developing 
 novel biochemical approaches to characterize and control 
 intracellular pathways and modify the action of cells and organisms 
 forms a core goal of biology and medicine .
ultimately , the synthesis and intracellular delivery of molecules 
 with spatial and temporal control provides a powerful combination . 

light - activation offers a means to maintain external spatial and temporal 
 control , demonstrated originally in 1978 by kaplan and co - workers 
 with light - activated  caged  atp .
however , many of the limitations of the original approach 
 that require the specific chemical modification of each molecule to 
 be delivered and the need for high energy uv light to trigger activation 
 still remain .
plasmonic gold nanoparticles 
 have attractive applications in biomolecule 
 delivery owing to their ease of surface functionalization and strong 
 absorption of physiologically friendly , near - infrared light .
spatially and temporally controlled cargo release 
 from gold nanoparticles has been shown with oligonucleotides for gene 
 regulation and expression and small molecules for anticancer therapy , using illumination wavelengths corresponding to the plasmon resonance . 

proteins carry out and regulate the majority of the known cellular 
 functions , and changes in their primary sequence or expression levels 
 lead to diverse diseases .
although extracellular 
 protein therapeutics have been remarkably successful ( e.g. , insulin , 
 recombinant factor ix , erythropoietin , and interferon ) , replacing or modulating the function of intracellular proteins 
 remains an unmet challenge .
the next 
 frontier of protein therapeutics aims to modulate a wide range of 
 intracellular metabolic , signaling , and regulatory activities , as 
 well as guiding stem cell differentiation by transcriptional network 
 perturbation . for a therapeutic protein to reach the site of action 
 inside the cell it must cross the plasma membrane and retain its biological 
 function .
transmembrane and endocytic delivery are 
 each confounded by the large macromolecular size , variable net charge , 
 and complex structure of proteins .
the 
 tendency for proteins to become trapped and degraded inside endosomes 
 and lysosomes , the last mile problem , 
 has been approached by using protein transduction domains , endosomal disrupting agents , degradable 
 polymeric carriers , and membrane interacting supramolecular 
 particles . despite these , endosomal escape 
 remains inefficient , and furthermore , 
 many of these methods can not be confined to a specific cell type or 
 area , or the timing of release is uncontrolled . for example , electroporation requires high concentrations 
 of protein and is more commonly used to 
 deliver nucleic acids .
techniques for protein delivery to individual 
 cells in a two - dimensional culture format are limited to ( 1 ) microinjection , which has low throughput and is not compatible 
 with three - dimensional tissues , and ( 2 ) 
 light activated polymer degradable methods , which respond to uv rather 
 than nir irradiation . here
we report the design 
 of a modular plasmonic nanocarrier platform 
 that delivers his - tagged proteins into the cytosol of cells , based 
 on a novel photocleavable oligonucleotide linker system ( scheme 1 ) .
our molecular linker design uses nickel - chelating 
 groups to overcome limitations posed by covalent immobilization of 
 proteins , thus allowing on - demand temporal control over release of 
 functional proteins from hollow gold nanoshells ( hgn ) tuned to absorb 
 in the biological  optical  window at 800 nm . by incorporating 
 peptides to induce receptor - mediated endocytosis
, we observed efficient 
 and specific cellular targeting that is immediately applicable to 
 ongoing efforts in drug delivery and tissue engineering .
endosomal 
 escape and cargo release were activated through an integration of 
 targeted nanocarriers and two - photon ( pulsed laser ) patterning microscopy , 
 taking advantage of the exquisite spatial control offered by the laser 
 scanning system to achieve subcellular resolution and real - time monitoring 
 of the protein transduction process .
( a ) diagram of the modular 
 plasmonic nanocarrier : a cell - targeting peptide motif ( rparpar ) is 
 fused to his - tag modified green fluorescent protein ( r - gfp ) , which 
 is assembled on dna - coated hgn .
( b ) schematic representation of r - gfp - hgn 
 cellular uptake and protein release .
laser excitation matching the nanocarrier 
 plasmon resonance releases r - gfp into the cytosol .
( c ) nanocarrier 
 synthetic steps : hgn are coated with single stranded thiol - dna - amine 
 and reacted with nhs - peg4-maleimide .
hgn 
 were synthesized by galvanic replacement of silver seeds , as described 
 previously .
silver seed particles were 
 prepared by reducing a stirred solution of 500 ml of 0.2 mm agno3 ( sigma ) and 0.5 mm sodium citrate ( sigma ) in deionized water 
 with 0.5 ml of 1.0 m nabh4 ( emd ) at 60 c .
the solution 
 was stirred for 2 h and then cooled to room temperature before growing 
 the seed particles to a final target size for use as a sacrificial 
 template for the gold nanoshells growth with the addition of 0.75 
 ml of 2 m nh2ohhcl ( sigma ) and 1.75 ml of 0.1 m agno3 and stirred overnight at room temperature .
the galvanic replacement 
 of the silver template particles with gold was optimized to have an 
 absorbance peak at around 800 nm by rapid addition of 3.2 ml of 25 
 mm haucl4 ( sigma ) at 60 c .
single stranded 
 thiol modified 25mer dna of sequence 3-nh2-(ch2)7-gccaccacgtctacttgaagtccca - peg18-(ch2)6-ss-(ch2)6-oh-5 
 was purchased from biosearch technologies and adsorbed onto hgn by 
 the method described by zhang et al .
one 
 hundred micromolar dna was incubated with 10 mm tris(2-carboxyethyl)phosphine 
 hydrochloride ( tcep ) ( sigma ) for 30 min and then dialyzed against 
 te buffer ph 8.0 ( idt dna ) overnight at 4 c .
a final concentration 
 of 3 m tcep - treated dna was added to 32 pm hgn at which point 
 low ph adsorption was induced with the addition of 10 mm sodium citrate 
 to a final ph of 3.1 .
after 10 min the ph was neutralized to 7.4 with 
 the addition of 1 m hepes ( sigma ) , and the nacl concentration was 
 brought to 500 mm .
the hgn were then pelleted by centrifugation three 
 times at 9000  g for 10 min , decanted , and 
 the pellet resuspended in 500 mm nacl in 10 mm phosphate buffer at 
 ph 7.4 to remove excess dna .
n-[n,n-bis(carboxymethyl)-l - lysine]-12-mercaptododecanamide 
 ( nta ) ( sigma ) was then conjugated to the 3 amine of the dna 
 scaffold with the addition of 1 mg / ml nhs - peg4-maleimide 
 linker ( quanta biodesign ) and incubated for 20 min at room temperature . 

