dendrimers provide an alternative and potent means for drug delivery due to their nanometer size and the ability to incorporate various functionalities on their interior and exterior .
modern chemical synthesis capabilities allow various functionalities to be incorporated on the dendrimer exterior in order to optimize performance in terms of drug binding , transport , targeting , delivery , and biocompatibility [ 13 ] .
polyamidoamine ( pamam ) dendrimers , for example , have been tailored for enhanced drug solubility , retention time , targeting , and efficacy [ 13 ] .
in addition to optimizing delivery , another issue is the reduction or elimination of cytotoxicity , which has been addressed by masking the terminal functional groups and charge .
this can be accomplished by adding biologically compatible terminal groups such as carboxylate , hydroxyl , acetamide , lipid , or polyethylene glycol ( peg ) . among these
, peg is the most widely used due to its minimal or nontoxicity , nonimmunogenicity , and nonantigenicity and has been approved by the fda in oral intravenous and pulmonary pharmaceutical formulations [ 58 ] .
it is known that peg chains adopt a variety of conformations , lengths , and packing density and that these structural presentations directly affect biocompatibility [ 911 ] . in some cases
, peg alters dendrimer 's drug loading capacity , retention time , and thus their delivery performance [ 8 , 12 , 13 ] .
therefore , the characterization of peg coating prior to animal testing is of great importance .
while computational studies have been carried out to probe peg conformation on dendrimers surfaces , experimental studies are lacking due to the difficulties in obtaining high - resolution structural characterization of peg when bound to soft materials , such as dendrimers [ 11 , 14 ] .
our prior work shows that high - resolution structural characterization of simple dendrimers such as pamam can be achieved by advanced sample preparation and combined atomic force and scanning tunneling microscopy ( afm and stm ) imaging [ 15 , 16 ] .
encouraged by this initial success , this work reports extending our approach to pegylated pamam dendrimers .
from the high - resolution images , the molecular conformation , packing density , and distribution of peg on the surface of individual dendrimers can be obtained .
this knowledge of the peg presentation on dendrimers provides important insights for understanding structure - delivery performance correlation , which could guide the design , optimization , and development of the next generation of dendrimers and reduce usage of animal tests .
the following materials were used as received : methoxy polyethylene glycol 1000  da ( peg1000 ) ( sigma - aldrich ) , triethylamine ( tea ) ( 99.5% , sigma - aldrich ) , pyridine ( 99.8% , sigma - aldrich ) p - nitrophenyl chloroformate ( pnpcf ) ( 98% , sigma - aldrich ) , 2,5-dihydroxybenzoic acid ( 2,5-dhb ) ( sigma - aldrich ) , anhydrous dichloromethane ( dcm ) ( sigma - aldrich ) , dimethyl sulfoxide ( dmso ) ( acros geel ) , k2ptcl4 ( min .
42.4% pt , alfa aesar ) , n - octanethiol ( c8 ) ( 98% , sigma - aldrich ) , deuterium oxide ( d , 99.96% , cambridge isotope laboratories ) , and phosphate buffer ( 10x , lief technologies ) .
fourth generation amine terminated pamam dendrimers were purchased as 10% by weight solutions in methanol ( sigma - aldrich , st . louis , mo ) .
silica gel 60  a 230400  mesh atsm ( whatman inc ) and silica gel 60 f254 plastic sheets ( tlc ) ( merck kgaa ) were used for column and thin layer chromatography , respectively .
ultrapure water ( 18  mcm , millipore milli - q ) and 200 proof ethanol ( gold shield chemical co. ) were used for dilution and washing .
( 98% , air gas co. ) and h2 ( 99.99% , praxair , inc . )
au slugs ( 99.99% , alpha aesar premion co. ) and mica ( clear ruby muscovite , mica new york corp . ) were used for au thin film preparation .
the following materials were used as received : methoxy polyethylene glycol 1000  da ( peg1000 ) ( sigma - aldrich , st .
louis , mo ) , triethylamine ( tea ) ( 99.5% , sigma - aldrich , st .
louis , mo ) p - nitrophenyl chloroformate ( pnpcf ) ( 98% , sigma - aldrich , st .
louis , mo ) , 2,5-dihydroxybenzoic acid ( 2,5-dhb ) ( sigma - aldrich , st .
louis , mo ) , anhydrous dichloromethane ( dcm ) ( sigma - aldrich , st .
louis , mo ) , dimethyl sulfoxide ( dmso ) ( 99.7% , acros , geel , belgium ) , k2ptcl4 ( min .
