a high - density brush polymer might be resistant to 
 proteolysis but still able to efficiently penetrate and carry a cargo 
 into cells . here , we show that polymerized tat and arg8 are at least 
 as efficient at penetrating cells as individual cpps . additionally , 
 we generate 1050 nm diameter spherical micellar assemblies 
 of a cpp - containing brush polymer and demonstrate that this formulation 
 also penetrates cells efficiently . importantly , both polymer and particle 
 formulations of the cpps are resistant to proteolysis under conditions 
 that freely degrade the peptides . therefore , in a critical demonstration 
 of this concept , the materials still penetrate cells after exposure 
 to multiple proteases unlike the standard cpps , which are inactivated 
 by cleavage . the generality of this approach was tested by polymerizing 
 two 
 additional peptide substrates for two different classes of enzymes : 
 a matrix metalloprotease ( mt1-mmp ) and a serine protease ( thrombin ) . these studies further confirm that polymerized peptides are more resistant 
 to proteolytic digestion than their monomeric analogues . we conjectured 
 that this resistance is derived from the high packing density of the 
 peptides in the polymer brush and we tested this concept through both 
 experimental and computational analyses of blend copolymers with varying 
 densities of peptide . computational studies validated experimental 
 findings and supported the notion that copolymerization of the peptide 
 monomer with a monomer containing a short oligoethylene glycol ( oeg ) 
 sequence alleviates steric congestion , and potentially noncovalent 
 interactions among the peptide strands . these phenomena otherwise 
 prevent access to the active site of a protease , and are features 
 that typically impart proteolytic resistance to globular protein structures . together , these data suggest that bioactive peptides can be packaged 
 as polymers to attenuate proteolytic degradation in a tunable fashion , 
 and , since the identity of the amino acid sequence is unaltered , the 
 inherent function of the peptide will , in many cases , be preserved . validation 
 of the proteolytic resistance and bioactivity of polymerized cpps 
 required preparation of well - defined brush polymers with low dispersity 
 via a living polymerization method . high - density brush polymers of 
 known cell - penetrating peptides , tat and arg8 , together with appropriate 
 control polymers , were prepared via living romp by a popular initiator , 
 ( ( h2imes)(pyr)2(cl)2ru = chph ) ( 1 , figure 1 ) . romp by this initiator was selected for preparation of these materials 
 for a variety of reasons . first , the initiator exhibits fast initiation 
 and slower propagation kinetics , which typically afford polymers with 
 exceptionally narrow molecular weight distributions . second , it is 
 highly functional group tolerant , enabling the incorporation of a 
 wide range of chemical functionality via polymerization of groups 
 pendant to a norbornene moiety , including fluorophores , drugs , sugars , oligonucleotides , and peptides . we note that very few polymerization techniques have been shown 
 to incorporate peptides directly by graft - through polymerization from 
 a peptide - containing monomer . reports on graft - through polymerization 
 of peptides by reversible addition  fragmentation chain transfer 
 ( raft ) or free - radical polymerizations describe only blend 
 polymers with less than 50% incorporation of peptides . additionally , 
 the polymers produced by these methods generally have broader molecular 
 weight distributions than those typically afforded by romp . furthermore , a high degree of functionality 
 and complexity can be readily generated on a single polymer via romp 
 by preparing multiblock copolymers of appropriately functionalized 
 norbornene monomers or via the use of chain transfer agents to end - label 
 a polymer through a single cross metathesis event upon complete consumption 
 of monomers . peptides used to generate romp monomers were prepared 
 by solid phase peptide synthesis ( spps ) using standard fluorenylmethyloxycarbonyl 
 ( fmoc ) chemistry . peptide monomers were prepared by coupling a carboxylic 
 acid - modified norbornene to the n - terminus of the 
 desired peptide sequence on resin . we have shown previously that peptide 
 monomers with side chain protecting groups and a five - carbon linker 
 between the peptide and the norbornene unit generally polymerize at 
 faster rates than those with shorter linkers or those that possess 
 no protecting groups . therefore , we prepared 
 all peptide monomers with this linker and used an arg8 monomer with 
 side chains protected . however , efforts to prepare and polymerize 
 the protected tat peptide were thwarted by poor solubility of the 
 protected material in solvents compatible with the romp initiator . 
 therefore , the tat peptide monomer was prepared without protecting 
 groups ( see figure s1 in the supporting information for chemical structures 
 of monomers ) . all peptides that were incorporated into polymers were 
 also separately prepared ( without norbornyl - groups ) as fluorescein - labeled 
 peptides via conjugation of 5/6-carboxyfluorescein to the -amino 
 group of an n - terminal lysine , for use as controls 
 to evaluate the cellular uptake efficiency of the peptides alone versus 
 polymerized materials . ( see figure s2s3 
 and table s1 for characterization data of the peptide controls . ) routes to the preparation of ( a ) homopolymers and ( b ) 
 block copolymers . note that the guanidinium moiety and arg8 peptide 
 are polymerized with protecting groups and deprotected after polymerization 
 by treatment of the polymers with a tfa solution . for each polymer , m and n are the degrees of polymerization 
 ( dps ) given in table 1 . each peptide - based monomer was polymerized , and the resulting 
 polymers 
 were end - labeled with fluorescein to enable tracking of the uptake 
 of the material and to serve as a model cargo ( figure 1a ) . in addition to polymers containing the canonical tat and 
 arg8 cpps , several control polymers were prepared , including a polymer 
 of an uncharged , nonpeptide unit of oligoethylene glycol ( oeg ) and 
 another consisting of a peptide side chain that did not contain any 
 charged residues ( gsgsg ) . for comparison , a polymer composed of monomers 
 bearing a single guanidinium moiety was 
 prepared as a graft - through analogue of polymers prepared via the 
 graft - to technique employed in other studies . we note that these graft - to guanidinium - containing polymers are 
 the only other cell penetrating polymers prepared by romp techniques 
 that have been reported to date . the graft - to guanidinium polymer 
 prepared in this work displayed poor solubility as a homopolymer and 
 was therefore prepared as a block copolymer with a water - solubilizing 
 oeg monomer ( figure 1b , where r1 = oeg and r2 = guanidinium ) . after polymerization , the 
 polymers were characterized by size - exclusion chromatography with 
 multiangle light scattering ( sec - mals ) to ascertain degree of polymerization 
 ( dp ) and molecular weight distribution ( dispersity or mw / mn ) ( table 1 and chromatograms in figure s4 ) . good agreement between the obtained dp and the theoretical dp 
 based on the initial monomer - to - initiator ratio ( [ m]0/[i]0 ) was observed . further , all dispersities were less than 1.11 , 
 indicating the expected narrow molecular weight distributions . block m and n refer to the first and second block to be 
 polymerized 
 as shown in figure 1 . each polymer is named 
 according to the identity of the monomer polymerized as drawn in figure 1 . the 
 dispersity of each block . experimentally determined degree 
 of polymerization for block m and block n as denoted in figure 1b , with theoretical 
 values based on the amount of material used , in parentheses . amphiphilic polymers were formulated 
 into nanoparticles , denoted in the text as the gsgsg particle and 
 tat particle . all data were obtained by sec - mals , except for those 
 describing the tat polymer , which did not elute on the sec column 
 and was instead characterized in a cuvette using batch - mode static 
 light scattering . without the sec component , no information on the 
 molecular weight distribution of this polymer was obtained . however , 
 the amphiphilic polymer that contains tat ( phenyl - b - tat ) eluted well on sec and yielded close to the predicted dp in 
 low dispersity . repeated polymerizations of peptide - containing homopolymers 
 yield consistent numerical values . the tat peptide - containing 
 homopolymer , lacking side - chain protecting 
 groups , performed poorly on sec - mals , presumably due to unfavorable 
 interactions of the peptide with the size exclusion column . therefore , 
 no information on the molecular weight distribution of these polymers 
 was obtained , but a molecular weight determination was achieved by 
 measuring the bulk light scattering of the solution in a cuvette ( without 
 the size exclusion column ) ; and complete consumption of the tat monomer 
 after polymerization was verified by h nmr ( figure s5 ) . given the complexity of the 