the maleimide terminated hgn were pelleted 3 times at 9000  g for 10 min at 4 c , and 120 m nta was added 
 and then incubated for 3 h at room temperature .
after pelleting 3 
 times , the solution was stored at 4 c until use .
oligonucleotides encoding the rparpar peptides were 
 synthesized and ligated downstream of gfp with a glycine - serine linker 
 placed in between in the backbone prset - emgfp ( invitrogen ) .
prset - sox2 
 was subcloned by replacing gfp with sox2 in the rparpar - modified gfp 
 construct ( r - gfp ) .
all recombinant proteins were expressed in escherichia coli bl21(de3 ) ( novagen ) and purified using 
 nickel - nitrilotriacetic acid affinity chromatography .
gfp protein 
 was purified under native conditions , whereas sox2 and p53 under denaturing 
 conditions and refolded .
the protein of interest ( poi ) was loaded 
 onto the hgn at a 100,000 to one poi / hgn molar ratio in the presence 
 of 400 m nicl2 and incubated for 30 min on ice . 

the hgn were pelleted by centrifugation at 9000  g for 10 min at 4 c for a minimum of 5 times to remove excess 
 protein and resuspended at a final concentration of 320 pm of poi - hgn 
 construct .
samples were irradiated with pulses 
 generated from a femtosecond 
 ti : sapphire regenerative amplifier ( spectraphysics spitfire ) running 
 at 1 khz repetition rate .
the laser beam was collimated by a galilean 
 telescope to achieve a gaussian diameter of 2.3 mm . in experiments 
 without collimation ,
pulse duration 
 was monitored by a home - built single - shot optical autocorrelator and 
 was kept at about 130 fs .
the spectral full width at half - maximum 
 of the laser radiation was 12 nm centered around 800 nm .
the laser 
 beam was directed onto the sample by a series of mirrors , and no focusing 
 optics were used .
the energy of the optical pulse was controlled by 
 schott neutral density glass filters .
ppc-1 was maintained in high glucose dulbecco s modified 
 eagle medium ( dmem ) with phenol red ( hyclone ) supplemented with 10% 
 fetal bovine serum ( hyclone ) at 37 c in 5% co2 .
for 
 spatial and temporal controlled release experiments , ppc-1 cells were 
 grown on an 8-well chambered glass slide ( thermo labtek ii ) at an 
 initial seeding density of 40,000 cells per well for 24 h 37 c 
 in 5% co2 in complete media .
one to 10 l of 
 gfp - hgn at 320 pm was added per 100 l of medium .
after 2 h 
 of incubation at 37 c in 5% co2 atmosphere , the cells 
 were rinsed with hank s balanced salt solution ( hbss ) ( thermo ) 
 prior to imaging .
fluorescence measurements of gfp were carried 
 out using a tecan infinite 
 200 pro microplate reader , exciting at 450 nm ( 9 nm bandwidth ) and 
 reading emission spectra at 490 to 600 nm ( 20 nm bandwidth ) or at 
 a single point at 510 nm .
the amount of gfp loaded onto nta - hgn was 
 determined by chemically competing for the nickel - nta sites of 3.2 
 pm nanoparticles with 100 mm imidazole in pbs and incubation for 30 
 min .
the particles were spun down at 12,000  g for 10 min , and the supernatant was loaded into a 96-well flat clear 
 bottom plastic microtiter plate for fluorescence readout .
efficiency 
 of gfp release by laser was examined by irradiating a number of samples 
 with particle concentration of 3.2 pm r - gfp - hgn with variable laser 
 powers and exposure times .
hgn were then centrifuged at 12,000  g for 10 min and the supernatants transferred to a 96-well 
 flat clear bottom plastic plate for fluorescence readout .
the pellets 
 were then treated with 100 mm imidazole in pbs and then spun down 
 to extract the supernatant containing the gfp retained on the hgn 
 after laser excitation .
the supernatant was loaded into a 96-well 
 flat clear bottom plastic microtiter plate for fluorescence readout .
cellular gfp concentration was determined by incubating 32 pm r - gfp - hgn 
 to a suspension of 500,000 cells for 2 h at 37 c and 5% co2 .
the cells were lysed with a solution of triton x-100 in 
 pbs and centrifuged to a pellet containing the hgn .
the pellet was 
 treated with 100 mm imidazole in pbs and centrifuged to transfer the 
 supernatant containing liberated gfp to a 96-well plate to measure 
 the fluorescence .
standard curves for gfp concentration quantification 
 in test tubes 
 and in cells were generated by measuring the fluorescence intensity 
 of known concentrations of gfp ( determined by absorption spectroscopy 
 with a nanodrop 1000 ( thermo ) using the extinction coefficient of 
 emerald gfp of 57,500 m cm at 487 nm ) serially diluted in the 
 proper experimental buffer .
cbqca protein staining of sox2 was 
 performed as directed in the 
 manufacturer s protocol ( invitrogen ) .
fluorescence analysis 
 was performed on a black 96-well plate with the tecan infinite 200 
 pro plate reader exciting at 465 nm ( 9 nm bandwidth ) and reading emission 
 at 550 nm ( 20 nm bandwidth ) .
following laser 
 excitation , hgn loaded with sox2 or p53 was pelleted by centrifugation 
 at 14,000  g for 10 min .
a 1 
 solution of laemmli sample buffer ( bio - rad ) was added to the samples 
 and then boiled for 10 min .
the samples were run on a 12% sds - page 
 gel and then transferred to a polyvinyl difluoride ( pvdf ) membrane 
 ( bio - rad ) .
the membrane was stained with a mouse anti - his tag monoclonal 
 antibody ( abgent ) at a 1:500 dilution at 4 c overnight .
after 
 stringent wash , the membrane was then stained with alexa fluor 488 
 goat antimouse igg secondary antibody ( invitrogen ) for 3 h at room 
 temperature .
five thousand 
 cells in 100 l of complete medium were seeded in a 96-well 
 plate and grown overnight at 37 c and 5% co2 .
the 
 cells were treated with r - gfp - hgn using the methods described above . 

the cells were irradiated using an 800 nm pulse - pumped femtosecond 
 ti : sapphire regenerative amplifier running at 1 khz repetition rate 
 using a 5 mm full beam diameter with variable exposure times and powers . 

after 72 h the cell viability was determined by prestoblue ( life technologies ) 
 according to the manufacturer s protocol .
the viability was 
 analyzed using a tecan infinite 200 pro microplate reader , exciting 
 at 560 nm ( 9 nm bandwidth ) and reading emission at 590 nm ( 20 nm bandwidth ) .
living cells were excited with a mode - locked ti : sapphire tunable ( 6901020 
 nm ) femtosecond pulsed laser ( 100 fs pulse duration , 80 mhz repetition 
 rate , mai tai hp , newport - spectra physics ) regulated with a modulator 
 linked to the fluoview software . a 25 water immersion objective 
 with a numerical aperture of 1.05
gfp fluorescence in cells 
 plated on an 8-well glass slide was imaged with a 15 mw blue laser 
 diode exciting at 473 nm raster scanning at a speed of 80,000 hz . 

