document: the specific targeting of nanoparticles to target cells in an organ of interest has been a long - term goal of many laboratories interested in using nanoparticles to mediate the functional delivery of biopharmaceutical agents ( including nucleic acids ) to defined target cells . 
   one of the main problems in using targeting ligands in conjunction with nanoparticles is the competition between specific - binding mediated cell entry and non - specific enhanced cell uptake mechanisms ( andreu et al , 2008 ) . 
   previously , we have observed that non - specific effects can be completely overwhelming ( waterhouse et al , 2005 ) , an observation that has led to a series of further studies ( wang et al , 2009 ) intended to determine and characterize those factors that might influence non - specific enhanced cell uptake mechanisms such as nanoparticle size and heterogeneity , surface hydrophilicity and charge , not to forget other factors such as ligand conformation , points of attachment and surface density . 
   recently , we described how ph - triggered , pegylated sirna nanoparticles ( also known as ph - triggered sirna - abc nanoparticles according to our recently described abcd nanoparticle structural paradigm ( kostarelos and miller , 2005 ; thanou et al , 2007 ; miller , 2008a ; miller , 2008b ) . 
   these nanoparticles with a surface - covering of 5mol% polyethylene glycol 2000 ( peg ) were able to mediate the passive targeting of sirna to liver cells in vivo , thereby providing proof of concept for an effective rnai therapeutic approach to the treatment of hepatitis b virus infections ( sifectplus nanoparticles ) ( carmona et al , 2009 ) . 
   in addition , pegylated sirna nanoparticles enabled for long - term circulation ( ltc sirna - abc nanoparticles ; lesirna nanoparticles ) were shown to do the same to tumour cells in vivo , thereby providing proof of concept for an effective rnai therapeutic approach to cancer treatment ( kenny et al , 2011 ) . in the case of 
   the ph - triggered , pegylated sirna nanoparticles , the peg coating required for the biological stabilization of these nanoparticles was introduced by a purpose - designed post - coupling methodology . 
   here , we report how this methodology has now been adapted to introduce integrin - targeting peptide motifs onto liposome and nanoparticle surfaces and so enable integrin receptor - mediated cellular uptake of agents to cells . there have been some spectacular data obtained in recent years using v6 integrin - receptor binding motifs for receptor - mediated delivery of different imaging agents to cells in vivo ( dicara et al , 2007 ; hausner et al , 2007 ; dicara et al , 2008 ; hausner et al , 2009 ) . 
   we have described previously the use of integrin - receptor targeting peptides to promote functional gene delivery to cells ( harbottle et al , 1998 ; cooper et al , 1999 ) . here , we now report on the use of an alternative cyclic pentapeptide construct that makes use of a single d - tyrosyl amino acid residue to present the arginine - glycine - aspartate ( rgd ) amino acid residue triad in an " active " conformation capable of mediating intracellular delivery through v3/5 receptor binding and internalization ( chen et al , 2004 ) . 
   arginine - glycine - glutamate ( rge ) controls are also reported in order to provide the means to demonstrate pure integrin - receptor mediated cellular uptake by rgd presenting imaging nanoparticles . 
   dioleoyl l--phosphatidylethanolamine ( dope ) 1 , dimyristoyl l--phosphatidylcholine ( dmpc ) 2 ( sigma ) , dope - lissamine - rhodamine b ( dope - rhoda ) 3 were obtained from avanti polar lipids ( usa ) ( figure 1 ) . 
   general synthetic procedures were performed as described previously ( carmona et al , 2009 ; mvel et al , 2010 ) . 
   syntheses of n - cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine ( cdan ) 4 and cholesteryl - aminoxy ( ca ) lipid 5 were performed as described previously ( keller et al , 2003 ; oliver et al , 2004 ; carmona et al , 2009 ) ( figure 1 ) . 
   the synthesis of other compounds necessary for our experiments were performed as described ( scheme 1 ) . 
