the use of visible 
 and near - infrared ( nir ) light , penetrable to 
 deep tissue , is an attractive method of spatiotemporally controlling 
 drug release from various drug delivery forms , such as prodrugs , liposomes , 
 polymers , and other nano- and macrodelivery systems .
however , because of its lower energy , it is difficult to 
 directly cleave a chemical bond using such light .
thus , novel mechanisms 
 using lower energy light to trigger the release of biologically active 
 compounds have been a major topic of interest . the photodynamic process and the unique chemistry of singlet oxygen 
 ( so ) were adopted to mediate lower energy light release of drugs .
so is formed during the photodynamic process and reacts with electron - rich 
 olefins to form unstable dioxetanes .
we named the cleavage of aminoacrylate by so 
  photo - unclick 
 chemistry  , and demonstrated the release of the anticancer 
 drug combretastatin a-4 ( ca4 ) from its prodrug using this method . 

we prepared cmp - l - ca4 , a ca4 prodrug that can be activated by far - red 
 light ( 690 nm ) , by combining a so - labile aminoacrylate linker , a core - modified 
 porphyrin ( cmp ) photosensitizer , and ca4 .
the prodrug released ca4 and enhanced cytotoxicity upon illumination . 
 the released ca4 damaged cancer cells , demonstrating our system s 
 ability to generate bystander effects in vitro .
we 
 found that the antitumor effects of cmp - l - ca4 were superior to the 
 effects of its pseudo - prodrug cmp - ncl - ca4 ( ncl = noncleavable linker ) . 

the ability to optically image the light - activatable 
 prodrug is useful for determining an illumination time when the prodrug 
 is at its maximum concentration at the target site .
if the prodrug accumulates in the target 
 sites , we can use optical imaging to detect the target areas , as well 
 as to treat the disease .
we expected that we could optically image 
 our photounclickable prodrug in vivo using a fluorescent 
 photosensitizer .
thus , we could track the prodrugs and then treat 
 the tumor with the combined effects of photodynamic therapy ( pdt ) 
 and local chemotherapy ( figure 1a ) .
the released 
 drug could damage the cancer cells that survived the initial pdt damage 
 through bystander effects ( figure 1b ) .
( a ) multifunctional 
 prodrug for optical imaging and combined treatment 
 with pdt and local chemotherapy .
( b ) bystander effects from the released 
 drugs can kill cancer cells that survive pdt damage . [ the lifetime 
 of so is short ( submicrosecond scale ) .
thus , direct cell damage by 
 so occurs only during illumination . the light dose used for
] we generated pc-(l - ca4)2 , an advanced multifunctioning 
 ca4 prodrug , for both fluorescence optical imaging and combination 
 therapy with pdt and released ca4 .
we chose phthalocyanine ( pc ) because 
 pc is a fluorescent photosensitizer that can generate both fluorescence 
 and singlet oxygen .
although fluorescence emission and so generation 
 are competing processes , pc has uniquely balanced yields of both functionality 
 ( i.e. , si - pc : o2 = 0.22 and f = 0.4 ) with a high molar extinction coefficient (  ) .
its brightness ( bt ) is greater than that of cmp ( e.g. ,  = 
 150,000 m cm at 675 nm , bt 
 = 6000 m cm for pc vs  
 = 5000 at 690 nm , bt = 50 m cm for cmp ) .
this pseudo - prodrug is similar to 
 pc-(l - ca4)2 in structure , but can not release ca4 upon illumination . 