nir excitation of r - gfp - hgn was performed using the femtosecond pulsed 
 mai - tai laser tuned to 800 nm at a raster scan speed of 125,000 hz 
 up to 35 full - frame cycles .
spatially controlled release experiments were performed 
 by selecting a region of interest using the fluoview software to scan 
 only the selected area with the femtosecond pulsed laser .
ratio quantification for figure 3 was performed using the ratio plus plugin in imagej 
 to calculate the pixel ratio between images corresponding to after 
 and before nir excitation .
corrected total cell 
 fluorescence ( figure 4 ) was calculated from 
 the integrated density within an roi enclosing a specific cell ( determined 
 by imagej ) according to the formula : ctcf = intden  ( area 
 of cell  mean fluorescence of background ) .
differences between the 
 viability of the two groups with different laser power intensity and 
 exposure time were analyzed by two - way anova with bonferroni s 
 post - tests using graphpad prism 5 software ( graphpad software , la 
 jolla , usa ) .
the means  standard deviations of triplicate samples 
 are reported , and a p  0.05 was considered 
 statistically significant .
differences between the mean fluorescence 
 for cells irradiated by the laser versus nonirradiated ( figure 4f ) were analyzed by 2-sample kolmogorov 
exposure of hgn to femtosecond pulsed near - infrared 
 ( nir ) laser 
 light generates hot electrons that can cleave a thiol - gold surface 
 bond , without damaging an attached nucleic acid cargo . also , the laser - supplied energy 
 induces plasmonic heating of a thin ( nanometer - scale ) shell of water 
 surrounding the metal surface , occurring on a time scale of picoseconds 
 that is too short for heat dissipation to the bulk fluid , thus inducing generation of nanobubbles . when hgn are localized 
 intracellularly in endosomes
this local heating effect has been harnessed to deliver nucleic 
 acids in previous reports , but protein delivery has not been described 
 using such plasmonic nanostructures . herein , we introduce a protein 
 delivery platform based on hgn coated by a densely packed dna layer 
 that serves both as anchor for proteins and as a release switch when 
 irradiated by pulsed nir light matching the hgn plasmon resonance 
 wavelength ( scheme 1 ) .
hgn of 40  15 nm diameter and 
 5  2 nm shell thickness were synthesized via the galvanic replacement 
 of gold from sacrificial silver templates as described previously 
 ( figure s1 , supporting information ) .
the hgn diameter and shell thickness were optimized to provide a 
 plasmon absorption peak at 800 nm since nir ( 7502500 
 nm ) light is physiologically friendly as it penetrates cells , blood , 
 and soft tissues at depths up to several centimeters without damage .
proteins were attached to the hgn using the histidine ( his ) tag , 
 which typically consists of five or six his residues added to the 
 n- or c - terminus of the protein by recombinant engineering .
protein tag sequences have been instrumental for the efficient purification , 
 detection , and surface immobilization of a variety of proteins , and 
 the his - tag is one of the most commonly used methods for immobilization 
 of functional proteins .
the imidazole 
 moieties of the his residues chelate the free coordination sites of 
 divalent metal ions ( e.g. , ni and co ) , which 
 in turn can be immobilized as chelate complexes of nitrilotriacetic 
 acid ( nta ) bound to a solid support via various linkers . nta chelates 
 ni strongly , while additional sites on the ni metal center 
 remain open for chelating a his - tag . to secure the nta - ni handles on hgn , we first assembled 
 a densely packed monolayer of single stranded thiol - dna - amine onto 
 the hgn surface by low - ph adsorption .
the number of linker dna - amines attached to the hgn was 3000 , 
 determined by fluorescence labeling of the amine with nhs - fluorescein 
 following a previously described method .
the dna layer stabilized the hgn against aggregation , while providing 
 a high density of terminal amine groups used to attach the ni and protein chelator , the nta - containing 
 moiety n-[n,n-bis(carboxymethyl)-l - lysine]-12-mercaptododecanamide , 
 via a hydrophilic and flexible nhs - peg - maleimide linker ( scheme 1 ) .
these steps resulted in a novel , generalized 
 carriage system for his - tagged proteins based on a photoreleasing 
 hgn core ( nta - hgn ) .
the affinity of individual nta - ni to his - tags is relatively 
 low , with kd of 10 m at neutral ph .
however , when a support 
 surface has a high density of immobilized nta - ni , multiple 
 linkages and  rebinding  effects by a his tagged protein 
 provide a greater affinity and long - lasting association .
it is with this valency effect 