   hplc purification of final products required the use of vydac c-4 reversed phase preparative column with 1ml / min flow rate : mobile phases as follows used trifluoroacetic acid ( tfa ) ; a : h2o ( 0.1% , v / v , tfa ) ; b : mecn ( 0.1% , v / v , tfa ) ; c : meoh ( 0.1% , v / v , tfa ) . program set at : 0 - 15.0min ( 100% , a ) , 15.1 - 25.0min ( 0 - 100% , b ) , 25.1 - 45min ( 100% , c ) , 45.1 - 55min ( 100% , a ) . 
   elution parameters : rgd - peg - cho 13a rt 15.2min , rge - peg - cho 13b rt 14.8min , prnhco - peg - cho 14 rt 15.8min , ca lipid 5 rt 23.8min , rgd - peg - ca 15a product rt 24.1min , rge - peg - ca 15b product rt 23.8min , and prnhco - peg - ca 16 product rt 26.3min ; mass spectrometry : m / z ( esi ) 1878 ( m - h , rgd - peg - cho 13a ) , 1917 ( mna , rge - peg - cho 13b ) , 1326 ( mh , prnhco - peg - cho 14 ) , 2406 ( mh , rgd - peg - ca 15a ) , 2443 ( mna , rge - peg - ca 15b ) , 1869 ( mna , prnhco - peg - ca 16 ) ( figure 1 ) . 
   main lipids were used to prepare liposomes cl1 and cl2 and hence 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles . 
   syntheses of -terminally modified - peg - chos : i ) a ) side chain protected , fluorenylmethyloxycarbonyl ( fmoc ) amino acid residue n - terminal coupling of l - arg(npbf ) , l - lys(boc ) , d - tyr(otbu ) and finally l - asp(otbu ) [ or l - glu(otbu ) ] to glycyl-2-chlorotrityl resin 6 using 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) with di - isopropylethylamine ( dipea ) to mediate residue coupling and 2% ( v / v ) each of 1,8-diazabicyclo[5.4.0]undec-7-ene ( dbu ) and piperidine in dimethylformamide ( dmf ) for fmoc deprotection between rounds of peptide bond formation : b ) resin release with 20% ( v / v ) each of acetic acid and trifluoroethanol ( tfe ) in ch2cl2 , 31 - 78% ; ii ) a ) 3eqv diphenylphosphoryl azide ( dppa ) in dry dmf , 5 eqv nahco3 , 0c  ambient temperature , ph 8.5 , 74 - 80% : 
   b ) 2.5% ( v / v ) each h2o and triisopropylsilane ( tis ) in trifluoroacetic acid ( tfa ) , 80 - 100% ; iii ) a ) ho - c2h4-oh , al2o3 , ccl4 , reflux , 27% : b ) pentafluorophenol ( pfpoh ) , n , n'-dicyclohexylcarbodiimide ( dcc ) , etoac , 71% ; iv ) a ) amino - peg - propionic acid , 4 eqv triethylamine ( tea ) , chcl3 , reflux , 84% : b ) 1.2 eqv pfpoh , 1.2 eqv dcc , etoac , 76% ; v ) 0.2 m na2hpo4 , 0.1 m naoh , 69 - 100% ; vi ) 10% ( v / v ) tfa in h2o , 100% ; vii ) a ) propylamine ( prnh2 ) , 4 eqv tea , chcl3 , reflux , 100% : b ) 10% ( v / v ) tfa in h2o , 51% ( v / v ) . 
   designated lipids were dissolved in chloroform at 5mg / ml and then appropriate aliquots were combined in a presilanized round - bottom flask ( 5ml ) . in each case , the organic solvent was evaporated to dryness to form an even thin lipid film that was further purged with a stream of argon gas to remove residual traces of the organic solvent . 
   the lipid film was hydrated using double distilled h2o ( ddh2o ) to give a multilamellar liposome suspension that was subsequently subjected to sonication in a water bath at 40c for 30min ( sonomatic water bath , langford ultrasonics , 33 khz ultrasound frequency ) . 
