since the particle size is 
 a key consideration in nuclear transport , three nps of different sizes 
 ( 15.0  1.2 , 8.5  0.8 , and 5.5  0.8 nm , respectively ) 
 were synthesized to study the size effect of the nanoparticle on nuclear 
 internalization ( see tem images in figure s1 ) .
after modification with a dense shell of the das , the hydrodynamic 
 diameters of these nps ( termed 15-np , 8.5-np , and 5.5-np ) , as measured 
 by dynamic light scattering ( dls ) , were 37.8  1.3 , 18.2  
 1.2 , and 11.7  1.5 nm , respectively . to prevent mutual interference 
 between tumor targeting and nanodrug capture ,
a directional modification 
 was performed on the nrs ( 65 nm  20 nm ) , which means the aptamer 
 and the cs were separately modified on the end faces and the side 
 face of the nr , rather than being randomly spread .
the principle of 
 this selective modification process was based on the preference of 
 ctab to accumulate on the side face of nr , which makes its end faces 
 more reactive to thiolated dnas .
specifically , 
 the aptamer at a relatively low concentration was first used to occupy 
 the nr ends .
subsequently , the cs at a high concentration was added 
 to replace ctab on the nr side face . to make the resulting nanomaterial 
 biocompatible , thiolated peg was used , followed by several washing 
 steps . via dna hybridization ,
as characterized by tem ( figure 1 ) , nps were exactly immobilized onto the nr sides , 
 leaving the nr ends bald , indicating successful fabrication of the 
 side - assembly nanostructures ( termed 15-np / nr , 8.5-np / nr , and 5.5-np / nr ) . 

the self - assembly process was further investigated with dls and uv  vis 
 absorption spectroscopy .
the results from different angles all demonstrated 
 the successful assembly of nps and nrs resulting from dna hybridization 
 ( see details in figures s2a and s2b ) .
tem images 
 of 15-np / nrs , 8.5-np / nrs , and 5.5-np / nrs .
15-nps , 8.5-nps , and 5.5-nps 
 mostly attach onto the nr sides , but not the nr ends , indicating the 
 successful fabrication of the side - assembly nanostructures .
the 
 scale bar represents 50 nm . after synthesizing and characterizing these 
 nanoassemblies , we next investigated their nir - response and drug - carrying 
 capability .
the efficient photothermal effect of nrs was verified 
 by the rapid temperature rise of the nr medium when irradiated by 
 the nir laser ( see details in figure s3a ) .
then the nir - activated release of nps from nrs was demonstrated 
 by the reduced average size ( dls ) and the disassembled structure ( tem ) 
 of the 15-np / nrs after exposure to the laser ( figure s3b ) . to fabricate
the nuclear - uptake nanodrug 
 delivery system , doxorubicin ( dox ) , a widely used anticancer drug , 
 was chosen as the model and loaded on the nps by intercalating into 
 the ( cgt)6/(acg)6 duplexes of the das ( table s1 ) .
the drug payload of nps was measured 
 by monitoring the dox fluorescence from the supernatant after nps 
 were centrifuged down . as shown in figure 2a , a gradual decrease of dox fluorescence
was observed when increasing 
 the molar ratio of nps . by interpolating from a standard calibration 
 curve ,
the dox payload of each 15- , 8.5- , and 5.5-np was 450  
 19 , 277  28 , and 40  13 molecules , respectively .
the stability 
 of np / dox complexes was evaluated via a drug leakage experiment using 
 mini dialysis units . as shown in figure 2b , 

the release of dox from nps was rather slow , with less than 30% of 
 the entire payload detected in the solution after 60 h , in comparison 
 to the rapid diffusion of free dox , indicating high stability of the 
 np / dox complexes .
furthermore , the nanoasemblies stored at 4 c 
 for 8 weeks remained mostly intact .
( a ) dox fluorescence spectra of the supernatant after centrifuging 
 to precipitate the das - modified nps , the dox concentration was fixed 
 at 2 m when increasing the 15-np - das / dox mole ratio .
( b ) dox 
 leakage dynamics from np - dox complexes ( dox : 10 m ) .