 in mind that we designed our photoactivated hgn platform .
a densely 
 packed nta - ni linker layer strongly binds the cargo protein to the 
 hgn . upon laser treatment
the linkers release from the hgn surface 
 and each one , acting on its own , reverts to the much reduced affinity 
 ( kd ) of the individual nta - ni - his bond .
hence , the linker moieties 
 and his - tag are expected to dissociate rapidly and diffuse away , leaving 
 the protein free and in its active state .
as proof of concept 
 for protein delivery to cells , we designed 
 a model protein with an n - terminal 6-his - tag and a c - terminal cell - targeting 
 peptide motif ( rparpar ) , on a green fluorescent protein ( gfp ) backbone , 
 designated r - gfp .
this cell - penetrating gfp variant was generated 
 using recombinant protein engineering , as is possible for a wide variety 
 of potential his - tagged protein cargo .
the peptide motif rparpar at 
 the c - terminus , which follows the rxxr sequence found in the c - end 
 rule ( cendr ) , facilitates specific endosome internalization into neuropilin-1 
 ( nrp-1 ) expressing cancer cells .
nrp-1 is often 
 overexpressed in tumors and is associated with poor prognosis , generating significant interest 
 as a receptor for targeted drug delivery .
importantly , r - gfp serves 
 as both fluorescent tracer and as an indicator of the mildness of 
 the photorelease event since it fluoresces only if its tertiary structure 
 is not denatured . to assemble the protein onto the nanoparticles , 
 r - gfp was incubated with the hgn - nta at 100,000:1 r - gfp / hgn molar 
 ratio in the presence of nicl2 .
the large excess of protein 
 was chosen to ensure maximal loading onto the particles and resulted 
 in colloidally stable complexes ( r - gfp - hgn ) .
after washing , the amount 
 of r - gfp successfully loaded onto the hgn was determined spectroscopically 
 by his - tag elution with imidazole ( figure 1a ) .
imidazole is known to compete with the nta : ni for 
 the his - tag , and we found that this treatment completely released 
 r - gfp from hgn ( figure 1a ) .
the loading of 
 r - gfp on hgn caused a reversible 80% quenching of the protein 
 fluorescence , relative to free r - gfp ( figure 1a ) , attributed to the well - known fluorescence quenching properties 
 of gold .
the dequenching of r - gfp upon release from hgn was used to 
 characterize the treatment efficiency under various laser conditions 
 ( figure 1b ) .
( a ) r - gfp fluorescence 
 was partially quenched when bound to hgn ; however , fluorescence increased 
 upon exposure to laser excitation power of 1.5 w / cm for 
 30 s. imidazole competes with histidine for the linker chelator groups 
 on the hgn , chemically releasing the protein from the nanocarrier , 
 resulting in maximum dequenching .
r - gfp - hgn uv  vis absorbance 
 decreased after laser exposure , indicative of changes to the core 
 structure .
( b ) quantification of r - gfp release from hgn after laser 
 treatment at various laser powers and exposure times .
r - gfp remaining 
 on the hgn after laser exposure is released by imidazole competition 
 and presented as the  retained  value , which is greater 
 for lower laser powers and exposure times .
error bars represent the 
 standard deviation . the expected sum of laser followed by imidazole 
 is 100% .
reabsorption of r - gfp onto freshly exposed gold surfaces 
 and partial protein denaturation may explain the incomplete release 
 under certain conditions , as nonspecifically bound proteins would 
 not be susceptible to imidazole competition .
( c ) r - gfp - hgn was internalized 
 into ppc-1 cells , which were then treated with various combinations 
 of laser power and exposure time .
decreased viability was observed 
 in cells exposed to 2.3 w / cm fluence for 60 s ( p < 0.05 , * ) , well above the optimal release conditions . 
 no loss in cell viability was observed with laser in the absence of 
 hgn .
our optimized nanoplatform carried 
 800 r - gfp per hgn ( figure 
 s2 , supporting information ) , which , if 
 we consider binding to occur only to the exterior of the hgn , indicates 
 a footprint of 7 nm per protein .
this is a reasonable 
 estimate since gfp is a barrel - shaped protein 4.2 nm long and 2.4 
 nm in diameter and has a cross - sectional area of 4.5 nm . assuming that each amine ( 3000 per hgn ) was converted 
 to an nta - ni during synthesis
we note that this value is above the 
 expected minimum of two nta - ni groups per his tag required for stable , 
 avidity - enhanced association . furthermore , 
 we observed minimal nonspecific binding in the absence of ni ions ( figure s3 , supporting information ) , substantiating the specificity of the nta - ni linkage . to characterize release of the protein , 

