over many millions 
 of years , viruses have evolved elegant and sophisticated 
 mechanisms for selectively delivering biological payloads into host 
 cells by performing a sequence of molecular functions in a coordinated 
 and systematic manner . for example , adeno - associated 
 viruses ( aav ) selectively adsorb onto subsets of host cells that express 
 specific surface markers . then , the aav is internalized 
 by these host cells via endocytosis , after which it makes its escape 
 from the endosome and releases its protein and dna contents into the 
 host cell . indeed , its excellent delivery efficiency , coupled with 
 the minimal pathogenicity of the virus , have made aav a powerful biotechnological 
 tool for delivering foreign dna and other biomolecules into host organisms . the past decade has witnessed considerable effort to devise 
 synthetic 
 systems that mimic these natural molecular machines to achieve more 
 effective drug delivery with fewer side effects . the 
 systems that have made the greatest progress in the clinic to date 
 are constructed by directly conjugating a targeting reagent ( e.g. , 
 an antibody ) to a drug . food and drug administration 
 ( fda ) , and dozens more are now in clinical trials . although most of the work to date has utilized monoclonal 
 antibodies as the targeting reagent , nucleic acid aptamers are an 
 alternative that offer additional advantages , including smaller size , 
 chemically defined synthesis , and lower immunogenicity . accordingly , several research efforts have demonstrated the potential 
 of aptamers as a targeting moiety . for example , 
 the tan group has shown that aptamer - drug conjugates can facilitate 
 drug uptake by specific target cells expressing an appropriate surface 
 marker . although these constructs that directly 
 couple affinity reagent 
 to drugs have shown promise thus far , we envision the potential for 
 further gains in safety and efficacy through the development of  multifunctional  
 vehicles that akin to a virus  achieve targeted cell 
 entry and selective payload release in a stepwise fashion . more specifically , such a delivery mechanism would entail : ( i ) prolonged 
 systemic circulation while carrying therapeutic agents in an inactive 
 state , ( ii ) efficient tissue penetration to reach target cells , ( iii ) 
 specific binding and internalization into target cells , and ( iv ) selective 
 activation and release of therapeutic payload upon internalization . these features would be particularly valuable in the context of cancer 
 therapeutics , where the drugs involved are typically highly toxic 
 and the aim is to selectively eliminate subpopulations of tumor cells 
 without harming healthy cells in the adjacent tissue . ( a ) a pegylated 
 aph carrying multiple deactivated drug molecules binds a cancer target 
 cell via aptamer - mediated recognition of nucleolin . ( c ) esterases within the endosome 
 induce specific cleavage that ( d ) releases drug molecules from the 
 aph scaffold , after which ( e ) the now - active small - molecule drugs 
 can diffuse into the cytoplasm . we conjugated a nucleolin - specific aptamer modified with 
 cy5 at its 5 end to a peg - based block copolymer via an efficient , 
 ligand - accelerated cuaac reaction . this polymer is loaded with multiple 
 payload molecules , which are released within target cells via esterase - mediated 
 cleavage . toward this end , the integration 
 of aptamers with different classes 
 of specialized biomaterials into a single , multifunctional molecular 
 construct offers a compelling strategy for synthetic delivery vehicles . for example , the langer and farokhzad laboratories have demonstrated 
 the potential utility of aptamers in this context , working extensively 
 with drug - loaded polymeric nanoparticles coated with aptamers targeting 
 tumor - specific membrane proteins and have shown that such constructs 
 can enhance selective killing of tumor cells . we believe 
 block copolymers constitute an especially promising class of polymers 