( c ) clsm images of cem cells after 
 incubation with 15-nps , 8.5-nps , and 5.5-nps ( which represent the 
 das - modified nps of 15 , 8.5 , and 5.5 nm , respectively ) at 37 c 
 for 10 or 22 h. the green and blue fluorescence arises from the tamra 
 fluorophore labeled on the 3 end of das and hoechst 33342 , 
 respectively .
the scale bar represents 5 m . having confirmed 
 the potential of using the np / nr nanoassemblies as nir - responsive 
 drug nanocarriers in buffer solution , we proceeded to test their performance 
 in cells . a nondrug - resistant leukemia cell line , cem ,
was first used 
 as the cell model . to monitor the cellular uptake and intracellular 
 distribution of nps of different sizes ,
the das was labeled with a 
 5-end tamra fluorophore ( das - tmr ) .
after modification with 
 das - tmr , the fluorescent particles were incubated with the cem cells 
 at 37 c for different lengths of time .
then confocal laser scanning 
 microscopy ( clsm ) measurements were performed , and the results are 
 shown in figure 2c .
nps of 15 nm were mainly 
 localized in the cytoplasm , even after 22-h incubation , as indicated 
 by the tamra fluorescence signal outside the nucleus . in contrast , 
 5.5-nps accumulated in the nucleus after incubation for 10 h , demonstrating 
 rapid nuclear uptake . for 8.5-nps ,
no obvious signal was observed 
 in the nucleus for 10-h incubation , but the nucleus emitted tamra 
 fluorescence after incubation for 22 h , indicating that 8.5-np - dass 
 can enter the nucleus starting from 10 h. to strike a balance between 
 drug loading capability and nuclear translocation efficiency , the 
 8.5-np was used as a model nuclear - uptake nanoscaffold of dox in the 
 following study . to achieve active tumor targeting , sgc8 , an 
 aptamer that can specifically bind to protein tyrosine kinase 7 ( ptk7 , kd = 0.8 nm ) which is overexpressed on 
 the membrane of cem cells but not ramos cells ,
the specific binding of sgc8 , nr - sgc8 , and np / nr - sgc8 
 to the target cem cells , rather than nontarget ramos cells , was demonstrated 
 by flow cytometry ( figure 3a ) .
also , the specific 
 cellular uptake and cytoplasmic location of nr - sgc8s were visualized 
 with clsm ( figure s4 ) .
moreover , the amount 
 of the internalized nps delivered by the nr - sgc8s was higher than 
 that of equivalent free nps ( figure s5 ) , 
 which may have resulted from the high payload and the favorable cell - uptake 
 size of the nanoassembly , as well as the promotion of the cell - internalizing 
 aptamer .
specific cell binding and photocontrolled intracellular distribution 
 of np / nr - sgc8s.(a ) flow cytometry assay proving the specific binding 
 of sgc8 , nr - sgc8 , and np / nr - sgc8 to target cem cells not to nontarget 
 ramos cells ( lib represents a random library sequence ) .
( b ) clsm images 
 of cem cells after treatment with 15-np / nr - sgc8s without ( i ) and with 
 ( ii ) nir irradiation or after treatment with 8.5-np / nr - sgc8s without 
 ( iii ) and with ( iv ) nir irradiation . from left to right : fluorescence 
 image for np - tmr , nr - cy5 , and overlay of the np - tmr , nr - cy5 , and hoechst 
 33342 fluorescence channels plus the bright field channel .
the scale 
 bar represents 5 m . the nir - responsive behavior of 8.5-np / nr - sgc8s and 15-np / nr - sgc8s 
 in cem cells was investigated with clsm . without laser treatment , 
 the 8.5-np signal was observed in the cytoplasm and overlapped with 
 the nr - sgc8 signal ( figure 3b ) .
however , upon 
 nir irradiation , 8.5-nps were found in the nuclei , while nr - sgc8s 
 remained in the cytoplasm , indicating that 8.5-nps were released from 
 nrs after exposure to the laser and then diffused into the nuclei . 