a wide - beam pulsed laser 
 ( diameter 5 mm ) was directed at r - gfp - hgn in solution .
we 
 found that the r - gfp fluorescence increased in a laser power and exposure 
 time - dependent manner , indicating a controlled detachment of the r - gfp 
 from the hgn .
thiol bonds holding the dna and , by extension , cause the 
 nta - ni handles to release from the hgn .
the linker length 
 was chosen to position the protein several nanometers from the surface 
 ( scheme 1 ) so that protein temperature remains 
 near ambient during laser release , and 
 thermal denaturation should be minimal .
a release of 55% r - gfp , as compared to imidazole release , 
 was possible for the optimal conditions ( figure 1b ) : irradiation at 800 nm at 0.91.5 w / cm ( corresponding 
 to a pulse fluence of 0.91.5 mj / cm , using 130 
 fs pulses at 1 khz pulse repetition rate ) , for 3060 s. for 
 3 s exposure at 1.5 w / cm a moderate fraction of 35% 
 r - gfp was released . since gfp fluorescence is known to directly correlate 
 with having a natively folded structure , the fluorescence we observe after laser release supports a conclusion 
 that the protein was released intact .
we found that the remaining 
 bound fraction of r - gfp was releasable by subsequent imidazole treatment 
 of the particles ( figure 1b ) , and the sum of 
 laser and imidazole released r - gfp approximately matched that from 
 using imidazole alone .
we conclude that at least 50% of the r - gfp cargo could be 
 released from hgn , with optical control , and without significant protein 
 denaturation .
we demonstrate that our nanoparticle assembly 
 can also support 
 the loading and release of transcription factors sox2 and p53 ( figure 2 ) , establishing the versatility of the nanocarrier 
 platform . sox2-carrying hgn ( sox2-hgn )
was subjected to laser irradiation 
 and release assessed to be up to 60% of total protein , using the protein - reactive 
 fluorescent dye 3-(4-carboxybenzoyl ) quinoline-2-carboxaldehyde ( cbqca ) , 
 as shown in figure 2a .
following laser treatment 
 and hgn centrifugation of sox2-hgn and p53-hgn , the pellet was etched 
 using kcn , to quantify the amount of protein retained on the nanocarrier 
 by western blot and densitometry analysis ( figure 2b , c ) .
a sharp extra sox2 band is observed indicating site 
 specific degradation of the protein as a result of the extensive processing 
 during the sample analysis and lack of protease inhibitors .
similar 
 degradation was observed in previous reports and suggests that degradation 
 is not dependent on the laser .
sox2 shows 
 a laser power - dependent release with approximately 40% of the protein 
 retained on the hgn after irradiation with 2.3 w / cm for 
 30 s ( figure 2a ) , confirming the quantification 
 done by cbqca . finally , we demonstrate that p53 shows a similar release 
 pattern upon laser irradiation ( figure 2c ) .
( a ) bar graph 
 showing the fraction of sox2 that could be released from sox2-hgn 
 by each of the consecutive steps : laser , imidazole , and kcn 
 etching of the hgn . for laser
the fraction of sox2 released 
 by kcn was similar to the + laser step .
sox2 release from sox2-hgn 
 was determined by labeling with an amine reactive dye cbqca after 
 laser exposure at 2.3 w / cm for 30 s. the amount of sox2 
 that remained on the hgn was evaluated by pelleting and treating with 
 imidazole and kcn , respectively .
( b ) anti - his western blot of sox2 
 retained on hgn after laser irradiation . upon laser irradiation
, samples 
 were pelleted and washed , then hgn was etched with kcn , and solutions 
 were run on a 12% sds page gel . etching the hgn with kcn after laser 
 excitation showed decreasing band intensity with increasing laser 
 power density revealing release of sox2 from the nanoparticle surface . 