 for the expansion of this strategy because of the diverse chemical 
 functionalities that can be incorporated ; furthermore , these molecules 
 can be designed to incorporate large numbers of drug molecules in 
 a chemically defined manner . in order to realize 
 this potential , we have developed a mild yet 
 highly effective synthesis strategy based on  click chemistry  
 that seamlessly integrates aptamers and block copolymers into a single , 
 chemically defined molecular construct . aptamer - polymer 
 hybrids ( aphs) that combine the targeting aptamer with a drug - loaded 
 multifunctional block copolymer , which secures its therapeutic payload 
 in an inactive state ( figure 1a ) . upon binding 
 the target cell , the aph is internalized via endocytosis , at which 
 point the covalent bonds linking the payload to the polymer scaffold 
 are cleaved by the enzymatic milieu of the endosome . this selectively 
 activates and releases the drug , which then diffuses into the cytoplasm 
 of the target cell . our synthesis scheme efficiently conjugates the 
 two material components with a high yield without compromising the 
 targeting efficiency of the aptamer or the programmed drug release 
 functionality of the block copolymer ( figure 1b ) . as a model , we demonstrate aph constructs that specifically target 
 tumor cells that overexpress the nucleolin surface marker . after confirming 
 the coordinated molecular function of the aph based on the successful 
 delivery of the fluorescent dye coumarin , we show that these aph constructs 
 can selectively deliver the chemotherapeutic agent doxorubicin ( dox ) 
 into nucleolin - expressing tumor cells , enabling targeted killing of 
 these cells . furthermore , we show that our synthetic approach can 
 be utilized with other aptamer sequences to achieve flexibility in 
 binding to different surface markers , demonstrating the generality 
 of our system for targeted drug delivery . we designed our aph with a dna aptamer that specifically binds to tumor cells that 
 express nucleolin , a well - known tumor surface marker . because nucleolin is known to undergo receptor cycling , 
 it enables internalization of the aph into the tumor cells through 
 endocytosis . the nucleolin aptamer is covalently 
 conjugated to a block copolymer with repeat units of ethylene glycol 
 and ethylene glycol vinyl glycidyl ether ( egvge ) using a we chose 
 this polymer scaffold ( 11.3 kda , see scheme 1 ) because it offers excellent biocompatibility , prolonged circulation , and desirable size for efficient tissue penetration 
 ( radius 10 nm ) . the cell viabiliy 
 assay also confirmed that this polymer scaffold is not cytotoxic to 
 the cells ( see figure s1 ) . importantly , 
 we designed the polymer backbone with multiple orthogonal reactive 
 groups to accommodate multiple dox molecules that are tethered through 
 enzyme - cleavable linkers ( see scheme s1 ) . these linkers are designed to be efficiently cleaved by esterases 
 that are endogenous to the endosome of mammalian cells . once cleaved , dox efficiently escapes the endosome 
 due to its small size and rapidly diffuses in the cytoplasm to reach 
 the nucleus . aph synthesis requires efficient , site - specific 
 conjugation of 
 the dna aptamer to the polymer scaffold without loss of function or 
 binding affinity . to this end , we developed an improved synthetic 
 strategy for coupling dna aptamers to synthetic polymers based on 
 the novel tricarboxylate ligand ( bimc4a)3 , which 
 stabilizes cu ( i ) during the copper - catalyzed azide  alkyne 
 cycloaddition ( cuaac ) ( scheme 1 ) . in comparison 
 to traditional cuaac ligands , the use of ( bimc4a)3 does not cause irreversible damage to the aptamers , leads to higher 
 yield , and offers superior water solubility compared to tris(benzyltriazolylmethyl)amine 
 ( tbta ) and other ligands . the synthesis 
 of our aph involves coupling of the nucleolin aptamer , which features 
 a single azide at its 3 terminus ( see table s1 ) , with an -alkyne - functionalized polyether 