the 15-nps remained in the cytoplasm irrespective of nir irradiation , 
 corresponding well with their inability to undergo nuclear internalization . 

the photocontrolled nuclear internalization of 8.5-nps was further 
 confirmed by using inductively coupled plasma atomic emission spectrometry 
 ( icp - aes , figure s6 ) .
on the basis 
 of the fluorescence quenching of dox by intercalating into the gc 
 duplex , the intracellular distribution of dox was investigated by 
 treating cem cells with nonfluorophore - labeled 8.5-np - dox / nr - sgc8s . 

after nir irradiation and then incubation for another 22 h , the recovered 
 fluorescence of dox was highly accumulated in the nuclei , with a relatively 
 small amount in the cytoplasm , indicating that most dox were released 
 in the nuclei ( figure s7 ) .
however , without 
 nir treatment , the intranuclear dox fluorescence was rather weak , 
 showing a slow and sustained release of dox in the cytoplasm .
as a 
 control , cells were treated with free dox , and the dox signal was 
 found throughout the cells , resulting from concentration - gradient 
 diffusion .
furthermore , the negligible influence of the laser irradiation 
 on the stability of the np - dox complex was confirmed by the small 
 dox signal change from the 8.5-np - dox / nr - sgc8s - treated cem cells before 
 and right after laser exposure ( figure s8 ) .
these results show great potential of the 8.5-np - dox / nr - sgc8 system 
 for nir - controlled intranuclear drug delivery .
the therapeutic 
 effect of this 8.5-np - dox / nr - sgc8 system on cem and ramos cells was 
 tested by mts assay . as shown in figure 4 and figure s9 , the nanomaterials themselves and
the 
 pure nir irradiation had little negative impact on either cem or ramos 
 cells ( the cell viability of both cell lines remained above 95% ) , 
 indicating excellent biocompatibility of these nanomaterials and the 
 laser .
for free dox , a dose - dependent cytotoxicity was observed on 
 both cem and ramos cells .
however , when treated with 8.5-np - dox / nr - sgc8s , 
 only cem cells showed dose - dependent cell inactivity , indicating the 
 selective cytotoxicity of dox delivered by this nanoassembly platform . 

after nir irradiation , a dramatic decrease of cell viability on cem 
 cells was caused by 8.5-np - dox / nr - sgc8s with a nearly 3-fold lower 
 ic50 of 0.36 m compared to that without nir irradiation 
 ( 1.22 m ) . to verify whether the enhanced therapeutic efficacy 
 originated from the synergy of dox , photothermal effect , and np / nr 
 nanocomplexes , cem cells were incubated with free dox and non - dox - loaded 
 8.5-np / nr - scg8c ( the dox loading site of das was replaced with a common 
 dna duplex ) together and then irradiated with the nir laser .
the therapeutic 
 effect of this case was lower than that of the nir - activated 8.5-np - dox / nr - scg8c , 
 indicating that the synergistic effect was not an important consideration 
 in this system ( figure s10 ) .
thus , it is 
 reasonable to attribute the enhanced killing efficiency to the released 
 8.5-np - dox from the nanotruck . to ensure that the nuclear accumulation 
 of nanodrug induced higher cell apoptosis ,
the nir - activated 15-np - dox / nr - sgc8 
 system was used as a non - nuclear - uptake control .
as shown in figure s11 , with nir irradiation , the cytotoxicity 
 of 8.5-np - dox / nr - sgc8s was 24% higher than that of 15-np - dox / nr - scg8c , 
 while no obvious difference was observed in either case without nir 
 irradiation .
these results demonstrate that this nuclear - uptake nanodrug 
 delivery system can greatly enhance the therapeutic efficacy on the 
 target cancer cells .
viability of cem cells ( a ) and ramos 
 cells ( b ) with different treatments .
the error bars represent the 
 standard deviation of three independent experiments . to investigate the ability 
 of this nuclear - uptake nanodrug delivery system to address the mdr 
 problem , k562/d , a drug - resistant cancer cell line with overexpression 
 of p - gp ( figure s12 ) ,
was used , while its 
 specific internalizing aptamer , kk1b10 , was used as the targeting ligand to functionalize the nanotruck . 