 tat and arg8 peptide - containing polymers 
 ( i.e. , multiple charged and nucleophilic side chains ) , we investigated 
 whether romp of these materials proceeds in a living fashion , in order 
 to ensure that well - defined and well - ordered structures , devoid of 
 cross - metathesis or premature termination , could regularly be accessed 
 by this strategy . confirming the living nature of the polymerization , 
 a plot of mn ( obtained by sec - mals ) vs 
 [ m]0/[i]0 for the arg8 monomer yields a linear 
 fit for [ m]0/[i]0 less than 9 ( figure 2a and table 2 ) . at larger 
 [ m]0/[i]0 ratios , propagation ceased , presumably 
 due to steric hindrance encountered from assembling multiple copies 
 of the long , side - chain protected peptide sequence , whose molecular 
 weight as a monomer is 3.5 kda . a similar plot was obtained from data 
 gathered for polymerization of the tat polymer , collected by static 
 light scattering ( sls ) in a cuvette ( figure 2b and table 2 ) . therefore , we conclude that 
 both cpp monomers are polymerized in a living fashion to a dp of < 9 , 
 despite the complexity and functionality of their side chains , making 
 this an exceptionally convenient strategy for predictably generating 
 polymeric architectures from peptide monomers . in addition to 
 exploring the activity of a single polymer chain , 
 we also aimed to examine the proteolytic resistance and bioactivity 
 of large assemblies of peptide - containing polymers . we envisioned 
 that nanoscale assemblies of multiple peptide  polymers would 
 be large enough to avoid renal clearance thresholds in future applications 
 that might otherwise prevent long circulation times of peptides or 
 lower molecular weight polymers , but it was unclear whether these 
 large assemblies would resist proteolysis , or enter cells . to generate 
 nanoparticles , we prepared amphiphilic polymers of two peptides : tat 
 and a gsgsg control peptide ( figure 1b . note : 
 arg8-based nanoparticles could not be generated with consistently 
 spherical morphologies matching gsgsg controls or tat systems and 
 hence were excluded from this study ) . the design of these amphiphiles 
 is such that the phenyl - modified norbornene monomers operate as hydrophobic 
 moieties to drive self - assembly of the amphiphiles into micellar nanoparticles 
 containing many copies of polymer . to prepare these amphiphiles , a 
 hydrophobic monomer was polymerized to completion prior to addition 
 of the peptide monomer to the living polymer ( figure 1b , where r1 is phenyl and r2 is gsgsg 
 or tat ) . self - assembly of these amphiphilic polymers into a nanoscale 
 structure was then accomplished by slow dialysis of the material from 
 an organic cosolvent , in which the amphiphile is completely dissolved 
 ( dmf ) , into a selective solvent , in which only the peptide brush is 
 soluble ( aqueous phosphate - buffered saline , pbs ) . the amphiphilic 
 polymers of tat and gsgsg were found , by dls and tem , to form spherical 
 micelles of 1050 nm diameter ( figure 3 ) . plots correlating the number - average molecular weight ( mn ) with the initial monomer  to - catalyst 
 ratio ( [ m0/i0 ] for the polymerization of the 
 arg8 monomer ( a ) and the tat monomer ( b ) . all 
 data for the arg8 monomer polymerization were collected by sec - mals . data for the tat monomer polymerization were obtained in a cuvette 
 via static light scattering . the data reported represent values from 
 a single batch of polymerizations performed on the same day . tem images and dls 
 data for the tat particle ( a , c ) and the gsgsg particle ( b , d ) . fluorescence - based in vitro 
 assays were performed in hela cells to compare the cellular uptake 
 of the peptide controls , polymers , and nanoparticles . the goal was 
 to determine whether polymerization of the cpps had an impact on their 
 ability to facilitate cellular entry or on their mechanisms of cellular 
 uptake . in these studies , flow cytometry was used to quantify the 
 amount of cellular uptake , and live - cell confocal microscopy was used 
 to verify internalization and examine the localization of the internalized 
 material . in flow cytometry experiments , relative to the vehicle 
 control ( pbs ) , all cpp - containing peptide polymers and particles gave 
 robust fluorescent counts , approximately 2-fold higher than those 
 of the peptides alone ( figure 4 and individual 
 histograms in figures s6s8 ) . these 
 data verify that cpps maintain or have enhanced function when incorporated 
 into brush polymers or larger polymeric assemblies . we also note that 
 the tat and arg8 polymers gave responses similar to those of the guanidinium 
 polymer , which is an analogue of the only other cell - penetrating romp 
 polymer reported to date . quantitative comparison of cellular uptake of 
 peptides , polymers 
 and particles at 2.5 m after 30 min incubation with hela cells 
 by flow cytometry . on the y - axis , normalized mean 
 fluorescence refers to the mean fluorescence counts detected for the 
 material divided by the mean fluorescence counts exhibited by the 
 vehicle control ( pbs ) . representative histograms showing the fluorescence 
 counts for each material are given in figures 
 s6s8 . to probe whether the cellular uptake of the polymerized materials 
 was due to the peptide amino acid sequence and not the polymer backbone 
 itself , or the result of the arrangement of any peptide into a brush 
 polymer , we investigated the uptake of polymeric materials containing 
 an oeg brush and a gsgsg brush , both of which do not enter cells as 
 their monomer units . the control materials showed negligible fluorescence 
 signals ( less than a 2-fold increase in fluorescence relative to vehicle ) , 
 similar to the small molecule fluorescein tag itself . therefore , these 
 data indicate that the amino acid sequences of tat and arg8 drive 
 the internalization of the polymers . live - cell confocal images of peptides , 
 polymers and nanoparticles 
 labeled with fluorescein . scale bars are 50 m . 
 for the tat variants , the six individual z - slices that were used for 
 the averaged images shown in this figure are provided in figures s9s11 . it was also important to confirm that the fluorescence observed 
 in the initial studies resided within the cytoplasm , rather than on 
 the cell s external surface . to this end , we chose to perform 
 live - cell confocal microscopy , because fixation of cells by formaldehyde , 
 methanol or other agents , can cause artifacts due to the release of 
 fluorescently labeled materials entrapped in endosomes . in particular , we performed z - stack analyses at 1 m step 
 sizes on live cells treated with each peptide - based material . across 
 multiple z - slices for cells treated with all tat- , arg8- and guanidinium- 
 containing materials , at the same concentration used in flow cytometry 
 experiments , a combination of punctate and diffuse fluorescence was 
 observed ( figure 5 , for individual z - slices , 
 see figures s9s11 ) , indicative 
 of compartmentalized and cytosolic localization , respectively . by 
 contrast , no fluorescence was seen for any of the negative controls 
 ( gsgsg and oeg polymers ) , which do not contain cationic moieties and 
 do not penetrate cells in any detectable manner . we also verified 
 that polymerization did not render the peptides 
 toxic to cells . the viability of cells treated with the tat and gsgsg 
 peptide , polymer and nanoparticles was assayed via the celltiter - blue 
 assay . when compared to the vehicle control , hela cells treated with 
 all formulations of the materials at 5 m , twice the concentration 
 used in the uptake studies described above , remained > 92% viable 
 for 
 48 h ( figure s12 ) . there is 
 much debate in the literature over the mechanism of entry of cpps . however , it is generally agreed that the cellular uptake of these 
 materials requires association with anionic species at the cell membrane 
 ( i.e. , sulfated proteoglycans or phospholipid polar headgroups ) followed 
 by internalization via endocytosis or membrane disruption . to investigate whether the monomeric , polymeric , and nanoparticle 
 formulations of the cpps follow similar internalization routes , we 
 subjected cells to thermal inhibition and common pharmacological compounds 
 that disrupt different aspects of membrane trafficking and endocytosis . this resulted 
 in a dramatic decrease in the fluorescent signals for the tat , arg8 , 
 and guanidinium polymers and nanoparticles by flow cytometry , but 
 had no influence on the values from the gsgsg controls ( figure 6a ) . similar effects were seen with an inhibitor 
 of dynamin - dependent endocytosis ( dynasore ) and also with methyl--cyclodextrin ( mcd ) , an agent known to remove membrane cholesterol , and thereby alters 
 the fluidity of the membrane . each condition resulted in no change 
 in the fluorescence values obtained for the gsgsg controls , which 
 is consistent with the notion that these uncharged materials do not 
 internalize . the polymers containing guanidinium , tat and arg8 side 
 chains all showed uptake , as is expected for these modes of inhibition . 