42.4%  pt , alfa aesar , ward hill , massachusetts ) , n - octanethiol ( c8 ) ( 98% , sigma - aldrich , st .
louis , mo ) , deuterium oxide ( d , 99.96% , cambridge isotope laboratories , tewksbury , ma ) , and phosphate buffer ( 10x , lief technologies , grand island , ny ) .
louis , mo ) . silica gel 60a 230400 mesh atsm ( whatman inc , pittsburgh , pa ) and silica gel 60 f254 plastic sheets ( tlc ) ( merck kgaa , darmstadt , germany ) were used for column and thin layer chromatography , respectively .
ultrapure water ( 18mcm , milliporemilli - q , billerica , ma ) and 200 proof ethanol ( gold shield chemical co. , hayward , ca ) were used for dilution and washing
( 98% , air gas co. , woodland , ca ) and h2 ( 99.99% , praxair inc . , sacramento , ca )
, 99.95% , california fine wire co. , grover beach , ca ) was used to make stm tips .
au slugs ( 99.99% , alpha aesar premion co. , ward hill ma ) and mica ( clear ruby muscovite , mica new york corp . , new york , ny ) were used for au thin film preparation .
g4-pamam - nh2 dendrimers were pegylated according to previous reports with some modifications . briefly ,
peg1000 was first modified by reaction with tea and a catalyst amount of pyridine in anhydrous dcm with peg1000 . to the organic solution
the g4-pamam - npeg1000 was synthesized by adding an anhydrous dmso solution of g4-pamam - nh2 dropwise to a solution of peg1000 carbonate in anhydrous dmso .
the reaction system was then stirred 24 hours for g4-pamam-6peg1000 , or 72 hours for g4-pamam-50peg1000 .
the resulting g4-pamam - npeg1000 was purified by centrifugal filter ( mwco = 10  kda ) until thin layer chromatography ( dcm / methanol = 80/20 , v / v ) showed no unreacted peg1000 carbonate or byproduct p - nitrophenol . the molecular weight of the modified and unmodified dendrimers was determined by matrix assisted laser desorption ionization time - of - flight mass spectrometry ( maldi - tof - ms ) ( ultraflex , bruker ) .
the conjugates were dissolved in deionized water at a concentration of 1.0  mg / ml .
10  l of the conjugate solution was mixed with 10  l of the dhb solution .
2  l of the sample was spotted on a maldi target plate ( mtp 384 , bruker daltonics , inc . ) .
the g4-pamam - nh2 , g4-pamam-6peg1000 , and g4-pamam-50peg1000 dendrimers were found to have a molecular weight of 14,579 , 22,080 , and 75,929  da , respectively .
the number of peg chains per dendrimer was determined by h nmr spectroscopy ( mr-400 , agilent ) using deuterated solvent .
the deuterated solvent peak ( dmso_d6 : 2.483 ; d2o : 4.577 ) in h nmr was set as a reference peak .
the pegylation resulted in 5.9  peg per dendrimer , referred to as g4-pamam-6peg1000 , and 50.5  peg per dendrimer , referred to as g4-pamam-50peg1000 .
the hydrodynamic diameter ( hd ) of samples was measured using dynamic light scattering ( dls ) ( zetasizer nano zs , melvern instruments ) .
the sample ( 1.0  mg / ml ) was dissolved in phosphate buffer solution ( 0.1  m , ph 7.4 ) to maintain the ph during the measurement .