   post - sonication , small unilamellar vesicles ( 50 - 80 nm ) ( b - component , cl1 or cl2 ) were diluted to 1.5mg / ml and incubated at room temperature for 15min . 
   thereafter , appropriate aliquots of -terminally modified - peg - cho 13a , 13b or 14 ( 1mg / ml in water , cd molecules ) were introduced for post - coupling such that their final composition was between 1 and 10mol% of total lipid . 
   the ph of solution was monitored by ph boy ( camlab ltd , cambridgeshire , uk ) and adjusted to ph 4 if required by addition of small aliquots of aqueous solutions of naoh ( 0.99 m ) or hcl ( 0.99 m ) . on completion of the reaction ( approximately 16 - 24hr ) ( as judged by hplc analysis ) , the solution ph was accurately readjusted to ph 7 resulting in 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles , respectively , ready for use . 
   1 - 5mol% pegylated abcd2 nanoparticles were prepared by combining 1 - 5mol% pegylated bcd2 nanoparticles with various volumes of aqueous 4 mm hepes , ph 7.0 - 7.4 , sirna solution ( 50m a - component ) under heavy vortex conditions ( final [ sirna ] typically 100g / ml ; 7m ) using a lipid : sirna ratio of 12:1 ( w / w ) that correlates with a lipid - nucleic acid n / p charge ratio of approximately 2 . 
   lipid compositions of two liposome formulations prepared in double distilled water with final diameters of 50 - 80 nm . for lipid structures see figure 1 . 
   these experiments were performed on a facs calibur instrument ( bd biosciences ) . in each set of indicated experiments 1 , 2 or 5mol% pegylated abcd2 nano - particles ( 14060 nm ) 
   were prepared with sirna ( [ sirna ] 80pmoles / well ) and uptake studies performed with huvec cells previously grown to 80% confluent at 37c , 10% co2 . 
   dioleoyl l--phosphatidylethanolamine ( dope ) 1 , dimyristoyl l--phosphatidylcholine ( dmpc ) 2 ( sigma ) , dope - lissamine - rhodamine b ( dope - rhoda ) 3 were obtained from avanti polar lipids ( usa ) ( figure 1 ) . 
   general synthetic procedures were performed as described previously ( carmona et al , 2009 ; mvel et al , 2010 ) . 
   syntheses of n - cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine ( cdan ) 4 and cholesteryl - aminoxy ( ca ) lipid 5 were performed as described previously ( keller et al , 2003 ; oliver et al , 2004 ; carmona et al , 2009 ) ( figure 1 ) . 
   the synthesis of other compounds necessary for our experiments were performed as described ( scheme 1 ) . 
   hplc purification of final products required the use of vydac c-4 reversed phase preparative column with 1ml / min flow rate : mobile phases as follows used trifluoroacetic acid ( tfa ) ; a : h2o ( 0.1% , v / v , tfa ) ; b : mecn ( 0.1% , v / v , tfa ) ; c : meoh ( 0.1% , v / v , tfa ) . program set at : 0 - 15.0min ( 100% , a ) , 15.1 - 25.0min ( 0 - 100% , b ) , 25.1 - 45min ( 100% , c ) , 45.1 - 55min ( 100% , a ) . 
   elution parameters : rgd - peg - cho 13a rt 15.2min , rge - peg - cho 13b rt 14.8min , prnhco - peg - cho 14 rt 15.8min , ca lipid 5 rt 23.8min , rgd - peg - ca 15a product rt 24.1min , rge - peg - ca 15b product rt 23.8min , and prnhco - peg - ca 16 product rt 26.3min ; mass spectrometry : m / z ( esi ) 1878 ( m - h , rgd - peg - cho 13a ) , 1917 ( mna , rge - peg - cho 13b ) , 1326 ( mh , prnhco - peg - cho 14 ) , 2406 ( mh , rgd - peg - ca 15a ) , 2443 ( mna , rge - peg - ca 15b ) , 1869 ( mna , prnhco - peg - ca 16 ) ( figure 1 ) . 