 note that , in all cases , pharmacological and thermal inhibition exhibit 
 only a small effect on the flow cytometry readings of the tat and 
 arg8 peptides . this behavior for cpp - type peptides has been observed 
 by others . specifically , dowdy and co - workers have suggested that 
 the apparent temperature - independence of peptide uptake is due to 
 false - positive readings resulting from membrane - bound , noninternalized 
 peptides . to circumvent this problem , the cells could be washed with heparin , a polyanionic saccharide , 
 which competes with the cell membrane for binding of the polycationic 
 cpps . in the dowdy work , heparin washes significantly reduced the 
 aberrant signals seen in flow cytometry recordings during incubation 
 at 4 c , but did not completely abolish them , as observed in 
 our studies ( figure 6a , tat and arg8 peptide 
 data ) . together , these results suggest that cell penetration of the 
 peptides ( individual cpps ) is due , in part , to membrane disruption 
 or endocytotic processes and that these mechanisms of entry are maintained 
 or enhanced upon polymerization . to verify that the wide range 
 of components found in fetal bovine 
 serum ( fbs ) do not play a role in facilitating or inhibiting cellular 
 entry of the materials , no significant difference in mean fluorescence from any material 
 was observed by flow cytometry in the presence or absence of fbs , 
 suggesting that fbs components , such as growth factors , lipids , hormones , 
 etc . , do not influence uptake of these materials ( figure s13 ) . in order to examine how the concentration 
 of the tat peptide - containing 
 materials impacted their cellular uptake , we performed flow cytometry 
 experiments at several concentrations of material . it is important 
 to note that concentration in these experiments was with respect to 
 the fluorophore , where there is one fluorophore per peptide or polymer , 
 but many copies of fluorophore per particle . ( for concentration determination , 
 see figure s14 . ) in general , the peptide 
 is less competent in cell penetration than the polymer or particle 
 formulations , with cellular uptake of the peptide nearly abolished 
 at 1.25 m . this is in contrast to the polymer and nanoparticles 
 that were still taken up by cells at concentrations as low as 0.5 
 m ( figure 6b ) . hela cells were 
 pretreated with mcd ( 9.5 mm ) or dynasore ( 80 m ) for 
 30 min prior to incubation with the material of interest or preincubated 
 at 4 c . all reported flow cytometry data are described as 
 a fold - shift relative to the vehicle control . studies on linear peptides have 
 shown that 816 guanidiniums 
 are optimal for cell penetration , with activity dramatically decreasing 
 when over 16 guanidiniums are used . likewise , polynorbornyl polymers bearing guanidinium 
 moieties showed decreased internalization when 25 guanidiniums were 
 incorporated , compared to when 10 were employed . therefore , it is somewhat surprising that we see such efficient 
 penetration for the tat side chain 5-mer homopolymer and nanoparticle , 
 which contains at least 30 guanindinium units ( figure 6b ) . in fact , the polymer penetrates cells as efficiently ( within 
 a factor of 2 fluorescence counts ) as the relevant peptide analogue , 
 even in scenarios in which 5-fold fewer fluorophores are present to 
 achieve the same effective concentration of peptide ( such as 2.5 m 
 tat polymer and 12.5 m tat peptide ) . these data suggest that 
 the arrangement of the brush polymer may aid in the cellular uptake 
 mechanism , which could require assembly of multiple cpps for proper 
 transport across the membrane . bundles and direct transportation of these bundles across the membrane 
 has been proposed for many years as the so - called  carpet mechanism  . alternatively , efficient cellular entry could be due to tangled 
 pendant peptide chains presenting a lower effective number of charged 
 residues to the cell membrane . we next assessed whether these cell - penetrating 
 materials were resistant to proteolysis , as hypothesized . we focused 
 on the proteolytic cleavage of materials containing the tat peptide , 
 given that it has a more diverse amino acid sequence than the arg8 
 peptide and would therefore have more unique cleavage sites . tat - containing materials , at the same concentration used in flow 
 cytometery and confocal microscopy studies described above ( 2.5 m ) , 
 were challenged for 20 min with various proteases at high enzyme concentration 
 ( 1 m ) prior to determining the extent of proteolytic 
 cleavage and residual bioactivity . such activity was assayed by three 
 separate methods : reverse - phase high - performance liquid chromatography 
 ( rp - hplc ) , flow cytometry , and confocal microscopy . in these assays , rp - hplc was used to determine the degree to which proteolytic treatment 
 degrades the integrity of the peptide as a monomer or as part of a 
 polymeric formulation . the bioactivity of enzymatically digested materials 
 was then assessed in cellular assays by both flow cytometry and confocal 
 microscopy . to determine whether the location of the peptide cleavage 
 site(s ) affects the sensitivity of the peptide to enzymatic cleavage , several different proteases were tested : trypsin ( 7 predicted cleavage 
 sites ) , chymotrypsin ( 2 predicted cleavage sites ) , and the protease 
 cocktail pronase , which has the potential to digest the peptide backbone 
 at every amino acid position . we employed rp - hplc to assess 
 the percent of intact material following 
 enzymatic digestion ( figure 7a ) . standard curves 
 were generated that compared peak areas of the uncleaved tat peptide , 
 polymer , or nanoparticle at an appropriate concentration range , such 
 that the concentration of material remaining after incubation with 
 enzyme could be estimated ( figure s15 ) . note , this is only an estimate of polymer or particle concentration , 
 as the absorbance measurements of the intact polymer will be affected 
 by the norbornyl polymer backbone , the phenyl coblock , and the fluorescein 
 end - label , which will still be present after proteolytic digestion . 