the specific binding of kk1b10 , nr - kk1b10 , and np / nr - kk1b10 with k562/d 
 cells was proven by flow cytometry ( figure s13 ) .
as shown in figure 5 and figure s14 , enhanced killing efficiency was achieved by incubating 
 k562/d cells with 8.5-np - dox / nr - kk1b10s with nir irradiation , while 
 a much lower therapeutic effect of free dox was detected for this 
 cancer cell line . to confirm that the enhanced therapeutic effect 
 originates from the intracellular accumulation of dox by this nir - responsive 
 nanodrug delivery system
, flow assay was performed to measure the 
 dox signal in k562/d cells under different treatments . upon nir irradiation , 
 the dox fluorescence from the sample incubated with 8.5-np - dox / nr - kk1b10s 
 was 2.5-fold higher than that from the sample incubated with 
 free dox ( figure s15 ) .
in contrast , without 
 laser treatment , the cells incubated with 8.5-np - dox / nr - kk1b10s produced 
 a modest dox signal .
these results demonstrate that this nuclear - uptake 
 nanodrug delivery system can recover the chemotherapeutic sensitivity 
 of k562/d to dox by bypassing cell membrane - expressed p - gp .
viability of k562/d cells treated with free dox or nir - activated 
 8.5-np - dox / nr - kk1b10s .
in summary , we have developed a dna - based 
 nanoassembly platform for cancer therapy . unlike traditional intranuclear 

transport strategies of nanoparticles , no nuclear 
 localization signal ( nls ) peptides are required in our design , and 
 the nuclear uptake of nanodrugs is mainly attributed to small particle 
 size , thus avoiding complicated nls modification processes and maintaining 
 the valid occupancy of drug - loading probes on the np surface . by using 
 this photocontrolled , size - transformable nanosystem , nanodrugs can 
 be efficiently transported across the cell membrane and enter the 
 nucleus in a coordinated and harmonious manner .
furthermore , this 
 dna - based nanoassembly platform can accumulate chemotherapeutic drugs 
 in the nuclei , thus greatly enhancing their therapeutic efficacy against 
 drug - resistant cancer cells by effectively bypassing p - gp .
this proof - of - concept 
 structure also opens a new door in the use of nanoassemblies for the 
 design of drug delivery systems for biological and clinical research . 
 to comprehensively evaluate the superiority of this nuclear - uptake 
 nanodrug delivery system
, further efforts are being made on the testing 
 in tumor - bearing mice models .
on the other hand , since the tissue 
 penetration of the nir laser is limited to around ten millimeters , 
 an alternative strategy for activatable dissociation of the nanoassemblies 
 are needed to apply this system to treatment of drug - resistant metastatic 
 tumors .
all dna strands were synthesized on an abi 3400 dna synthesizer ( applied 
 biosystems , foster city , ca , usa ) , and the specific sequences are 
 listed in table s1 . both the synthesis 
 and the deprotection processes were conducted as described by the 
 reagent manufacturers .
then , the dnas were precipitated by high - salt 
 ethanol in a freezer at 20 c for 30 min and collected 
 by centrifugation at 4000 rpm for 30 min .
subsequently , the dna precipitates 
 were dissolved with 400 l of 0.2 m triethylamine / acetate ( glen 
 research corp ) .
the purification step was performed by hplc ( prostar , 
 varian , walnut creek , ca , usa ) with a c18 column ( 5 m , 250 
 mm  4.6 mm , alltech ) using acetonitrile and 0.1 m triethylammonium 
 acetate ( teaa ) aqueous solution as the mobile phase . after being dried 
 by a rotary evaporator
, the purified dnas were detritylated with 80% 
 acetic acid , precipitated with cold salted ethanol , collected by centrifugation , 
 and dried by vacuum .
finally , the dna products were obtained , and 
 their concentrations were measured with a uv  vis spectrometer 
 ( cary bio-300 , varian ) .
nps of 5.5 and 8.5 nm were synthesized by a 
 seed - mediated growth method and then 
 washed by centrifugation to remove hexadecyltrimethylammonium bromide 
 ( ctab ) .