   main lipids were used to prepare liposomes cl1 and cl2 and hence 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles . 
   syntheses of -terminally modified - peg - chos : i ) a ) side chain protected , fluorenylmethyloxycarbonyl ( fmoc ) amino acid residue n - terminal coupling of l - arg(npbf ) , l - lys(boc ) , d - tyr(otbu ) and finally l - asp(otbu ) [ or l - glu(otbu ) ] to glycyl-2-chlorotrityl resin 6 using 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) with di - isopropylethylamine ( dipea ) to mediate residue coupling and 2% ( v / v ) each of 1,8-diazabicyclo[5.4.0]undec-7-ene ( dbu ) and piperidine in dimethylformamide ( dmf ) for fmoc deprotection between rounds of peptide bond formation : b ) resin release with 20% ( v / v ) each of acetic acid and trifluoroethanol ( tfe ) in ch2cl2 , 31 - 78% ; ii ) a ) 3eqv diphenylphosphoryl azide ( dppa ) in dry dmf , 5 eqv nahco3 , 0c  ambient temperature , ph 8.5 , 74 - 80% : 
   b ) 2.5% ( v / v ) each h2o and triisopropylsilane ( tis ) in trifluoroacetic acid ( tfa ) , 80 - 100% ; iii ) a ) ho - c2h4-oh , al2o3 , ccl4 , reflux , 27% : b ) pentafluorophenol ( pfpoh ) , n , n'-dicyclohexylcarbodiimide ( dcc ) , etoac , 71% ; iv ) a ) amino - peg - propionic acid , 4 eqv triethylamine ( tea ) , chcl3 , reflux , 84% : b ) 1.2 eqv pfpoh , 1.2 eqv dcc , etoac , 76% ; v ) 0.2 m na2hpo4 , 0.1 m naoh , 69 - 100% ; vi ) 10% ( v / v ) tfa in h2o , 100% ; vii ) a ) propylamine ( prnh2 ) , 4 eqv tea , chcl3 , reflux , 100% : b ) 10% ( v / v ) tfa in h2o , 51% ( v / v ) . 
   designated lipids were dissolved in chloroform at 5mg / ml and then appropriate aliquots were combined in a presilanized round - bottom flask ( 5ml ) . in each case , the organic solvent was evaporated to dryness to form an even thin lipid film that was further purged with a stream of argon gas to remove residual traces of the organic solvent . 
   the lipid film was hydrated using double distilled h2o ( ddh2o ) to give a multilamellar liposome suspension that was subsequently subjected to sonication in a water bath at 40c for 30min ( sonomatic water bath , langford ultrasonics , 33 khz ultrasound frequency ) . 
   post - sonication , small unilamellar vesicles ( 50 - 80 nm ) ( b - component , cl1 or cl2 ) were diluted to 1.5mg / ml and incubated at room temperature for 15min . 
   thereafter , appropriate aliquots of -terminally modified - peg - cho 13a , 13b or 14 ( 1mg / ml in water , cd molecules ) were introduced for post - coupling such that their final composition was between 1 and 10mol% of total lipid . 
   the ph of solution was monitored by ph boy ( camlab ltd , cambridgeshire , uk ) and adjusted to ph 4 if required by addition of small aliquots of aqueous solutions of naoh ( 0.99 m ) or hcl ( 0.99 m ) . on completion of the reaction ( approximately 16 - 24hr ) ( as judged by hplc analysis ) , the solution ph was accurately readjusted to ph 7 resulting in 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles , respectively , ready for use . 