 ( scheme 1 and methods for details ) . after purification , dynamic light scattering of the 
 aphs with concentrations ranging from 0.5 to 10 m showed that 
 the resulting aph consistently exhibited 10  2 nm in radius 
 ( figure 2a ) , and we found negligible evidence 
 of micelle formation or aggregation at the measured concentrations 
 ( figure 2a ) . this was clearly evident in the electrophoresis shown in figure 2b . specifically , aphs synthesized using conventional 
 conjugation schemes with tbta or the tbta analogue ( bim)3 caused significant damage to the aptamer , 
 which is evident in the heavy smearing of the band due to the fragmentation 
 of dna ( figure 2b , lane 3 ) . in contrast , aph 
 synthesis with ( bimc4a)3 gave rise to a sharply 
 defined single band , indicating minimal damage to the dna aptamer 
 ( lane 4 ) . as controls , the unconjugated aptamer ( lane 2 ) formed a 
 single band of the predicted size , whereas the unconjugated polymer 
 scaffold exhibited no electrophoretic mobility due to its neutral 
 charge and remained in the loading well ( lane 1 ) . interestingly , when 
 the polymer was loaded with coumarin dye molecules , it migrated toward 
 the cathode due to the slightly positive charge resulting from protonation 
 of coumarin ( figure s2 ) . characterizing functional 
 integrity of the aph construct . ( a ) dynamic 
 light scattering measurements revealed a narrow radius distribution 
 of 10 nm with negligible aggregation in phosphate - buffered 
 saline buffer . ( b ) gel electrophoresis shows that aph conjugation 
 via ( bimc4a)3 ligand - accelerated cuaac greatly 
 minimizes dna damage . lanes : 1 , unconjugated polymer ; 2 , unconjugated 
 aptamer ( 45 nt ) ; 3 , aph synthesized with ( bim)3 ; 4 , aph 
 synthesized with ( bimc4a)3 ; 5 , 20-bp ladder . ( c ) affinity measurements from a bead - based nucleolin - binding assay 
 show that aptamer target affinity is preserved within the aph construct . the unconjugated aptamer exhibits a kd of 8.37  0.75 nm ( black ) , while aph molecules consisting of 
 cy5-labeled aptamer and coumarin - labeled polymer scaffold display 
 similar kds of 5.18  0.72 and 4.01 
  0.72 nm based on cy5 ( red ) and coumarin ( blue ) intensities , 
 respectively . untreated aphs emit a red - shifted , self - quenched 
 signal ( red ) , but esterase treatment shifts the peak fluorescence 
 wavelength to that of free coumarin ( blue ) , with a signal that increases 
 over time as more coumarin is released ( dashed lines ) . we further confirmed 
 the retention of aptamer function by comparing the equilibrium dissociation 
 constant ( kd ) of the aptamer in solution 
 to that of the aph . a bead - based binding assay ( see methods ) revealed a kd of 8.37  0.75 nm for cy5-labeled nucleolin 
 aptamer versus 5.18  0.72 nm for the fully conjugated aph ( figure 2c ) , confirming that the conjugation scheme described 
 above does not compromise the binding affinity of the aptamer . importantly , 
 the efficiency of quantitative polymerase chain reaction ( qpcr ) was 
 comparable for aptamers conjugated to the polymer scaffold and unconjugated 
 aptamers in solution ( figure s3 ) . these 
 data offer compelling evidence that our aph synthesis scheme using 
 ( bimc4a)3 does not affect aptamer functionality . 
 finally , we confirmed that the fully assembled aph retains the affinity 
 of the aptamer by loading the polymer moiety with coumarin dye . this 
 construct exhibited a kd of 4.01  
 0.72 nm ( figure 2c , blue ) in our bead - based 
 binding assay , essentially equivalent to that of the aptamer alone 
 or the aph in the absence of a payload . as expected , the coumarin - loaded 
 polymer scaffold showed negligible affinity for nucleolin in the absence 
 of aptamer conjugation ( figure 2c , gray ) . our aph synthetic process can also be generalized to other aptamers 
 as a means for targeting different cell - surface markers . to demonstrate 
 this important feature , we used the same chemistry to conjugate dna 
 aptamers for thrombin and immunoglobulin 
 e ( ige ) to the same polymer scaffold . 