- nh2 , g4-pamam-6peg1000 , and g4-pamam-50peg1000 dendrimers were found to have a hd of 4.4  1.4 , 5.8  2.1 , and 12.2  4.4  nm , respectively .
the solutions were prepared following previously established procedures , including metal ion doping to facilitate stm imaging [ 1517 ] . in short ,
k2ptcl4 was then added to obtain molar ratios of 1  :  70 , or 1  :  700 dendrimer  :  pt .
solutions were kept at room temperature for 48 hours in order to allow for sufficient pt - amine coordination within dendrimers .
for the surface deposition of dendrimers , 1  cm pieces of gold films were h2-flamed and allowed to cool for 10 minutes under clean ambient conditions . then
, a 60100  l drop of the dendrimer solution was deposited resulting in a droplet with a contact diameter of 0.70.9  cm .
after 1.25 minutes the surface was washed with 2  ml of pure water , dried with n2 , washed with 2  ml of ethanol , and dried again with n2 .
a 60100  l drop of 1.0  mm c8 solution was then applied to the substrate for 4 minutes , washed with 2  ml of ethanol , and dried with n2 .
c8 sams are incorporated in order to replace weakly adsorbed molecules , confine dendrimers laterally , and prevent mobility during scanning .
this process results in a clean , high coverage submonolayer of dendrimers on the surface .
afm images were acquired using a mfp3d - sa system ( asylum research ) , which includes a closed loop capability .
the probe has a typical force constant of k = 1.0  n / m as measured by the thermal noise method [ 16 , 18 , 20 , 21 ] . during tapping mode imaging ,
the cantilever was modulated by a driving frequency of 74  khz and amplitude of 87  nm ( 1.0  v ) . during nanoshaving to displace adsorbates such as dendrimers or alkanethiolates ,
tips were placed in contact with the surface with increasing load beyond threshold [ 22 , 23 ] .
image processing and data analysis were performed using gwyddion ( version 2.33 , http://gwyddion.net/ ) .
stm images were taken using a walker - type scanner ( uhv 300 , rhk technologies , inc . ) , under ambient pressure and temperature [ 18 , 19 ] .
stm tips were prepared by etching tungsten wires electrochemically at 2.0  v in 3.0  m naoh solutions using a homemade potentiostat to monitor the etching process [ 18 , 19 ] .
all stm images were acquired in constant current mode with typical bias voltages ranging from 0.4 to 0.9  v and tunneling currents from 10 to 40  pa .
the scanner was calibrated laterally using an octanethiol sam lattice constant of 0.50  nm and vertically using au(111 ) single atomic step ( 0.235  nm ) .
image processing and data analysis were performed using xpmpro ( version 2.0.0.8 , rhk technologies , inc . ) .
surface contact area measurements were carried out using imagej ( version 1.47v , wayne rasband , nih , http://imagej.nih.gov/ij/index.html ) .
the characteristic afm tapping mode images of g4-pamam-50peg1000 , are shown in figures 1(a ) and 1(b ) .
the two images are of the same area but under two different damping set points during tapping mode afm imaging . in the case of g4-pamam-50peg1000
, the topographic images were found to vary with the set point values . in figure 1(a ) , at 42% damping , each dendrimer appears as a ring . reducing the damping to 23% , that is , gentler tapping ,
we have varied damping ( from 0% to 100% ) and found that dendrimers appear as either rings as shown in figure 1(a ) or ellipsoidal caps as shown in figure 1(b ) .
we assigned the two features to pamam core and peg shell ( or coating ) , respectively .
the assignment is based on three observations . first , the damping - dependence was not present for non - pegylated dendrimers , such as g4-pamam - nh2 , as revealed in figures 1(c ) and 1(d ) .
regardless of tapping conditions , the g4-pamam - nh2 dendrimers always appear as ellipsoidal caps , consistent with prior studies .
secondly , the core region of the g4-pamam-50peg1000 ring structures is commensurate in size with that of the g4-pamam - nh2 core , as shown in cursor 1 of figure 1 .
thirdly , damping dependence in tapping mode afm imaging typically indicates variation in materials ' viscoelastic property , for example , how materials respond to periodical tapping of the afm tip [ 2427 ] .
the distinct differences in the case of g4-pamam-50peg1000 dendrimers are consistent with a tight and elastic dendrimer core and a relatively loose and viscoelastic peg coating .
the peg regions could exhibit high responses at specific damping conditions , revealing rings more evidently .
the width of the rings , measured as full width at half of the maximum height , varies from 5.0 to 12.4  nm , with an average value of 6.9  2.3  nm .