 in these assays , no differences in the peak area or retention time 
 of the polymer or particle were observed after treatment with any 
 of the proteases tested and the rp - hplc chromatograms are identical 
 with and without enzyme treatment ( i.e. no new peaks 
 formed in the chromatogram ) ( figure s16 ) , suggesting that the tat polymers and particles are resistant to 
 proteolysis . by contrast , complete consumption of the tat peptide 
 was detected , along with the appearance of new peaks in the rp - hplc 
 chromatograms . correspondingly , the mean fluorescence counts of the 
 polymer or particle measured by flow cytometry are largely unaffected 
 by protease treatment ( presumably since the peptide chains have not 
 been digested ) . however , proteolytic digestion of the tat peptide 
 diminished the intensity of the fluorescence signal to less than 10% 
 of the value obtained prior to enzymatic digestion ( figure 7b ) . the same trends were observed by both rp - hplc 
 and flow cytometry when the peptide concentration was kept uniform 
 ( 12.5 m peptide , 2.5 m polymer ) to normalize the number 
 of potential cleavage events . this is an especially notable difference 
 given that the peptides had 5 times the number of fluorescein ( 1 per 
 peptide ) equivalents per peptide than the polymer ( 1 per 5 peptides ) . furthermore , a time - course plot of rp - hplc and flow cytometry data 
 reveal that the tat polymer is stable to chymotrypsin treatment over 
 14 h and the material retains the ability to enter cells after incubation 
 with the enzyme ( figure s17 ) . finally , 
 confocal microscopy was used to verify trends observed 
 by rp - hplc and flow cytometry . in these experiments , the tat peptide , 
 tat polymer , and tat nanoparticle were pretreated with chymotrypsin 
 ( under identical conditions as used in the flow cytometry and rp - hplc 
 assays ) prior to incubation with hela cells . these cells were then 
 imaged by live - cell confocal microscopy alongside cells incubated 
 with the same materials that had not been subjected to the enzyme 
 pretreatment . a dramatic comparison emerges in which cells treated 
 with protease - digested tat peptides show minimal fluorescence relative 
 to those treated with undigested peptides ( figure 7c ) . in stark contrast , the tat polymer and particles give 
 identical fluorescence images with or without enzyme treatment . assessment 
 of the effects of protease treatment on the integrity 
 and bioactivity of the tat peptide , polymer , and particle by three 
 methods . ( a ) rp - hplc data showing the quantity of remaining material 
 postenzymatic treatment . standard curves comparing peak area to the 
 concentration of the intact peptide , polymer , or particle were prepared 
 to determine percent cleavage ( figure s15 ) . data is reported as the percentage of fluorescence seen after enzyme 
 treatment relative to the value seen without treatment . ( c ) confocal 
 microscopy images comparing cells incubated with materials that have 
 been pretreated with chymotrypsin alongside cells incubated with materials 
 that have not received this pretreatment . scale bars are 50 
 m . in all cases tat - containing materials at the indicated concentrations were treated with 1 m of protease for 20 min at 37 c . to test whether our strategy could be 
 extended to different proteases and to peptide sequences other than 
 the highly charged tat and arg8 sequences , we polymerized two additional 
 peptide substrates . importantly , the two peptides each have a more 
 extensive sampling of amino acid side chains compared to the cpp sequences 
 and are optimized substrates for degradation by two different enzymes : 
 a serine protease and a metalloprotease . we first examined the 
 generality of our approach by preparing a peptide substrate for thrombin , 
 a coagulation factor protease . a monomer bearing the thrombin substrate 
 sequence ( galvprgs ) was readily prepared via spps with a short , water - solubilizing 
 peptide sequence ( gerdg ) at the c - terminus ( figure s18 ) , and was polymerized by romp to several 
 degrees of polymerization ( characterization data for polymers are 
 given in figure s19 and table s2 ) . the 
 monomer peptide and homopolymers were treated with thrombin , and the 
 resulting product mixture was analyzed by rp - hplc ( figure s20 ) . these analyses indicate that the monomeric peptide 
 was readily degraded by thrombin , as evidenced by the disappearance 
 of the monomer peak and corresponding appearance of product peaks , 
 however , homopolymers at several degrees of polymerization were resistant 
 to cleavage relative to the monomer , albeit not completely shut off 
 from proteolysis , confirming the generality of the approach ( figure 8a , b ) . chemical structures and cleavage kinetics of 
 monomeric and polymeric 
 peptide substrates for thrombin and the matrix metalloprotease ( mt1-mmp ) . ( a ) structures of a set of homopolymers containing a thrombin peptide 
 substrate . ( b ) cleavage kinetics of thrombin - sensitive monomers and 
 homopolymers at dp = 10 , 20 , and 30 . ( d ) cleavage kinetics of the fluorogenic homopolymer 
 relative to monomer , by multiple proteases in addition to the protease 
 for which the substrate is optimized , mt1-mmp . we next examined an optimized peptide substrate sequence 
 for a 
 cancer - associated membrane - bound matrix metalloprotease ( mmp ) . here , we omitted the n - terminal 
 cys residue from the optimized peptide sequence , crpahlrdsg , because 
 free thiols ( lacking protecting groups ) are difficult to polymerize 
 by romp , due to coordination to the initiators . since this peptide was not expected to function in an orthogonal 
 bioactivity assay , as for the cpp studies above , we prepared the sequence 
 as a fluorogenic substrate , to readily and rapidly quantify cleavage 
 events at low concentrations of material to obtain detailed kinetic 
 information . for more information on the preparation of monomers , 
 polymers and additional assays and kinetics details , see the supporting information and also figures s21s29 
 and tables s3s4 . in kinetic assays , the fluorogenic 
 monomer was readily cleaved 
 by an assortment of proteases ( figure 8c , d ) , 
 but not by mmp-9 for which it is not a substrate ( figure s28 ) . in contrast , the homopolymer ( dp = 20 ) exhibits 
 very little cleavage upon treatment with multiple proteases , as seen 
 in the 24-h time course plots ( figure 8b ) . 
 initial enzymatic reaction rates ( v0 ) , 
 obtained by monitoring each reaction for the first 40 min ( less than 
 25% cleavage seen for all materials ) , indicate that the monomer is 
 cleaved 17- to 95-fold faster than the homopolymer at comparable peptide 
 concentrations ( table 3 ) . menten 
 plots were obtained for the cleavage of the monomer by mt1-mmp , yielding 
 a specificity constant ( kcat / km ) of 0.52  mmin and a km of 11 m 
 ( figure 9a ) . a michaelis  menten plot 
 of the homopolymer time - course data reveals that saturation kinetics 
 have not been reached at 7 times the calculated km of the monomer , as suggested by the near linear fit 
 to the data obtained ( figure 9b ) . note that 
 the fluorescence observed in assays of the homopolymer approached 
 the lower limit of detection , resulting in large standard errors in 
 the data . nevertheless , 
 these data clearly indicate that the protease exhibits lower affinity 
 for and activity on the peptide substrate when it is incorporated 
 into a brush polymer . the fluorogenic 
 peptide is optimized so that it is not a substrate for mmp-9 . proteolysis of 
 ( a ) the fluorogenic 
 monomer where kcat = 5.7  0.4 min , km = 11  2 m , 
 and kcat / km = 0.52  mmin and ( b ) the fluorogenic homopolymer by mt1-mmp , where the estimated km is > 70 m . we were curious about the origin of proteolytic 
 resistance and also recognized that , in certain circumstances , it 
 might be disadvantageous to render a peptide entirely refractory to 
 proteolysis , for example , in the case of a peptide sensor for a protease , or when designing a device that targets tissue or releases a drug 
 in response to proteolytic digestion . therefore , 
 we sought to tune the proteolytic susceptibility of the fluorogenic 
 substrate for mt1-mmp described in the previous section . we envisioned 
 that the proteolytic resistance of homopolymers might result from 
 packing or other stabilizing peptide  peptide interactions , 
 leading to steric protection against enzymatic cleavage . this picture 
 led to the hypothesis that proteolytic susceptibility would be restored 
 by spacing the peptides out along the polymer backbone . spacing was 
 accomplished by preparation of random blend copolymers that incorporated 
 a monomer  spacer  or  diluent  at varying 
 blend ratios ( figure 10 ) . the spacer we chose 
 was a water - soluble oeg monomer , which is inert to proteolytic enzymes . random blend copolymers ( total dp = 20 ) were prepared at substrate 
 to oeg ratios of 1:19 , 5:15 , 10:10 , and 15:5 ( figure 10a ) . a detailed description of the preparation and characterization 
 of these blend polymers is given in the supporting 
 information ( figure s30 and table s5 ) . a general trend emerged 
 in which proteolytic activity of mt1-mmp was greatest when more spacers 
 were incorporated . indeed , the 1:19 blend polymer proved to be as 
 susceptible to proteolytic degradation as the substrate monomer ( figure 10b ) . these data suggest that the protection from 
 proteolysis observed in our systems does not result simply from the 
 attachment of the peptide to a high molecular weight polymer , but 
 rather from its arrangement into a high - density peptide brush . chemical 
 structure of the random blend copolymers and cleavage 
 kinetics of the monomer , homopolymer , and a series of random blend 
 copolymers of the fluorogenic peptide substrate with oeg diluent . ( a ) structure of a series of random blend copolymers of the fluorogenic 
 peptide substrate monomer and an oeg monomer . ( b ) comparison of the 
 cleavage kinetics of the fluorogenic random blend copolymers , ( ratio 
 is m : n as shown in a , overall dp 
 = 20 ) homopolymer ( dp = 20 ) and monomer . to further examine the 
 proteolytic susceptibility trends observed with the random blend copolymers 
 of the fluorogenic substrate and oeg moiety , a series of molecular 
 dynamics simulations were performed , examining analogous blend copolymers 
 with discrete structures and no dispersity ( i.e. , a single molecular 
 entity was modeled for each structural analogue ) . for computational 
 simplicity , all polymers were constructed in silico to have a dp of 
 10 , instead of 20 , in four key arrangements meant to best simulate 
 idealized scenarios : a homopolymer of ten repeated fluorogenic substrates 
 ( figure 11a ) ; two blend copolymers with an 
 oeg : peptide ratio of 9:1 , one having the peptide at one end of the 
 polymer ( figure 11b ) , and the other having 
 the peptide at position five ( figure 11c ) ; 
 and one with an intermediate peptide : oeg ratio of 5:5 ( figure 11d ) . simulations were performed on each 
 structure , starting with the molecule in an artificial , extended conformation 
 with a straight norbornyl backbone , extended peptides , and oeg brushes 
 arrayed at right angles to the polymer backbone . in the simulations , 
 each structure was equilibrated for an initial 20 ns at 300 k , after 
 which each simulation was split and continued in two ways : one simulation 
 for each molecule was continued for 100 ns at 300 k and the other 
 was further randomized by a single heating ( 500 k ) and cooling ( 300 
 k ) cycle before continuing at 300 k for remainder of the 100 ns simulation . in every simulation , the initial extended structure of each molecule 
 collapsed quickly into a more compact conformational ensemble . representative 
 conformations of each construct were obtained by applying a root - mean - square 
 deviation ( rmsd)-based clustering algorithm to the last 40 ns of the 
 respective heat  cool simulations . in these structures , the 
 homopolymer and 5:5 copolymer have collapsed into an elongated globule , 
 with their peptide chains tangled around the polymer backbone . in 
 contrast , the single peptide chain in the 9:1 copolymers , are visible 
 as relatively isolated components at the surface of the constructs . 