   1 - 5mol% pegylated abcd2 nanoparticles were prepared by combining 1 - 5mol% pegylated bcd2 nanoparticles with various volumes of aqueous 4 mm hepes , ph 7.0 - 7.4 , sirna solution ( 50m a - component ) under heavy vortex conditions ( final [ sirna ] typically 100g / ml ; 7m ) using a lipid : sirna ratio of 12:1 ( w / w ) that correlates with a lipid - nucleic acid n / p charge ratio of approximately 2 . 
   lipid compositions of two liposome formulations prepared in double distilled water with final diameters of 50 - 80 nm . for lipid structures see figure 1 . 
   these experiments were performed on a facs calibur instrument ( bd biosciences ) . in each set of indicated experiments 1 , 2 or 5mol% pegylated abcd2 nano - particles ( 14060 nm ) 
   were prepared with sirna ( [ sirna ] 80pmoles / well ) and uptake studies performed with huvec cells previously grown to 80% confluent at 37c , 10% co2 . 
   post - coupling represents an optimal way to attach a biological targeting ligand of interest to a nanoparticle surface with controlled mol% attachment and ligand orientation ( scheme 2 ) . 
   our approach to the development of a post - coupling methodology has been to learn from the chemistry employed to create ph - triggered , pegylated sirna nanoparticles ( as mentioned in the introduction ) in order to design - hybrid targeting ligands ( l ) that comprise a peptide targeting moiety conjugated to a peg extension chain enabled for postcoupling to a nanoparticle surface ( scheme 2 ) . 
   rgd peptides and corresponding rge control peptides were prepared as follows ( scheme 1 ) . starting with glycyl-2-chlorotrityl solid - phase peptide resin 6 , 
   the desired pentapeptide rgd - targeting moiety was prepared in an open chain , protected form 7 by solid - peptide synthesis , then subject to cyclization and deprotection to give cyclic pentapeptide 8a . 
   a control cyclic - rge pentapeptide 8b ( with aspartate [ d ] replaced by glutamate [ e ] ) was prepared in an equivalent manner . 
   thereafter , the aldehyde functionality of 4-carboxybenzaldehyde 9 was acetal protected and the carboxy function was activated for coupling by means of pentafluorophenol ( pfpoh ) esterification giving 10 . 
   reagent 10 was then coupled to amino - peg - propionic acid and the free carboxylate activated once again for coupling by means of pfpoh esterification giving heterofunctional peg moiety 11 . 
   the coupling of 11 to 8a or 8b resulted in rgd acetal 12a and rge acetal 12b respectively . 
   routine acetal deprotection yielded the desired products rgd - peg - cho 13a and control rge - peg - cho 13b ( scheme 1 ) . 
   an alternative control aldehyde was obtained through combination of heterofunctional peg moiety 11 with propylamine followed by acetal deprotection resulting in the non - peptide control product prnhco - peg - cho 14 ( scheme 1 ) . 
   all three -terminally modified - peg - cho compounds prepared as described above were used in subsequent experiments described below . using all three -terminally modified - peg - cho compounds in turn , coupling reactions were performed individually with neutral cholesteryl - aminoxy lipid ( ca ) 5 ( figure 1 ) and then with neutral liposomes ( equivalent in composition to cl1 , table 1 ) . 
   reactions were monitored by hplc and mass spectrometry in water at ph4 and shown to reach completion after 16 - 24h stirring at ambient temperature giving 15a , 15b , or 16 as appropriate . 
   liposome - coupling experiments were then performed under similar conditions in order to couple rgd - peg - cho 13a ( or control aldehydes rge - peg - cho 13b and prnhco - peg - cho 14 ) to alternative liposome formulations cl1 or cl2 ( table 1 , figure 1 ) , making use of ca lipid 5 once again in order to present the aminoxy functional groups required for conjugation of liposome surfaces with each -terminally modified - peg - cho compound in turn ( scheme 2 ) . upon conclusion of these liposome - coupling reactions , 
   ph was raised to 7 in order to stabilize the resulting oxime bonds and the resulting pegylated liposomes . 