the measured ring width provides an approximate view regarding the extension , or conformation , of the peg molecules suggesting a variation in the peg conformation . according to afm imaging
the difference between the thickest and thinnest regions of the peg coating on a single dendrimer varies by 3.4% up to 47.6% .
this preliminary assessment of dendrimer morphology indicates that the peg density and extension are not uniform at the outer shell of individual dendrimers . to determine the fine structure of the peg coating , stm imaging
is performed and provides a more accurate visualization of the peg presentation , as will be discussed in detail in section 3.2 . to verify
that the features are not due to deformation upon surface immobilization , we have also imaged dendrimers at various surface coverages . at high surface coverage ,
as shown in figure 2 , afm topographs are again found to depend on tapping conditions .
overall , the trend is similar to that at low coverage , that is , solid ellipsoidal caps are observed under most tapping conditions , while rings are observed at greater dampening .
this indicates that the ring - like features are intrinsic to the dendrimer structure instead of surface deformation .
one specific difference was seen under 42% damping , at which ring contrast was observed previously ; the morphology appears fragmented , analogous to flower pedals , as shown in figure 2(a ) .
this overlapping region can extend the entire thickness of the peg coating up to the pamam core as shown in the cursor profile ( red ) . at 23% damping
, peg - coated g4 dendrimers appear as individual ellipsoidal caps with a clear depression between adjacent dendrimers , as shown in figure 2(b ) and the cursor ( blue ) of figure 2 . in the case of g4-pamam - nh2 dendrimers ,
these observations suggest that the peg chains could be interdigitated at close proximity , which could lead to stacking , interspersion , and merging among neighboring dendrimers .
this behavior is in sharp contrast to the pamam cores , where no overlapping or coalescing was seen .
these observations are important in the context of drug delivery in vivo , as aggregation due to high accumulations of dendrimers is of concern .
our previous work has demonstrated that the physical height of surface immobilized dendrimers can be measured using nanoshaving , an afm based technique , in conjunction with topographic imaging [ 1517 ] .
the surface was first surveyed to select a relatively flat 1.5  m  1.5  m region .
higher force was then applied during scanning of a central 0.5  m  0.5  m region to remove the dendrimer monolayer .
finally the entire area was imaged again in tapping mode to reveal both the freshly exposed surface region and the surrounding dendrimer monolayer in a single frame , shown in figure 3(a ) inset .
the physical height of individual g4-pamam - nh2 dendrimers relative to the bare substrate can be directly obtained from the cursor profile . as shown in figure 3(a ) , cursor 1 reveals the height of five individual dendrimers . the height taken from multiple experiments and a large number of g4-pamam - nh2 dendrimers
was found to be 2.2  0.3  nm , which is identical to 3 previous measurements of surface immobilized g4-pamam dendrimers [ 1517 ] .
this value is lower than 4.4  nm determined by dls which probes hydrodynamic diameter .
this is not uncommon due to the deformation of the dendrimer upon surface immobilization under ambient conditions [ 1517 , 2830 ] .
the dendrimers were displaced in the upper left region , enabling accurate height measurements , as shown for two representative g4-pamam-50peg1000 in cursor 2 .
the average height of g4-pamam-50peg1000 measured from two experiments and multiple images is 3.4  1.2  nm , which is 1.2  nm taller than the core , g4-pamam - nh2 . as indicated by the increased standard deviation relative to g4-pamam - nh2 , the pegylated dendrimers have a far greater distribution of height , indicating a greater range of size and/or conformation upon surface immobilization .
stm provides submolecular resolution characterization of pegylated dendrimers , revealing the morphology and structure of the peg chains . with the overall morphology of dendrimers established by afm
, we could use stm to provide a more detailed look at the intramolecular structure especially at peg region .
although dendrimers are not sufficiently conductive for direct stm imaging , our prior work has indicated that stm conductivity may be enhanced by coordinating metal ions into the dendrimer [ 1517 ] .
this work demonstrates that the same approach is effective to enable stm imaging of pegylated dendrimers .
individual dendrimers are clearly resolved , at the coverage of submonolayer with sufficient interparticle separation . at higher resolution shown in figure 4(b ) , it is apparent that the geometry of surface immobilized g4-pamam - nh2 adopts a very similar shape to the most commonly known pamam dendrimers , g4-pamam - oh , which are ellipsoidal caps [ 1517 ] .