 a detailed discussion of differences in radius of gyration of each 
 structure and also on conformational fluctuations in the structures 
 as quantified by rmsds is given in the supporting 
 information and figures s31s33 and table s6 . we examined the role of hydrogen bonding in facilitating the compression 
 or tangling of the peptide - containing structures by computing the 
 numbers of intrasolute hydrogen - bonds during the last 40 ns of each 
 heat  cool trajectory ( table s6 ) . the homopolymer averaged 0.5 amino acid - amino acid peptide bonds per 
 residue ( 88 interactions over 17 residues per monomer ) , which is about 
 half the ratio typically seen for a folded protein . the three copolymers averaged somewhat fewer ( 0.4 ) hydrogen 
 bonds per residue , with very few hydrogen bonds to the oeg moieties 
 ( less than four in all cases ) . overall , these hydrogen bond counts 
 are consistent with a view that , although the polymers have collapsed , 
 they are not as well structured as typical globular proteins and the 
 inclusion of oeg units leads to a decrease in hydrogen bonding . thus , 
 the addition of oeg units effectively  dilutes  the 
 density of the peptide - brush by reducing the overall degree of hydrogen 
 bonding in the polymer structure . moreover , the oeg moieties block 
 what would otherwise be stabilizing interactions among the peptides 
 brushes without compensating with new oeg - peptide interactions . representative 
 conformations and surface - accessibility data for 
 in silico models of the fluorogenic substrate / oeg copolymers . chemical 
 structure and a representative conformation of the discrete , monodisperse , 
 simulated polymers : homopolymer ( a ) ; the 9:1 ratio blend copolymer 
 with the peptide at the end of the polymer ( b ) ; the 9:1 ratio blend 
 copolymer with the peptide in the middle ( i.e. , position 5 ) ( c ) ; and 
 the intermediate 5:5 ratio blend copolymer ( d ) . ( e ) plots of the probe - accessible 
 surface area of the four structures , averaged over the last 40 ns 
 of each heat  cool cycle . blue bars represent the surface area 
 accessible per peptide to a spherical probe with a radius of 3.14 
 nm ( size on the order of a typical protease ) and the gray bars represent 
 the same measurement using a probe radius of 0.14 nm ( approximately 
 the size of a water molecule ) . finally , the accessibility of the peptide components of the 
 various 
 constructs to large and small molecules was examined by computing 
 their time - averaged probe - accessible surface area ( sa ) , using a large 
 ( 3.14 nm ) probe sphere whose size is similar to that of a protein , 
 and a small ( 0.14 nm ) water - sized probe sphere ( figure 11e ) . a general trend was seen with the large probe , where larger 
 surface accessibility was seen as the peptide content decreased . the 
 greatest accessibility is observed for the 9:1 copolymer with the 
 peptide at position 5 . this is the closest representation to the experimental 
 19:1 random blend copolymer ( based on the polymerization method , figure s30 and table s5 ) , which is nearly as 
 susceptible to proteolytic degradation as the peptide monomer in vitro . in contrast , the probe - accessible surface areas obtained with the 
 water - sized probe are relatively uniform across all constructs . these 
 results suggest that the peptides in all five constructs maintain 
 similar accessibility to small molecules , like water , but that the 
 tighter packing of the more peptide - rich constructs reduces accessibility 
 to protein - size molecules , such as the proteases examined experimentally . 
 moreover , these data suggest that peptides whose function depends 
 on interaction with a small molecule or a receptor with a relatively 
 accessible binding site will exhibit ample binding to the peptide 
 substrates in any of the constructs considered , including the homopolymer . the implication of this finding is that the bioactivity of many peptides 
 will be maintained after polymerization of the sequence into a high - density 
 brush . for peptides whose function depends on interaction with proteins 
 or macromolecules with tight or cramped binding pockets ( such as a 
 protease ) , polymerization into a high - density brush polymer may impede 
 function , as is consistent with the data presented in this work . in sum , the simulation results suggest that all of the constructs 
 studied here tend to collapse into fairly compact globular conformations , 
 and that a higher peptide content leads to formation of more stabilizing 
 intramolecular hydrogen bonds and reduced accessibility of the peptides 
 to proteins in solution . this picture is qualitatively consistent 
 with the experimental observation that constructs with high peptide 
 content are better protected from enzymatic degradation . although 
 the simulations are subject to error due to their limited duration 
 and uncertainties in the force field , they yield a usefully detailed 
 representation of the systems under study and offer a plausible explanation 
 for the key experimental results . electrostatic repulsion is 
 another potential contributor to the 
 proteolytic protection observed in these systems . wooley and co - workers 
 recently reported that charge - matched nanoparticles and proteases 
 show a decrease in proteolytic activity , presumably due to repulsion 
 at large charge densities . given the 
 range of proteases used in the present study , including protease cocktails , 
 it is difficult to correlate the proteolytic susceptibility of the 
 polymers with the pairing of the overall polymer charge with the isoelectric 
 point of each enzyme . however , tuning chargecharge repulsion 
 along with steric interactions could provide an additional route for 
 optimization of related systems in future work . herein we present a new , easily deployed methodology for formulating 
 peptides into well - defined brush polymers , which preserve bioactivity 
 but protect the peptides from proteolysis . our strategy involves the 
 direct ( graft - through ) polymerization of peptide - containing norbornene 
 monomers via romp . this obviates the need for the extra purification 
 steps required for more traditional synthetic routes to peptide - containing 
 polymers , which involve chemical conjugation of the peptide to a preformed 
 polymer , as would be required in a graft - to synthesis scheme . the 
 wide range of peptide substrates and proteases for which the present 
 strategy succeeds suggests that it could be applicable to many peptide 
 based therapeutic agents or biosensors that must retain function despite 
 exposure to harsh proteolytic milieu . this approach offers an attractive 
 alternative to existing methods of protecting peptides from proteolysis 
 because it is simple , does not alter the amino acid sequence of the 
 peptide , and enables easy functionalization with other useful moieties 
 or cargo . it is possible that polymerization of a peptide into 
 a high - density 
 brush could not only render the peptide resistant to proteolysis , 
 but also affect its function . the computational data suggests that 
 peptides whose functions rely upon interaction with a large molecule 
 or with a tight or cramped active site of an enzyme would encounter 
 significant steric hindrance that could inhibit function . however , 
 peptides that bind small molecules or accessible receptors on a cell 