 after verifying that our conjugation strategy preserves the integrity 
 of the thrombin and ige aptamers via gel electrophoresis ( figure s4 ) and qpcr ( figure 
 s5 ) , we measured the binding affinities of these aphs ( figures s6 and s7 ) . in both cases , the difference 
 in binding between the unconjugated aptamer and the aph was negligible , 
 clearly demonstrating that the conjugation scheme does not affect 
 the structure or function of the resulting hybrids . although the underlying 
 mechanism behind our ligand is not yet fully understood , we suspect 
 that the negative charge of the ( bimc4a)3 complex 
 electrostatically repels the negatively charged phosphate backbone 
 of dna and thereby prevents cu ( i ) from inflicting oxidative damage 
 on the aptamer . finally , we verified that the therapeutic payload 
 remains sequestered 
 in an inactive state within the aph until it is cleaved by the enzymes 
 in the endosome . to assess the efficiency of this cleavage reaction , 
 we measured the release of coumarin by porcine liver esterase ( ple ) 
 as a function of time . without ple , we 
 observe minimal fluorescence from the coumarin because it is tightly 
 sequestered within the aph ( figure 2d ) , even 
 after 3 h in either buffer or undiluted human serum ( figure s8 ) . this latter result demonstrates that drug payloads 
 should remain stably sequestered in the physiological environment 
 prior to internalization by target cells . however , in the presence 
 of ple , we observed a dramatic increase 
 in fluorescence at 467 nm , the expected peak for free coumarin . quantification 
 of the data showed that 50% of the payload is released within 30 min 
 at 15 u / ml ple ( figure s9 ) . as expected , 
 the rate of coumarin release increased proportionally with the enzyme 
 concentration ( figure s10 ) . we confirmed 
 that this process was enzyme dependent rather than chemically driven , 
 as exposure to the acidic ph typically found in the endosome was insufficient 
 to promote coumarin release ( figure s11 ) . taken together , these results confirm that our aph retains the 
 binding affinity of the parent aptamer and requires the enzymatic 
 activity of endosomal esterases to release their therapeutic payload . we 
 used confocal fluorescence microscopy to verify targeted aph binding , 
 internalization and payload release in live cells . as target cells , 
 we used tumorigenic human breast epithelial cells ( mcf-7 ) that overexpress 
 nucleolin on their membrane , with nontumorigenic human breast epithelial 
 cells ( mcf-10a ) that do not express nucleolin as negative control . in order to fluorescently track the payload 
 and aph independently inside cells , we used coumarin as the model 
 payload ( blue fluorescence ) and labeled the aptamer component of the 
 aph with cy5 ( red fluorescence ) . our aphs exhibited highly selective 
 binding to cells that overexpress nucleolin . with mcf-7 cells , we 
 observed aph binding at the cell surface immediately after incubation , 
 as indicated by strong red fluorescence along the cellular contour 
 ( figures 3 , top , and s12 ) . in contrast , negligible binding was observed with mcf-10a cells 
 ( figure 3 , bottom ) , confirming that nucleolin 
 expression is a prerequisite for specific binding . after 10 min , aph 
 is internalized into endosomes , based on the localization of red fluorescence 
 within the mcf-7 cells , and within 1 h , we observed evidence of cleavage 
 of the ester linkages and endosomal escape of coumarin based on the 
 permeation of blue fluorescence into the cytoplasm . after 4 h , efficient 
 endosomal escape of coumarin was observed , with widespread diffusion 
 throughout the cytoplasm . in contrast , the red fluorescence remained 
 localized within the endosome at this late time - point , indicating 
 that the polymer scaffold remained trapped , presumably due to its 
 larger size . these data show that endosomal internalization efficiently 
 triggers payload cleavage and release into the cellular interior . critically , we observed a complete lack of blue fluorescence within 
 mcf-10a cells even after 4 h , demonstrating that payload delivery 