an additional sox2 band is observed from nonlaser - dependent degradation 
 due to sample processing .
laser power densities were varied at a constant 
 exposure time of 30 s. ( c ) p53 release from p53-hgn was evaluated 
 by western blot staining as in panel b after laser exposure at 2.3 
 w / cm for 30 s. nrp-1 expressing ppc-1 
 cells were incubated with r - gfp - hgn optimized to allow maximal internalization 
 while minimizing the incubation time to 2 h ( yielding an estimated 
 count of 120 r - gfp - hgn per cell , see supporting 
 information for details ) .
the cells were exposed to the same 
 range of nir laser conditions used above for characterizing r - gfp , 
 then cell viability was assessed 72 h post - treatment ( figure 1c ) .
there was no significant reduction of cell viability 
 by gfp - hgn for exposure time of 60 s and 1.5 w / cm , a condition 
 giving maximum release in figure 1b .
r - gfp - hgn 
 in combination with laser treatment showed significant viability loss 
 ( 30% or more ) only for the highest laser power ( 2.3 w / cm ) and longest exposure times
the 
 nir treatment of cells without hgn showed negligible toxicity even 
 at the highest light intensity .
the nrp-1 deficient 
 melanoma cell line m21 served as a negative control for uptake ( figure 
 s4 , supporting information ) .
we quantified the gfp - hgn associated with ppc-1 
 by cell lysate fluorescence to be 10 gfp molecules 
 per cell or 100 nm gfp assuming 2 pl cell volume ( figure s6 , supporting information ) .
this is competitive to typical intracellular concentrations of transcription 
 factors such as p53 , which has copy numbers ranging from 10 to 10 depending on tissue type and within the active 
 concentration range of 10 nm to 10 m of transcription factors 
 transduced by peptide transduction domains .
next , we tested the ability of r - gfp - hgn 
 to release protein cargo from endosomes into the cytosol in response 
 to nir laser pulses . in figure 3 , we explored protein release in ppc-1 cells using 
 a laser scanning confocal two - photon microscope ( olympus fluoview 
 1000 mpe ) equipped with a tunable ( 6901020 nm ) fs pulsed laser , 
 as well as a 473 nm continuous wave laser .
the instrument allows switching 
 between these two modes of excitation , with fine control over imaging 
 and submicron resolution in laser patterning .
ppc-1 cells were cultured 
 on chambered glass slides and incubated with r - gfp - hgn for 2 h to 
 allow internalization to occur .
the field of view was irradiated with 800 nm 
 fs pulsed laser light ( 80 mhz ) , then the imaging mode was switched 
 back to single photon to compare with the initial image . by increasing 
 the power of the pulsed - nir laser we defined a working range for r - gfp 
 release from the hgn .
a laser power of 8  10 w / cm was chosen ( 0.1 mj / cm pulse fluence , using 100 
 fs pulses at 80 mhz pulse repetition rate , with beam diameter of 6 
 m ) , whereby an average exposure time of 280 s caused 
 a significant increase of r - gfp fluorescence intensity in cells , indicating 
 dequenching and release from hgn ( figure 3 ) . 
 furthermore , an additional irradiation cycle released more r - gfp from 
 the hgn but with diminishing returns ( figure 3 , second pass ) .
autofluorescence intensity from ppc-1 without r - gfp - hgn 
 did not change upon laser exposure ( figure s7 , supporting information ) .
quantification of r - gfp release in ppc-1 
 cells after exposure to 
 a focused nir pulsed laser .
( a ) single photon confocal microscopy 
 fluorescence image of ppc-1 cells incubated with r - gfp - hgn for 2 h , 
 prior to nir excitation ( initial ) , and after two rounds of 800 nm 
 fs pulsed laser exposure ( first pass and second pass ) , across the 
 full field of view with a total exposure of 280 s per irradiated 
 area for each pass .
r - gfp release and dequenching is indicated by 
 the overall increase in pixel brightness in almost all cells in the 
 first and second passes . scale bar is 50 m .
( b ) the increase 
 of pixel intensities before and after each pass of the laser was quantified 
 by the ratio of after / before and is represented by white pixels ( first 
 and second pass ratios ) .
most of the cells show r - gfp release , but 
 the ratio of increase was higher for the first pass than second pass , 
 indicating first pass rapid release under these laser conditions . 