 should retain bioactivity . certainly , the accessibility of polymeric 
 peptide brushes in terms of receptor binding is supported by studies 
 of polynorbornyl polymers containing rgd targeting peptides , which 
 bind integrin receptors more efficiently than their peptide analogues . as such , while we have provided a strategy for 
 adjusting the density of the polymer brush , one could imagine applications 
 or environments in which even more flexibility or design control is 
 needed . in these scenarios , for example , the incorporation of a longer linker between 
 the norbornyl polymer backbone and the peptide could provide a means 
 for separating the peptide from the backbone , thereby making the peptides 
 more flexible and sterically accessible . this could include linkers 
 that are cleavable under appropriate biological conditions or by exogenous 
 sources . indeed , appropriate ph- or uv - sensitive 
 linkers have been described for romp 
 polymers . in addition to proteolysis , there are two other problems 
 that often 
 limit the bioavailability and clinical efficacy of peptide - based therapeutics : 
 rapid renal clearance and inefficiencies in cellular uptake . regarding 
 the first , it should be noted that the exclusion limit for glomerular 
 filtration includes molecules or assemblies whose molecular weight 
 exceeds 50 kda . it is thus relevant that 
 the size of peptide constructs generated by romp can be controlled 
 by preparation of amphiphilic polymers that self - assemble into various 
 structures and sizes , based on their hydrophilic - to - hydrophobic ratios , 
 as exemplified by the 1050 nm spherical micelles generated 
 here with the tat peptide . such particles are not expected to be subject 
 to rapid renal elimination , and work in our laboratory is exploring 
 the requirements for in vivo retention of romp - derived materials in 
 general . regarding the second problem , we have shown here that the 
 romp copolymer strategy affords a robust ability to synthesize polymers 
 and polymer assemblies that cross cell membranes efficiently , and 
 we anticipate that these could transport a variety of cargo into cells . 
 for example , therapeutic peptides could be easily incorporated , as 
 multiple copies in a block copolymer ; or as a single copy in a polymer , 
 configured as an end - label . indeed , dual labeled polymers with a polymerized 
 cpp and a releasable therapeutic moiety could provide a unique approach 
 to addressing transmembrane delivery of materials into cells . in summary , we have presented a new method for packaging peptides , 
 which renders them resistant to proteolysis in a tunable fashion but 
 does not alter their amino acid sequence , and therefore preserves 
 their biological activities . we envision that this strategy can be 
 employed broadly to render therapeutic peptides or peptide - based sensors 
 resistant to proteolysis , thus enhancing their bioavailability and 
 clinical efficacy . all other materials were obtained from sigma - aldrich 
 and used without further purification unless otherwise noted . analytical scale rp - hplc was performed with 
 a jupiter proteo90a phenomenex column ( 150  4.60 mm ) using a 
 hitachi - elite lachrom l2130 pump with a uv  vis detector ( hitachi - elite 
 lachrome l-2420 ) monitoring at 214 nm . peptides were purified with 
 a jupiter proteo90a phenomenex column ( 2050  25.0 mm ) on a waters 
 deltaprep 300 system . for all rp - hplc assays , gradient solvent systems 
 were used in which buffer a was 0.1% tfa in water and buffer b was 
 0.1% tfa in acetonitrile . polymer dispersities and molecular weights 
 were determine by size - exclusion chromatography ( phenomenex phenogel 
 5 u 10 , 175 k , 300  7.80 mm in series with a phenomenex 
 phenogel 5 u 10 , 101000 k , 300  7.80 mm with 0.05 m 
 libr in dmf as the running buffer at a flow rate of 0.75 ml / min ) using 
 a shimadzu pump equipped with a multiangle light scattering detector 
 ( dawn - helio , wyatt technology ) and a refractive index detector ( hitachi 
 l2490 or a wyatt optilab t - rex detector ) normalized to a 30 k mw polystyrene 
 standard . for sec - mals chromatograms in which a multimodal distribution 
 is observed by light scattering but not in the ri chromatogram , we 
 analyzed only the peak width that has an associated ri component . tem images were obtained by depositing samples on carbon - formavar - coated 
 copper grids ( ted paella , inc . ) , which were then stained with 1% w / w 
 uranyl acetate and then imaged on a techanai g2 sphera operating at 
 an accelerating voltage of 200 kv . all concentrations of fluorescent 
 materials were obtained by measuring uv absorbance of the fluorophore 
 on a thermoscientific nanodrop 2000c and the data was fit to the standard 
 curves described in supporting information . fluorescent data was recorded on a fluorescence plate reader , perkinelmer 
 hts 7000 plus bio assay reader ( excitation : 340 nm ; emission : 465 
 nm ) , or on a photon technology international fluorescence reader . h ( 400 mhz ) and c ( 100 mhz ) nmr spectra were 
 recorded on a varian mercury plus spectrometer . chemical shifts are 
 reported in ppm relative to the dmf - d7 or cdcl3 residual peaks . a 10 ml round - bottom 
 flask equipped with a stir bar was charged with an amine - terminated 
 norbornene ( 2-(2-aminoethyl)-3a,4,7,7a - tetrahydro-1h-4,7-methanoisoindole-1,3-(2h)-dione ) ( 70 mg , 0.24 
 mmol , 1 equiv ) , which was prepared as described previously and dissolved in 4.5 ml of dry dmf under n2 ( g ) . to this was added n , n - bis(boc)-1-guanylpyrazole ( 105 mg , 0.34 mmol , 1 equiv ) 
 and diisopropylethylamine ( 120 l , 0.68 mmol , 2 equiv ) . the 
 reaction mixture was stirred at room temperature for 12 h. the solution 
 was concentrated to dryness and resuspended in 25 ml of ch2cl2 , then washed with water ( 3 ) and then brine . the ch2cl2 layer was collected , dried over na2so4 ( s ) and concentrated to dryness . the material 
 was then purified by flash column chromatography on silica gel ( 33% 
 etoac in hexanes ) to yield a white powder in 92% yield ( 140 mg , 0.31 
 mmol ) rf 0.37 ( 33% etoac 
 in hexanes ) : h nmr ( 400 mhz , cdcl3 , 298 k ) 
  11.43 ppm ( 1h , b ) , 8.45 ( 1h , b ) , 6.27 ( 2h , t , j = 1.5 hz ) , 3.71 ( 2h , dd , j = 7.0 , 4.4 ) , 3.64 ( 2h , 
 m ) , 3.25 ( 2h , d , j = 1.5 hz ) , 2.7 ( 2h , m ) , 1.51 ( 1h , d , j = 1.1 hz ) , 1.48 ( 9h , s ) , 1.47 ( 9h , s ) , 1.25 ( 1h , d , j = 1.8 hz ) ; c nmr ( 100 mhz , cdcl3 , 298 k ) 
  178.1 , 157.0 , 156.6 , 153.0 , 137.9 , 137.7 , 83.3 , 79.5 , 48.0 , 
 45.0 , 43.1 , 40.0 , 38.0 , 28.3 , 28.1 ; high - resolution ms analysis ( esi - tofms ) m / z calculated 449.2395 , found 449.2394 . peptides were synthesized using standard 
 solid phase peptide synthesis ( spps ) procedures on an aapptec focus 
 xc automated synthesizer . the arg8 peptide was synthesized with the 
 pbf protecting group left on the side chains by the use of highly 
 acid - sensitive sieber amide resin . all other peptides were prepared 
 protecting group - free on rink amide mbha resin . a typical spps procedure 
 involved fmoc deprotection with 20% methylpiperidine in dmf ( one 5 
 min deprotection followed by one 15 min deprotection ) , and 45 min 
 amide couplings using 3.75 equiv of the fmoc - protected , and side chain - protected 