the modification of nps with the drug - loading / attaching dna 
 strand ( das ) was conducted following a reported protocol .
the thiolated das ( 0.2 mm , 20 l ) was 
 deprotected by 10 mm tris(2-carboxyethyl ) phosphine ( tcep , neutral ph , thermo scientific ) at room temperature 
 for 60 min and then mixed with 1 ml of nps ( 20 nm ) .
after incubation 
 for 16 h , the mixture was salt - aged by slowly adding 200 l 
 of nacl ( 1 m ) and allowed to incubate for 16 h. then , the excess dnas 
 were twice removed by centrifugation , and the precipitate was resuspended 
 in 250 l of 1  pbs .
silver nanorods 
 were synthesized and washed according to the procedure in our previous 
 report , and the concentration was determined 
 through the longitudinal absorption band of the uv  vis spectrum .
the selective modification of nr was performed 
 according to a reported protocol with some adjustment .
briefly , nrs were incubated with targeting aptamers 
 ( nr / aptamer ratio was 1:100 ) in 2 mm ctab solution for 12 h. then , 
 the capture strands ( cs ) were added ( nr / cs ratio was 1:500 ) and allowed 
 to incubate for another 12 h. the adsorbed ctab was further displaced 
 with thiolated peg .
after that , the modified nr was salt aged by slowly 
 adding 1 m nacl to give a final 0.3 m concentration of na , and the mixture was held at room temperature for at least 12 h. 
 after washing 5 times by centrifugation , the resultant nrs were resuspended 
 in 1 pbs for further use . to fabricate the self - assembly 
 nanocomplexes , the das - modified nps of 5.5 , 8.5 , and 15 nm were mixed 
 with the modified nrs at a np / nr ratio of 20:1 , 15:1 , and 10:1 , respectively , 
 and incubated at room temperature for at least 24 h. the mixture was 
 then gently centrifuged to remove unbound nps . for dox loading
, nps were modified with das whose 
 3-end portion can form a ( cgt)6/(acg)6 duplex with a corresponding cdna .
the dox loading was conducted 
 by mixing dox with np - dass , incubating at room temperature for 30 
 min , and then centrifuging to collect the np / dox complexes .
the number 
 of dox per np was determined by measuring the fluorescence intensity 
 of unbound dox in the supernatant and then interpolating from a standard 
 linear calibration curve .
for the dox leakage assay , 150 l 
 of np / dox complexes was added to a mini dialysis unit [ 3.5 molecular 
 weight cutoff ( mwko ) , thermo scientific ] , and the equivalent of free 
 dox was used as a control .
each unit was immersed in 3 ml of 1 
 pbs in a 5 ml beaker with gentle stirring at 450 rpm . at each given 
 time point , a 100-l aliquot of the dialysis solution was collected 
 for dox fluorescence measurement . after that , the collected solution 
 was returned to the corresponding beaker .
cem ( human acute lymphoblastic leukemia ) and ramos ( human burkitt s 
 lymphoma ) were purchased from american type culture collection .
k562/d 
 ( doxorubicin - resistant chronic myelogenous leukemia ) was generously 
 provided by dr .
troy a. a. harkness of the 
 department of anatomy and cell biology , college of medicine , university 
 of saskatchewan .
cem , ramos , and k562/d cells were cultured in rpmi 
 1640 , rpmi 1640 , and imdm medium , respectively , supplemented with 
 10% fbs ( heat - inactivated ; gibco ) and 100 iu / ml penicillin 
cells ( 5  10 in 100 l of medium ) were 
 incubated with free dox , fluorescent nanoparticles , or nanocomplexes 
 at 37 c with 5% co2 for different time lengths . after 
 several centrifugation / washing steps , the cells were suspended in 
 1 pbs . for photoactivation , cells were irradiated with nir laser 
 ( 600 mw / cm ) for 10 min .
bisbenzimide hoechst 33342 ( sigma - aldrich ) 
 was used for nuclear staining by incubating with cells at 37 c 
 for 20 min .