 does not occur without nucleolin - mediated aph binding and internalization 
 ( figure 3 , bottom ) . live - cell imaging confirms 
 aph - mediated , targeted coumarin delivery 
 to nucleolin - expressing cells . we incubated aphs that couple a cy5-labeled 
 nucleolin aptamer ( red ) with a polymer scaffold loaded with coumarin 
 ( blue ) with mcf-7 ( top ) and mcf-10a ( bottom ) cells , which express 
 high and low levels of nucleolin , respectively . after 10 min , we washed 
 the cells thoroughly ; over the next 4 h , we observed nucleolin - mediated 
 cell - surface binding , aph internalization and cytoplasmic payload 
 release in mcf-7 but not mcf-10a cells . the power of this modular coupling 
 was clearly demonstrated through 
 a series of control experiments that show how each component of the 
 aph makes a critical contribution to its targeted delivery function . the deletion of the aptamer component rendered the polymer scaffold 
 incapable of target recognition , and we observed no internalization 
 by mcf-7 cells ( figure 4a ) . similarly , a mixture 
 of unconjugated aptamers and polymer scaffolds only showed localized 
 red fluorescence within endosomes due to aptamer uptake but no blue 
 fluorescence when incubated with mcf-7 cells ( figure 4b ) . replacing the nucleolin aptamer in the aph with a cy5-labeled 
 ige aptamer also prevented uptake by mcf-7 cells due to the lack of 
 ige receptors on their cell surface ( figure 4c ) . control experiments demonstrate that each component of the aph 
 contributes essentially to targeted delivery . ( a ) in the absence of 
 a nucleolin - targeting aptamer , the polymer scaffold is no longer bound 
 or internalized by mcf-7 cells . ( b ) mcf-7 cells incubated with an 
 unconjugated mixture of nucleolin aptamers and polymer scaffolds internalized 
 the aptamer but not the polymer . ( c ) aphs that target ige are not 
 bound or internalized by mcf-7 cells , which do not express this protein . ( d ) nucleolin - targeting aphs bind mcf-7 cells but are no longer internalized 
 at temperatures that inhibit endocytosis . ( e ) coumarin release is 
 greatly reduced when the payload is linked to the polymer scaffold 
 via esterase - resistant bonds . we confirmed that aph molecules are actively internalized 
 via endocytosis 
 rather than diffusion or another passive mechanism by incubating mcf-7 
 cells with nucleolin - targeting aphs at 4 c , a temperature at 
 which endocytotic pathways are inactive . even after 4 h , red fluorescence remained entirely localized at 
 the cellular surface , indicating that the aphs could successfully 
 bind nucleolin but were no longer being internalized at this temperature 
 ( figure 4d ) . finally , we verified that payload 
 release requires esterase - mediated cleavage in the endosome through 
 the synthesis of an alternate aph wherein coumarin was coupled to 
 the polymer scaffold through amide linkages ( scheme 
 s2 ) that can not be cleaved by esterases . in this case , only 
 minimal blue fluorescence could be detected after 4 h at 37 c 
 ( figure 4e ) with the magnitude of the signal 
 being dramatically reduced in comparison to aphs with esterase - cleavable 
 linkages ( figure 3 , top row ) . finally , we constructed 
 aph with the chemotherapeutic agent dox as the payload and used it 
 to selectively kill nucleolin - expressing target cells . to synthesize 
 the aph , we conjugated the hydroxyl group of dox to the polymer scaffold 
 via the esterase - cleavable linker described above ( scheme s1 ) . we found that dox - loaded aph molecules efficiently 
 killed mcf-7 cells at a concentration of 500 nm . we used a live / dead 
 cell viability assay ( see methods ) to measure 
 the cytotoxicity of the dox - loaded aphs ( figure 5 ) and determined that this treatment reduced cell viability by 64% . the potency of the aph was also readily apparent in microscopic images 
 of treated cells , in which viability could be visually ascertained 
 based on changes in cell morphology and adhesion characteristics ( figure s13a ) . however , the cytotoxicity of the 
 aph is lower than equivalent , free dox at a concentration of 5 m , 
 indicating that some ester linkages between the aph and the dox would 