 amino acid , 4 equiv of hbtu and 8 equiv of dipea . peptide couplings 
 that were incomplete by kaiser test were drained and then subjected 
 to fresh reagents . monomers were prepared by amide coupling to n-(hexanoic acid)-cis-5-norbornene - exo - dicarboximide ( prepared via a published protocol ) or to n-(glycine)-cis-5-norbornene - exo - dicarboximide for the fluorogenic substrates at the n - terminus of the peptide . fluorescein - labeled peptides 
 were assembled by addition of boc - lys(fmoc)-oh to the n - terminus of the peptide , followed by removal of the fmoc protecting 
 group and amide coupling to 5/6-carboxy fluorescein . the side - chain 
 protected arg8 peptide was cleaved from the sieber amide resin by 
 five 2 min rinses with 2% tfa in dcm . all other peptides were cleaved 
 and deprotected by treatment with tfa / h2o / tips in a 9.5:2.5:2.5 
 ratio for 2 h. the peptides were then precipitated in cold ether and 
 purified by rp - hplc . the identity of each peptide was confirmed by 
 esi - ms or maldi - ms and purities were verified by observation of a 
 single peak in analytical rp - hplc chromatograms ( figures s2 , s20a , and s22 and tables s1 and s3 ) . polymerizations were carried out in 
 a glovebox under n2 ( g ) . a typical protocol used to generate 
 a polymer with dp = 10 involved mixing the monomer ( 0.0125 mmol , 10 
 equiv , 25 mm ) with the catalyst ( 0.00125 mmol , 1 equiv , 2.5 mm ) in 
 dry dmf ( 0.5 ml ) . homopolymerizations that have not been reported 
 previously in the literature were preformed in dmf - d7 and followed by h nmr to confirm complete 
 consumption of the monomer and to determine the time period required 
 to reach completion . polymers for cell penetration studies were end - labeled 
 with a copy of fluorescein using a chain transfer agent ( 1.5 equiv ) 
 for 2 h as described previously , followed 
 by termination with ethyl vinyl ether ( 10 equiv ) for 1 h at room temperature . block copolymers were prepared by polymerizing the first monomer ( either 
 phenyl or peg ) to completion and then adding the second monomer ( a 
 peptide or the guanidinium group ) , followed by end labeling with the 
 fluorescein chain transfer agent and finally termination with ethyl 
 vinyl ether . fluorescein - labeled polymers were treated with nh4oh ( aq ) for 20 min to remove the pivolate protecting group , 
 as described previously . the side - chain 
 protected arg8 polymer was precipitated with cold ether and collected 
 by centrifugation . the resulting powder was dissolved in 2 ml of a 
 mixture of tfa / h2o / tips ( 95:2.5:2.5 ) and stirred for 4 
 h at room temperature . the product was precipitated with cold ether , 
 collected by centrifugation and dried . in preparation for in vitro 
 studies , all polymers were washed ( 3 ) with cold ether ( to remove 
 the ru catalyst ) and then dissolved in pbs and dialyzed in an effort 
 to remove any residual monomer or catalyst . the tat and gsgsg particles 
 were generated by dissolving the amphiphilic polymers in dmf , and 
 then diluting with an equivalent volume of pbs over 1 h and finally 
 dialyzing this solution into pbs over 48 h with 3 buffer changes using 
 dialysis cups of mwco 3500 ( thermo scientific , cat . # 69552 ) . the extent of proteolytic 
 degradation of the tat peptide , polymer and particle by trypsin ( gibco 
 life tech . , # 10165921001 ) 
 was assessed by comparison of chromatograms in rp - hplc . in these experiments , 
 the material at the indicated concentration was incubated with each 
 protease ( at 1 m ) for 20 min , and then the enzymes were heat 
 denatured at 65 c for 15 min , and the solution was immediately 
 injected onto an analytical rp - hplc . given that treatment with each 
 protease gives multiple fragments of the tat sequence , a standard 
 curve for each starting material was prepared to assess the percentage 
 of intact material remaining after proteolytic digestion ( figure s15 ) . note that the standard curves for 
 the polymer and particle will be biased due to the fact that after 
 cleavage , the polymer backbone and fluorophore should remain intact , 
 and will comprise part of the measured peak area . nevertheless , no 
 new peaks were seen in the chromatograms of the polymer or particle 
 post enzyme treatment ( figure s16 ) , suggesting 
 that these materials are not susceptible to cleavage by the proteases . cells were cultured at 37 c under 5% co2 in phenol - red- 
 containing dulbecco s modified eagle medium ( dmem ; gibco life 
 tech . , cat . # # fb02 ) and with 1 concentrations 
 of nonessential amino acids ( gibco life tech . , cat . # 35050061 ) , and the antibiotics 
 penicillin / streptomycin ( corning cellgro , cat . cells were grown in t75 culture flasks and subcultured at 7580% 
 confluency ( every 34 days ) . hela cells were plated at a density 
 of 90  000 cells per well of a 24-well plate 18 h prior to treatment . materials dissolved in dulbecco s phosphate buffered saline 
 ( dpbs without ca or mg ; corning cellgro , 
 cat . # 21031-cm ) at 10 the desired concentration were 
 added to the wells , and the plates were incubated for 30 min at 37 
 c . the medium was then removed , and the cells were washed twice 
 with dpbs and then incubated three times for 5 min with heparin ( 0.5 
 mg / ml in dpbs ; affymetrix , cat . the cells were then trypsinized ( 0.25% trypsin ; gibco life 
 tech . , cat . # 15090046 ) for 10 min , cold medium was added , 
 and the cells were transferred to eppendorfs , centrifuged to pellets 
 and then resuspended in a minimal amount of cold dpbs . fluorescence 
 activated cell sorting data ( 10  000 events on three separate 
 cultures ) was acquired on an accuri c6 flow cytometer set to default 
  3 blue 1 red  configuration with standard optics and 
 slow fluidics ( 14 l / min ) . for proteolysis studies , the indicated 
 concentration of tat peptide , homopolymer or particle was pretreated 
 with 1 m of trypsin , chymotrypsin or pronase for 20 min in 
 dpbs , after which the protease was heat denatured for 15 min at 65 
 c . the cells were then incubated , prepared and analyzed by flow 
 cytometry as described above . for mechanistic studies , cells were 
 preincubated with the indicated compound for 30 min at 37 c 
 prior to addition of the cell - penetrating material . the following 
 concentrations were used : 80 m dynasore ( enzo life sciences , 
 cat . # 270502-m005 ) and 9.5 mm mcd ( fischer scientific , 
 cat . for studies at reduced temperature , cells were 
 incubated at 4 c for 30 min prior to and during the incubation 
 with the compound of interest . all subsequent washes and manipulations 
 were also done with ice - cooled media and other materials . data is 
 reported as the normalized mean fluorescence , which is the mean fluorescence 
 yielded by the material / the mean fluorescence from the vehicle control . hela cells were seeded 
 on glass - bottom 24-well plates at a cell density of 90  000 
 cells per well 18 h prior to treatment . the medium was removed and 
 then replaced with medium lacking phenol red ( gibco life tech . , cat # 
 31053028 ) to minimize background fluorescence . materials dissolved 
 in dpbs ( at 10 the desired concentration ) were added to the 
 wells and the plates were incubated for 30 min at 37 c . the 
 washing procedure used in the flow cytometry experiments ( 2  
 dpbs , 3  heparin for 5 min , 1  dpbs ) was followed here . 