( c ) close up of the blue box region in panel a for three frames .
histogram 
 of ratios in the white box region , plotted as percent of pixels from 
 panel b. this plot shows the fluorescence intensity increases most 
 during the first pass of the laser . to understand the spatial release capability of the r - gfp in greater 
 detail , we next defined regions of interest ( rois ) for the focused 
 scanning laser to specifically illuminate ( figure 4 ) .
the laser was rastered 
 within the user - defined shapes , modulated by a beam deflector that 
 enables precise control over the exposure time applied at the pixel 
 ( sample volume ) level .
we incubated r - gfp - hgn with ppc-1 cells as 
 in figure 3 , and for the laser operation , we 
 defined rois that contained single cells , or parts of cells , that 
 were interspersed between negative control cells not to be exposed . 
 in the first example ( figure 4a ,
b ) individual 
 cells were targeted by roi to test the cell level accuracy of the 
 release process , whereas in figure 4c  f , 
 we targeted cells and parts of cells within a high - density cell cluster . 

for each experiment the pulsed laser was rastered exclusively across 
 the areas within each red roi ( figure 4a , c ) . 

single - photon confocal imaging ( 473 nm laser ) of r - gfp in the full 
 field of view , before and 1 min after pulsed laser , revealed 
 up to a 5-fold increase in intensity for the cells outlined by the 
 rois ( figure 4b , d ) .
figure 4b shows that for cells external to the laser - exposed rois , 
 one example being the cell within the blue oval ( not targeted by laser ) , 
 r - gfp was not released .
the separation between it and the closest 
 targeted roi was 5 m , indicating the laser patterning 
 technique indeed has high fidelity .
the cell in roi 1 was 
 separated from the control cell roi 2 by 5 m .
( b ) line profiles taken along the cell in roi 
 1 shows r - gfp fluorescence intensity increased up to 8-fold after 
 laser , whereas the neighboring control cell in roi 2 was unchanged . 

( c ) single photon confocal microscopy of r - gfp - hgn fluorescence before 
 and after nir laser exposure in the region denoted by the red square . 

( d ) intensity profile along lines 3 and 
 4 in panel c showing the gfp fluorescence increase after laser .
subcellular 
 release of r - gfp was elicited mostly (  ) , while the cellular 
 area (  ) , not exposed , showed only a slight increase .
laser 
 power density was 8  10 w / cm ( 0.1 mj / cm ) , and 300 s exposure time 
 in the region is denoted by the red square .
( e ) quantification of 
 corrected total cell fluorescence percent increase in selected cells 
 ( outlines shown in panel f ) above the initial intensity .
n = 6 for cells completely outside the roi , n = 5 
 for all other cases .
mean intensity for cells completely inside the 
 roi was significantly higher than cells completely outside ( kolmogorov  smirnov 
 test ; p < 0.005 ; * * * ) .
subcellular resolution over release was possible by defining 
 a 
 roi smaller than the cells . in figure 4c , d , 