while these dendrimers are considered to be nearly spherical in solution , the flattening and deformation from spherical geometry are due to dendrimer - surface interaction .
the dendrimer - surface contact areas , as highlighted in red , of the three dendrimers were measured as 7.7 , 12.0 , and 13.4  nm .
intraparticular features are also visible , such as the bright protrusions on the surface of each dendrimer , which likely correspond to individual nh2 termini , analogous to oh termini visualized previously . upon pegylation ,
the pamam cores of individual dendrimers are still visible , but the footprint or dendrimer - surface contact area is interspersed with peg chains spreading out and filling the space between dendrimers . at higher resolution
the peg are visualized more clearly , as shown in figure 4(d ) . unlike g4-pamam - nh2 ,
the dendrimers are no longer ellipsoidal , as the contact area adopts a more irregular geometry ( see red lines tracing the boundaries ) .
the periphery of these dendrimers was determined using multiple cursor profiles to accurately determine the boundaries of each dendrimer .
the contact area of the two pegylated dendrimers is much greater than the corresponding core particles , measuring 41.8 and 29.1  nm , respectively .
these high - resolution images reveal that the peg presentation varies from dendrimer to dendrimer , which leads to a large variation in contact upon surface immobilization . as shown in figure 4(d ) , the majority of the peg chains extend as groups to form peg  podia .
 the peg podia tend to fan out in regions of bare substrate and bunch up near adjacent dendrimer peg extensions .
these peg podia extend from 0.5 to 4.5  nm from the core contact region , which indicate variations in peg conformation .
previous work indicated several conformations adopted by peg chains attached to solid substrates [ 9 , 10 ] . to our knowledge ,
results shown in figure 4 represent the first observation of the peg conformational variations in the context of dendrimers . in order to determine the effect of the packing density of the peg at the dendrimer surfaces , various peg  :  core ratios were prepared .  
the overall morphology of g4-pamam-6peg1000 closely resembles that of g4-pamam - nh2 , as clearly defined ellipsoidal caps with intraparticular features and protrusions visible .
the degree of spreading observed is shown in figure 4(f ) , which falls in between g4-pamam-50peg1000 and g4-pamam - nh2 .
the dendrimer - surface contact area of the two representative g4-pamam-6peg1000 shown in figure 4(f ) measures 24.5 and 21.1  nm , from left to right .
peg podia appear shorter than that in high peg  :  core ratio cases with their extension 0.52.0  nm from the pamam core .
it is known that the packing density of peg chains on flat surfaces greatly impacts their conformation [ 9 , 13 ] .
our investigations indicate that this concept appears to be valid in the context of peg chains at the surface of pamam dendrimers .
the observed coverage dependence of peg conformation may be understood by taking into account peg - peg , peg - solution , and peg - surface interactions . on solid flat surfaces
three characteristic conformations have been described previously , known as pancake , mushroom , and brush , with the extension length of 0.5 , 1.0 , and 2.5  nm , respectively [ 810 ] .
peg chains on the dendrimer exterior differ from those on flat surfaces due to the curvature and mechanical flexibility of the core .
the curvature allows more space than that on flat surfaces under the same coverage , while the relatively soft core allows for greater deformation and flexibility upon surface immobilization .
computational studies on g3-pamam-8peg1000 indicate that peg chains fold in close contact with the dendrimer core in solution , due primarily to interactions between the ether groups of peg and the protonated amine termini of the dendrimers extending approximately 1  nm in solution [ 11 , 31 ] . in the case of dendrimers with lower coverage peg , such as in g4-pamam-6peg1000 , the peg chains seem to follow this theoretical prediction and exhibit tightly bound podia .
the extension is larger than predicted , extending from 0.5 to 2.5  nm , which indicates the presence of all three conformations described previously . at higher coverage , g4-pamam-50peg1000
the longest extensions exceed the brush conformation , which we attribute to the effect of dendrimer support and interchain interactions . considering
that peg - surface interactions could offset the intramolecular interactions in solution phase , local structural characterization is critical to reveal individual dendrimers ' structures .