 following removal of the final dpbs rinsate , for proteolysis studies , the indicated 
 concentration of tat peptide , homopolymer and particle were pretreated 
 with 1 m of trypsin , chymotrypsin or pronase for 20 min in 
 dpbs , after which the protease was heat denatured for 15 min at 65 
 c . the cells were then incubated , prepared and analyzed by confocal 
 microscopy as described above . the celltiter - blue assay ( promega , 
 cat . # hela cells were plated at a density of 4000 cells per 
 well of a 96-well plate 18 h prior to treatment . materials dissolved 
 in dpbs at 5 m were added to the wells along with a 10% dmso 
 positive control . the 
 medium was removed and 80 l of fresh media lacking phenol red 
 was added . to this was added 20 l of the celltiter - blue reagent 
 and the cells were then incubated for 2 h prior to measuring fluorescence 
 in a plate reader using 560 nm excitation and 590 nm emission . the 
 fluorescence measurements were corrected for background fluorescence 
 from the celltiter - blue reagent by subtracting the fluorescence reading 
 of wells treated with the reagent in the absence of cells . fluorescence 
 values were then referenced as a percentage of the value obtained 
 for the pbs vehicle control . polymerizations 
 were carried out in a glovebox under a n2  ( g ) atmosphere . 
 to generate the polymers containing the thrombin peptide sequence , 
 the monomer ( 0.007 mmol , 10 equiv , 23 mm for dp = 10 ; 0.013 mmol , 
 20 equiv , 45 mm for dp = 20 ; 0.021 mmol , 30 equiv , 70 mm for dp = 
 30 ) was mixed with the catalyst ( 0.0007 mmol , 1 equiv , 2.3 mm ) in 
 dmf - d7 ( 0.3 ml ) and monitored by h nmr to confirm complete consumption of the monomer and to 
 determine the time period required to reach completion . upon completion , 
 the polymers were quenched with ethyl vinyl ether for 10 min , and 
 then precipitated with cold ether and dried under a vacuum . the resulting 
 polymers were directly characterized by sec - mals . the extent 
 of proteolytic degradation of the thrombin peptide polymers by thrombin 
 ( sigma , cat . # t6884100un ) was assessed by comparison of chromatograms 
 in reverse - phase hplc . in these experiments , the monomer and polymers 
 were dissolved in pbs buffer ( 2.2 mm with respect to peptide ) . thrombin 
 ( 10 units ) was added to each sample and an hplc trace was immediately 
 obtained followed by subsequent hplc injections every 45 min . a standard 
 curve of the authentic c - terminal fragment was generated to convert 
 the peak area to percent cleavage . the fluorogenic peptide 
 norg - e(edans)rpahlrdsgk(dabcyl)gsgsg was prepared by spps 
 as described above where the edans was added as a modified glu ( fmoc - glu(edans)-oh ; 
 aaptec , cat . # afe150 ) while dabcyl ( anaspec , cat # 81800 ) was conjugated 
 to the -amino group of a lysine . this monomer was polymerized 
 into a homopolymer with dp = 20 , determined by bulk light scattering . note that the fluorogenic peptide sequence did not run as a polymer 
 on the sec column needed for sec - mals . blend copolymers with a peg 
 monomer were prepared by first assessing the rate of polymerization 
 of the two monomers . at the concentration of monomer studied , the 
 peg monomer was quick to polymerize ( complete within 15 min ) , while 
 the fluorogenic substrate polymerized at a rate of 1.78 monomers per 
 hr ( figure s30 ) . to ensure reasonable interdigitation 
 of the two monomers in the random blend copolymer , the peg monomer 
 was added via syringe pump at appropriate rates to prepare peptide : 
 peg polymers at a ratio of 1:19 , 5:15 , 10:10 , 15:5 and 19:1 as described 
 in table s5 . cleavage of the homopolymer 
 monomer and blend copolymers ( 40 m ) by the noted protease in 
 ( at 25 nm ) in reaction buffer ( 50 mm tris ( ph 7.4 ) , 1 mm zncl2 , 150 mm nacl , 5 mm cacl2 ) was monitored by measurement 
 of fluorescence in a plate reader or fluorimeter . # 476935 ) , mmp-9 ( catalytic domain ; 
 enzo life sciences cat . # # v4001 ) , trypsin ( gibco life tech . , cat . # 15090046 ) and 
 pronase ( roche , cat . details of the polymer constructs 
 that were simulated are as follows . in all cases , the polymer backbone 
 was composed of 10 norbornene units , flexibly linked by olefin bonds , 
 with a 1:1 mix of cis and trans units . 
 in the homopolymer , each norbornene residue is linked to the n - terminus 
 of the fluorogenic peptide norge(edans)rpahlrdsgk(dabcyl)gsgsg ) 
 ( figure 11a ) , where the edans and dabcyl fluorophores 
 are linked to e and k residues , respectively . the c - terminus of each 
 peptide was amide capped . in the 5:5 blend copolymer , five of these 
 peptide - dye chains were linked to norbornenes 1 , 2 , 5 , 6 , and 10 ( counting 
 from the end of the phenyl ring ) ; and five oeg chains , each with four 
 ethylene glycol units , were linked to the remaining norbornenes ( figure 11d ) . in the 9:1 blend copolymers , all positions 
 are occupied by oegs except that the tenth or fifth norbornene ( figure 11b or 11c ) is occupied by 
 the fluorogenic peptide . all - atom molecular dynamics simulations 
 were performed to study the conformations of the simulated polymers , 
 using the explicit water model tip3p and 
 the gromacs 4.6 software package . all 
 bonded and lennard  jones terms of the polymer backbone and 
 dye moieties were assigned by the general amber force field ( gaff ) and partial atomic charges were assigned using 
 am1-bcc . parameters from the amber ff99sb - ildn force 
 field were assigned to the peptide components . all simulations started from extended polymer backbone and peptide 
 configuration and were performed using periodic boundary conditions . each polymer construct was solvated in a cubic simulation box with 
 edge lengths set to the longest dimension of the molecule plus 2 nm . the systems 
 were first energy minimized with the steepest - descent algorithm , and 
 then equilibrated for 10 ns under constant volume and temperature 
 conditions and then another 10 ns under constant temperature and pressure 
 conditions . the particle  mesh ewald ( pme ) method was used for electrostatic interactions , and 
 the cutoff distance of the lennard jones ( lj ) interactions 
 was 10  . in some simulations , a heat  cool cycle was used 
 immediately after the equilibration phase to boost the systems out 
 of local energy minima and search for additional stable conformational 
 states . here , the temperature was increased from 300 to 500 k linearly 
 over 2 ns ; the simulation was run for 1 ns at 500 k ; and the temperature 
 was then reduced back to 300 k linearly over 2 ns , and kept at 300 
 k for 95 ns of production dynamics . for comparison , regular md simulations 
 at constant 300 k were also performed for 100 ns following the same 
 equilibration phase . we also simulated the two 9:1 blend copolymers 
 without the dye components ( see supporting information ) , to verify that the dye molecules do not influence our major conclusions . 
 for these simulated polymers ,
we 
 describe a strategy for rendering peptides resistant to proteolysis 
 by formulating them as high - density brush polymers . 
 the utility of 
 this approach is demonstrated by polymerizing well - established cell - penetrating 
 peptides ( cpps ) and showing that the resulting polymers are not only 
 resistant to proteolysis but also maintain their ability to enter 
 cells . 
 the scope of this design concept is explored by studying the 
 proteolytic resistance of brush polymers composed of peptides that 
 are substrates for either thrombin or a metalloprotease . 
 finally , 
 we demonstrate that the proteolytic susceptibility of peptide brush 
 polymers can be tuned by adjusting the density of the polymer brush 
 and offer in silico models to rationalize this finding . 
 we contend 
 that this strategy offers a plausible method of preparing peptides 
 for in vivo use , where rapid digestion by proteases has traditionally 
 restricted their utility .