the portion of the cell ( line scan 3 ) within the irradiated roi areas 
 showed increased intensity while the portion outside the roi was not 
 much increased .
in quantification for several cells , figure 4e , those cells completely within the red square 
 gave a total integrated cell fluorescence increase of 300% 
 greater than the initial intensity , in good agreement with decreased 
 r - gfp quenching in figure 4a , c .
two photon 
 real - time imaging during nir laser stimulation further confirmed an 
 inverse relationship between cytosolic ( diffuse ) and endosomal ( punctate ) 
 fluorescence intensity ( figure s8 , supporting 
 information ) .
cells that were completely outside the area ( figure 4e , f ) showed negligible fluorescence increase ( 8% ) 
 likely due to changes in the focal plane when switching from single 
 to multiphoton modality . for cells lying across the red lines ,
those 
 cellular areas within the roi showed increases of 300% , whereas 
 regions of the same cells that were outside the roi showed a moderate 
 fluorescence increase , 60% , attributable to diffusion of intracellularly 
 released r - gfp , occurring on a time scale of 1 min between 
 stimulation and imaging .
the reported intracellular diffusion coefficient 
 of gfp in mammalian cells is 25 m / s , although r - gfp may diffuse at a different rate 
 if the rparpar peptide motif interacts with cellular organelles , as 
 gfp fused with arginine rich hiv - derived tat peptide ( ygrkkrrqrrr ) 
 was reported to have an impaired diffusion coefficient of 5.9 m / s .
interestingly , the rparpar 
 peptide on the gfp seems to impart nuclear targeting properties since 
 we observed that when the protein was delivered into the cytosol the 
 nucleoli showed increased brightness relative to the nucleus as a 
 whole ( figure 4c ) .
this effect is not surprising 
 since rparpar resembles known nuclear localization sequences having 
 cationic residues , as well as the putative 
 histone binding peptide cqrppr .
further 
 studies may examine intracellular targeting of laser - released cargo 
 in more detail .
in summary , 
 we utilized a commercial two - photon microscope to provide 
 unprecedented control and real - time monitoring of payload release 
 by  painting  individual cells with nir light , providing 
 protein delivery with cell - level resolution with no additional equipment 
 or effort .
we demonstrate cellular delivery of a his - tagged protein 
 by a nanocarrier in response to a focused fs pulsed laser and show 
 that the protein cargo retains a functional conformation .
spatial 
 ( of the order of 1 m ) and temporal ( within seconds ) control 
 over delivery resulted from light absorption at the plasmon resonance 
 of an optimized hollow gold nanoshell .
we used a fusion protein containing 
 a cell targeting peptide to drive uptake into endosomes , here demonstrated 
 for the ppc-1 prostate cancer cells that express the nrp-1 receptor . 

the thiol - dna - nta anchoring system confers control over laser release ; 
 the gold 
thiol bonds are thermalized and released by the nir 
 laser pulses , presumably reducing the valency of the nta toward the 
 protein and ultimately separating the protein from the hgn carrier . 

the nir laser pulses also induce vapor bubble formation that leads 
 to rupture of the endosomes and release of the gfp throughout the 
 cell .
we observed nuclear accumulation of released r - gfp , as well 
 as increased fluorescence over distant cytosolic regions of the cell , 
 which together indicate successful r - gfp delivery throughout the cell . 

there were minimal adverse effects on cell viability due to either 
 the nanoparticles or irradiation with nir light .
this technique opens 
 the possibility of delivering various his - tagged proteins to the cytosol 
 of cells , individually or in concert , and could enable optical control 
 for stem cell differentiation , or for selective apoptosis .
although 
 different cells may be affected differently by the treatment , we anticipate 
 that the platform can be tuned to best suit each cell line s 
 specific requirements .
 while 
 a host of methods exist to deliver genetic materials or small 
 molecules to cells , very few are available for protein delivery to 
 the cytosol . we describe a modular , light - activated nanocarrier that 
 transports proteins into cells by receptor - mediated endocytosis and 
 delivers the cargo to the cytosol by light triggered endosomal escape . 
 
 the platform is based on hollow gold nanoshells ( hgn ) with polyhistidine 
 tagged proteins attached through an avidity - enhanced , nickel chelation 
 linking layer ; here , we used green fluorescent protein ( gfp ) as a 
 model deliverable cargo . 
 endosomal uptake of the gfp loaded nanocarrier 
 was mediated by a c - end rule ( cendr ) internalizing peptide fused to 
 the gfp . 
 focused femtosecond pulsed - laser excitation triggered protein 
 release from the nanocarrier and endosome disruption , and the released 
 protein was capable of targeting the nucleoli , a model intracellular 
 organelle . 
 we further demonstrate the generality of the approach by 
 loading and releasing sox2 and p53 . 
 this method for targeting of individual 
 cells , with resolution similar to microinjection , provides spatial 
 and temporal control over protein delivery . 
Introduction
Materials and Methods
Results
and Discussion
Conclusions