 be still intact within the endosome . mcf-7 cells incubated with dox - loaded aphs for 4 days at 37 
 c exhibited 36% viability , while the cells with free dox yielded 
 18% viability relative to untreated controls . similar treatment with 
 either unconjugated aptamers or dox - loaded polymers alone resulted 
 in minimal cytotoxicity . as demonstrated above with coumarin - loaded aphs , we found 
 that 
 each component of the aph plays an integral role in the targeted delivery 
 of chemotherapy to nucleolin - expressing cancer cells . equal concentrations 
 of unconjugated nucleolin aptamers ( figure s13b ) or dox - loaded polymers ( figure s13c ) 
 alone had negligible effect on mcf-7 cell viability , even after 4 
 days of incubation , with levels of cell death that were essentially 
 indistinguishable from untreated cells in culture media ( figure s13d ) . based on our cell viability assay , cells treated with unconjugated aptamers or dox - loaded polymers respectively 
 exhibited 100% and 91% viability relative to untreated cells 
 ( figure 5 ) ; the latter result confirms that 
 the polymer keeps the drug safely sequestered in an inactive state , 
 which is an important feature when delivering toxic therapeutics . in this work , we describe the design and 
 synthesis of multifunctional 
 drug - delivery vehicles that combine the advantages of aptamers and 
 functional polymers to enable controlled , targeted therapy . the resulting 
 aph constructs incorporate an aptamer component and a block copolymer 
 component . the aptamer serves to selectively bind cell - surface markers 
 specific to the target cell , such that the aph is subsequently internalized 
 via an active endocytotic mechanism . in parallel , the polymer component 
 incorporates multiple payload molecules , which are enzymatically cleaved 
 from the scaffold and subsequently diffuse into the cytoplasm only 
 after uptake of the aph in an endosomal compartment . as a result , 
 drug - mediated cytotoxicity is restricted to target cells that express 
 the surface marker of interest , and control experiments verified that 
 there is minimal drug release in the absence of cellular binding and 
 endosomal internalization . importantly , aph synthesis is achieved 
 via a click chemistry scheme 
 that offers reproducibility and high yield under mild conditions that 
 ensure the structural and functional integrity of both the aptamer 
 and polymer components . we showed that our synthesis strategy is modular 
 and can be used with a variety of aptamers to target other cell surface 
 markers . finally , given that our scheme could be readily adopted for 
 other polymer systems and functional nucleic acids such as dnazymes , rna riboswitches , and structure - switching aptamers , we believe 
 our strategy of integrating aptamer technology with functional , biocompatible 
 synthetic polymers into a single chemical platform may enable the 
 development of other useful functional materials for biomedical applications .
viruses 
 have evolved specialized mechanisms to efficiently transport 
 nucleic acids and other biomolecules into specific host cells . 
 they 
 achieve this by performing a coordinated series of complex functions , 
 resulting in delivery that is far more efficient than existing synthetic 
 delivery mechanisms . 
 inspired by these natural systems , we describe 
 a process for synthesizing chemically defined molecular constructs 
 that likewise achieve targeted delivery through a series of coordinated 
 functions . 
 we employ an efficient  click chemistry 
 technique to synthesize aptamer - polymer hybrids ( aphs ) , coupling cell - targeting 
 aptamers to block copolymers that secure a therapeutic payload in 
 an inactive state . upon recognizing the targeted cell - surface marker , 
 the aph enters the host cell via endocytosis , at which point the payload 
 is triggered to be released into the cytoplasm . after visualizing 
 this process with coumarin dye , we demonstrate targeted killing of 
 tumor cells with doxorubicin . 
 importantly , this process can be generalized 
 to yield aphs that specifically target different surface markers .