fluorescence imaging was performed on a leica tcs sp5 
 confocal microscope ( leica microsystems ) with a 63 oil - immersion 
 objective . in most cases
, the red color represents the fluorescence 
 of cy5 ( em = 670 nm ) , the pink color represents 
 the fluorescence of dox ( em = 600 nm ) , 
 and the green color represents the fluorescence of tamra ( em = 570 nm ) .
cem cells ( 1  10 ) were incubated with 8.5-np / nr - sgc8s 
 or 15-np / nr - sgc8s at 37 c with 5% co2 for 6 h. then 
 the cells were washed with pbs three times , irradiated with nir laser 
 for 0 or 10 min , and incubated at 37 c with 5% co2 for another 22 h. to extract nuclei , the cells were collected by 
 centrifugation , resuspended in 10 mm tris - hcl buffer ( ph 7.4 ) containing 
 100 mm nacl , 1 mm edta , and 1% triton x-100 at 4 c for 10 min 
 and finally centrifuged at 1000 g for 3 min .
after several centrifugation / washing 
 rounds to remove the adsorbed nanoparticles on the nuclear membrane , 
 the collected nuclei were lysed by a lysis solution containing 0.5% 
 triton x-100 and 1 m naoh with sonication .
subsequently , the nanoparticles 
 from the nuclei were digested by incubating with aqua regia at 65 c 
 overnight and diluted in 2% hno3 solution .
nps accumulated 
 in nuclei were measured by quantifying au element by inductively coupled 
 plasma atomic emission spectrometry ( icp - aes ) .
to evaluate the cell binding affinity of different nanocarriers , 
 the aptamer was labeled with a fluorescein ( fitc ) .
cells were incubated 
 with free aptamers , nr - aptamers , or 15-np / nr - aptamers at 4 c 
 for 30 min .
after removal of unbound materials by several centrifugation / washing 
 steps , the cells were analyzed on a facscan cytometer ( accuri c6 ) 
 by counting 20  000 events .
the cell viability under different treatments was determined by celltiter 
 96 cell proliferation assay ( promega ) .
cells were incubated with free 
 dox , np / nrs , or np - dox / nrs at 37 c with 5% co2 for 
 2 h and then centrifuged to precipitate .
the supernatant ( 80% ) was 
 removed , followed by adding equivalent fresh culture medium ( 10% fbs ) . 
 for nir - responsive regulation ,
cells were irradiated with a nir laser 
 ( 808 nm , 600 mw / cm ) for 10 min , followed by additional 
 incubation to allow further growth for 48 h. then , 80% of the medium 
 was removed and replaced with 20 l of mts reagent diluted in 
 100 l of rpmi 1640 .
the resulting cell samples were incubated 
 at 37 c for 12 h. finally , the absorbance at 490 nm 
 was collected using a tecan safire microplate reader , and cell viability 
 was calculated using the equation provided by the manufacturer .
 the development of multidrug resistance 
 ( mdr ) has become an increasingly serious problem in cancer therapy . 
 the cell - membrane overexpression of p - glycoprotein ( p - gp ) , which can 
 actively efflux various anticancer drugs from the cell , is a major 
 mechanism of mdr . 
 nuclear - uptake nanodrug delivery systems , which 
 enable intranuclear release of anticancer drugs , are expected to address 
 this challenge by bypassing p - gp . however , before entering the nucleus , 
 the nanocarrier must pass through the cell membrane , necessitating 
 coordination between intracellular and intranuclear delivery . 
 to accommodate 
 this requirement , we have used dna self - assembly to develop a nuclear - uptake 
 nanodrug system carried by a cell - targeted near - infrared ( nir)-responsive 
 nanotruck for drug - resistant cancer therapy . via dna hybridization , 
 small drug - loaded gold nanoparticles ( termed nanodrugs ) 
 can self - assemble 
 onto the side face of a silver  gold nanorod ( nr , termed nanotruck ) 
 whose end faces were modified with a cell type - specific internalizing 
 aptamer . by using this size - photocontrollable nanodrug delivery system , 
 
 anticancer drugs can be efficiently accumulated in the nuclei to effectively 
 kill the cancer cells . 