   resulting ligand - pegylated liposomes ( approx 100 nm in diameter ) were described as belonging to either bcd1 or bcd2 nanoparticle families depending upon whether the parent liposome formulation used in preparation was cl1 or cl2 , respectively . 
   initially bcd1 family members rgd - bcd1 , rge - bcd1 and prnhco - bcd1 were prepared with 1mol% of dope - rhoda 3 for fluorescence microscopy cell uptake studies . 
   the results demonstrate that only 10mol% pegylated rgd - bcd1 nanoparticles were capable of delivering measurable and observable levels of fluorescent - label to integrin - rich human umbilical vein endothelial cells ( huvec ) ( figure 2a ) . 
   this is very interesting in view of the physical properties of bcd1 nanoparticles , namely approx 100 nm in diameter , 10mol% pegylation with peg , and a -potential of essentially zero mv . 
   the lack of charge originates from the fact that the majority lipid components needed to prepare cl1 liposomes were neutral while the minority ca lipid 5 component possesses an aminoxy functional group unprotonated at ph 7 due to its pka value of about 4.5 ( compared with the pka values of more typical amine functional groups that are found around 10 ) ( castro , 1990 ) . 
   we would argue that such nanoparticle physical properties could be an ideal starting point from which to optimize receptor specific cellular uptake of nanoparticles relative to non - specific enhanced cell uptake . 
   subsequently , bcd2 family members rgd - bcd2 , rge - bcd2 and prnhco - bcd2 were prepared with 1.0 mol% of dope - rhoda 3 and up to 5mol% of each -terminally modified - peg - cho for fluorescence - activated cell sorting ( facs ) studies . 
   in addition these cationic liposome systems were further combined with a non - functional nucleic acid ( small interfering rna [ sirna ] ) ( a component ) in order to neutralize the positive charge and convert the bcd2 family members in to a varieties of corresponding low charge abcd2 nanoparticles with lipid : sirna 
   the physical properties of these nanoparticles were diameters of approximately 140 nm , up to 5mol% pegylation with peg , and a -potential of less than 10mv . in these cases , 
   facs studies were performed with low charge 1- , 2- and 5mol% pegylated rgd - abcd2 , rge - abcd2 and prnhco - abcd2 nanoparticles . in each case , the discrimination between rgd presenting and control nanoparticles was still clearly present and was found to increase as a function of the mol% pegylation coverage with peg ( figure 2b ) . 
   the fact that the discrimination was not as substantial as observed above ( figure 2a ) appears to be due to the lower mol% levels of pegylation coverage and the presence of residual nanoparticle charge . 
   therefore , control of both pegylation levels ( plus other peg - structure related criteria ) and residual charges are essential in order to optimize receptor mediated cell uptake in preference to non - specific enhanced cell uptake mechanisms . 
   schematic illustration of nanoparticle formulations involving the inclusion of an aminoxy coupling lipid by a formulation procedure known as premodification followed by the post coupling of -terminally ligand ( l)-modified peg aldehydes ( l - peg - cho ) . 
   biological receptor mediated functional delivery of imaging agents is well established ( dicara et al , 2007 ; dicara et al , 2008 ; hausner et al . 2007 ; 
   hausner et al , 2009 ) , even of genes ( harbottle et al , 1998 ; cooper et al , 1999 ) . 
   however , receptor - mediated delivery can not be taken as a given following the covalent attachment of a biological targeting ligand to a nanoparticle ( waterhouse et al , 2005 ; andreu et al , 2008 ) . 
   therefore , in our view , receptor - mediated delivery phenomena should never be treated with the casual assumption that receptor - mediation is inevitable once an appropriate receptor - specific ligand has been mounted on a nanoparticle surface . 
   any given receptor - specific ligand should in fact be subject to a standard set of trial experiments to ensure that the biophysical properties of a nanoparticle platform and the presentation of the receptor - specific ligand are optimized for bonefide receptor - mediated delivery of the corresponding nanoparticle . in this paper 
   we describe just such a set of trial experiments ( figure 2 ) with sufficient controls to demonstrate clearly that specific receptor - mediated uptake by cells has been enabled over background . 