results from stm investigations indicate that the incorporation of pt ions leads to sufficient conductivity for high - resolution stm imaging , despite pegylation , which in principle should hinder metal ion doping . in comparison
to pamam dendrimers , the incorporation of metal ions follows slower kinetics . in the case of g4-pamam - oh , for example
, each dendrimer was saturated by pt within 48  hrs , under 1  :  70 dendrimer  :  pt molar ratio , at a 1  m concentration . the resulting increase in tunneling probability allowed for high - resolution stm imaging , which could resolve individual dendrimer termini [ 1517 ] . under identical conditions ,
g4-pamam - nh2 also yielded high - resolution stm images , as shown in figures 4(b ) and 5(c ) . in the presence of a peg coating high - resolution
is still provided ; however , the apparent height , or happ , is lower , as shown in figures 5(a)5(c ) and cursors 13 .
the average happ of g4-pamam - nh2 is 0.42  0.11  nm , whereas the happ for g4-pamam-6peg1000 and g4-pamam-50peg1000 is 0.38  0.13  nm and 0.32  0.07  nm , respectively .
we infer that the metal ion concentration inside g4-pamam-50peg1000 and g4-pamam-6peg1000 dendrimers is lower than g4-pamam - nh2 , leading to a lower local density of states and therefore happ . to increase the metal ion coordination within the dendrimers , the dendrimer  :  pt molar ratio was increased from 1  :  70 to 1  :  700 , and the results are shown in figures 5(d)5(f ) and cursors 46 . at a higher pt ratio , the average happ of g4-pamam - nh2 , g4-pamam-6peg1000 , and g4-pamam-50peg1000 increased to 0.51  0.07  nm , 0.45  0.12  nm , and 0.56  0.04  nm , respectively . in the case of g4-pamam - nh2 ,
the average happ increases only 17% , indicating that the intraparticulate pt concentration is very similar under the two concentrations .
it is important to note here that a height increase was not observed , as measured by afm , for any of the dendrimers when the pt ratio was increased .
thus , by increasing the pt concentration , the metal ions were able to penetrate the peg coating and coordinate with the amines within the dendrimer core to a greater extent .
since the g4-pamam - nh2 dendrimer is already nearly saturated at the lower pt concentration the effect on happ is relatively small as compared to the highly pegylated dendrimer .
these observations should shed light when using dendrimers as drug delivery vehicles , as the coating may change the pharmacokinetic behavior .
using afm and stm , we have characterized the morphology and structure of pegylated dendrimers .
afm investigation allows for the visualization of individual dendrimers on surfaces and provides accurate height measurements .
in addition , afm studies reveal that the pamam core and peg shell can be visualized under tapping mode imaging to ascertain the uniformity and distribution of pegylation on individual dendrimers .
further , the results indicate that peg chains among adjacent dendrimers could interdigitate , in contrast to the dendrimer cores .
the peg chains at the exterior pamam cores adopt various conformations including pancake , mushroom , and brush , similar to that at the solid and flat surfaces .
unique to high coverage pegylated dendrimers , a greater variation in peg structure and degree of extension is observed with the peg podia , up to 4.5  nm from the core . to the best of our knowledge ,
this work is among the first to reveal high - resolution information on the local structure of pegylated dendrimers .
collectively , this investigation provides important insight into the structure of coated dendrimers , which shall be important to guide the design and development of better drug delivery vehicles .
 dendrimers have shown great promise as drug delivery vehicles in recent years because they can be synthesized with designed size and functionalities for optimal transportation , targeting , and biocompatibility . 
 one of the most well - known termini used for biocompatibility is polyethylene glycol ( peg ) , whose performance is affected by its actual conformation . however , the conformation of individual peg bound to soft materials such as dendrimers has not been directly observed . using atomic force microscopy ( afm ) and scanning tunneling microscopy ( stm ) , this work characterizes the structure adopted by pegylated dendrimers with the highest resolution reported to date . 
 afm imaging enables visualization of the individual dendrimers , as well as the differentiation and characterization of the dendrimer core and peg shell . 
 stm provides direct imaging of the peg extensions with high - resolution . 
 collectively , this investigation provides important insight into the structure of coated dendrimers , which is crucial for the design and development of better drug delivery vehicles . 
1. Introduction 
2. Materials and Methods
3. Results and Discussion
4. Conclusions