   accordingly , we would like to propose that both studies involving first the delivery of an imaging agent and then second a potential agent of pharmaceutical interest ( api ) , should be standard assays to perform and demonstrate before all else that a nanoparticle - attached ligand is truly a receptor - specific ligand in the context of the nanoparticle platform to which the ligand is covalently attached . if this is not shown , then nanoparticle biophysical properties , ligand attachment and orientation , and mol% ligand presentation should all be systematically altered until specific receptor - mediated cell uptake can be observed substantially over and above any non - specific enhanced cell uptake background ( kamaly et al , 2009 ; kamaly et al , 2010 ) . 
   fluorescence microscopy of huvec cells , previously grown to 80% confluent at 37c with 10% ( v / v ) co2 , treated with 10mol% pegylated bcd1 nanoparticles prepared from rgd - peg - cho 13a or rge - peg - cho 13b . 
   cells were in contact with nanoparticles for at least 4hr prior to mounting and observation of rhodamine fluorescence under a nikon 600 fluorescence microscope . 
   cell uptake was determined after 4hr using rhodamine fluorescence and detected using the fl-2 channel . 
   ab corresponds with simple sirna - lipoplex control nanoparticles , formulated from cl2 cationic liposomes and an equivalent final [ sirna ] . in our case here 
   , we can say with confidence that our post - coupling chemistry and methodology for the attachment of integrin - targeting rgd ligands has resulted in the successful formation of integrin - targeted imaging nanoparticles that can also mediate integrin - specific delivery of an api such as sirna to v3/5 integrin - receptor presenting cells . 
   therefore , this post - coupling chemistry and premodification - postcoupling methodology could be applicable to other nanoparticle platforms with equal success provided that the biophysical properties of the nanoparticle platform also conform to the following biophysical parameters : nanoparticle dimensions of approx 100 nm in diameter nanoparticle -potential values that converge on neutral ( 0 mv ) nanoparticle ligand surface coverage of approx 2 mol% ( or higher ) further research with this and other nanoparticle systems will now be needed to demonstrate if these three nanoparticle " rules " for receptor - mediated cell entry are indeed general rules or simply guidelines for receptor - mediated cellular uptake of nanoparticle systems by corresponding receptor expressing cells in vitro , ex vivo and/or in vivo . 
   the data described here represent the completion of a first study involving the preparation of ligand - mounted pegylated nanoparticles constructed by a bespoke pre - modification postcoupling methodology . 
   data suggest for the first time in our hands that this methodology may be used to ensure that receptor mediated cell uptake of attached nanoparticles can be " engineered " to dominate non - specific enhanced cell uptake mechanisms .
summary: a key goal of our research is the targeted delivery of functional biopharmaceutical agents of interest , such as small interfering rna ( sirna ) , to selected cells by means of receptor - mediated nanoparticle technologies . 
   recently , we described how ph - triggered , pegylated sirna - nanoparticles ( ph triggered sirna - abc nanoparticles ) were able to mediate the passive targeting of sirna to liver cells in vivo . 
   in addition , pegylated sirna nanoparticles enabled for long - term circulation ( ltc sirna - abc nanoparticles , lesirna nanoparticles ) were shown to do the same to tumour cells in vivo . 
   further gains in the efficiency of sirna delivery are expected to require active targeting with nanoparticles targeted for delivery and cellular uptake by means of attached biological ligands . 
   here we report on the development of a new synthetic chemistry and a bioconjugation methodology that allows for the controlled formulation of pegylated nanoparticles which surface - present integrin - targeting peptides unambiguously and so enable integrin receptor - mediated cellular uptake . 
   furthermore , we present delivery data that provide a clear preliminary demonstration of physical principles that we propose should underpin successful , bonefide receptor - mediated targeted delivery of therapeutic and/or imaging agents to cells .