it will mimic the pdt effects of pc-(l - ca4)2 , but can not 
 induce damage from released ca4 .
we evaluated the cytotoxic effects 
 of these two prodrugs with and without illumination , the inhibition 
 of tubulin polymerization , the in vitro bystander 
 effects , tumor localization using optical imaging , and the antitumor 
 effects .
we developed a synthetic 
 scheme using high - yield 
 reactions , such as esterification , nucleophilic substitution , and 
 the yne - amine reaction , to make the process easily adaptable to other 
 alcohol - containing drugs ( scheme 1 ) .
a nucleophilic substitution reaction 
 of silicon phthalocyanine dichloride ( pc - cl2 ) yielded compound 3 .
pc-(l - ca4)2 was synthesized through a click 
 ( yne - amine ) reaction of compounds 1 and 3 . under the basic condition , pc-(ncl - ca4)2
overall , 
 the synthesis was straightforward and all steps gave high yields ( > 73% 
 each step ) .
reagents and conditions : ( i ) 
 propynoic acid , dcc , dmap , ch2cl2 , room temp , 
 24 h ; ( ii ) 1,3-dibromopropane , anhydrous k2co3 , acetone , reflux , 12 h ; ( iii ) 2-(piperazin-1-yl)ethanol , pyridine , 
 toluene , reflux , 4 h ; ( iv ) 1 , anhydrous thf , 30 min ; 
 ( v ) 2 , anhydrous k2co3 , acetone , 
 reflux , 12 h. we 
 formulated the prodrugs using peg 
pla [ poly(ethylene glycol)-poly(d , l - lactide ) ] copolymer micelles to take advantage 
 of the enhanced permeability and retention ( epr ) effect to enhance 
 the delivery to tumor .
the nanosized 
 peg  pla polymer micelle was expected to provide three advantages : 
 ( 1 ) passive targeting to tumors via the epr effect , ( 2 ) prolonged circulation in the plasma , and ( 3 ) solubilization 
 of the nonpolar prodrug .
the zeta potentials and mean diameters 
 of the micelles of pc-(l - ca4)2 and pc-(ncl - ca4)2 were determined by dynamic light scattering ( dls ) ( zeta potential 
 = 11.64  1.38 mv , 16.81  1.67 mv and mean diameter = 71.96 
  1.34 nm , 75.07  1.45 nm , respectively ) . to visualize 
 the formation of the polymeric micelles ,
we used transmission electron 
 micrographs ( tem ) . tem images of the micelles showed consistent particle 
 sizes ( 6178 nm for pc-(l - ca4)2 and 6580 
 nm for pc-(ncl - ca4)2 micelles ) .
the stability 
 of the micelles was monitored by their particle sizes and zeta potentials 
 at 4 c under dark conditions .
these values remained within 95% 
 of the initial values for up to 21 days .
( a ) particle size distribution 
 and tem images ( inset ) of micelles 
 of ( a ) pc-(l - ca4)2 and ( b ) pc-(nlc - ca4)2 .
ca4 is known to inhibit tubulin 
 polymerization by binding to the colchicine binding pocket of tubulin . because the bulky groups ( pc - l and pc - ncl ) are attached to ca4 , 
 we expected lower inhibitory activity of tubulin polymerization .
we 
 determined the effects of these prodrugs using the tubulin polymerization 
 assay , in which fluorescence emission increases as tubulins polymerize 
 ( figure 3a ) .
consistent 
 with our data on the previous ca4 prodrug cmp - l - ca4 , both pc-(l - ca4)2 and pc-(ncl - ca4)2 had significantly ( p < 0.02 ) lower inhibitory 
 activity ( 23% and 17% , respectively ) than the parent drug ca4 ( 100% , 
 figure 3b ) .
effects of 3 m each of pcx , ca4 , 
 pc-(l - ca4)2 , 
 and pc-(ncl - ca4)2 on tubulin polymerization : ( a ) one data 
 set of representative kinetic traces ( data from two more experiments 
 are found in figure s1 , supporting information ) and ( b ) inhibition of tubulin polymerization by ca4 , pc-(l - ca4)2 , and pc-(ncl - ca4)2 after 1 h incubation ( sd 
 of three experiments ) . due to the dramatic reduction 
 of the inhibition of tubulin polymerization , it was expected that 
 pc-(l - ca4)2 and pc-(ncl - ca4)2 would have lower 
 dark toxicity ( cytotoxicity without illumination ) than ca4 . using 
 mcf-7 cells
, we found that the dark toxicity of the prodrugs decreased 
 by 19- and 101-fold [ ic50d values = 9 , 173 , and 916 nm 
 for ca4 , pc-(l - ca4)2 and pc-(ncl - ca4)2 , respectively ] . 

pc - l and pc - ncl reduced the cytotoxicity , presumably by interfering 
 with its binding to tubulin ( figure 3 ) .
however , 
 illumination enhanced the toxicity of both the prodrug and the pseudo - prodrug 
 [ ic50p = 6 nm and 34 nm for pc-(l - ca4)2 and 
 pc-(ncl - ca4)2 , respectively ] .
the difference 
 in the magnitudes of phototoxicity and dark toxicity effects may be 
 the result of each prodrug using a different mechanism . while pc-(ncl - ca4)2 can only kill cells via the pdt effects of so , pc-(l - ca4)2 can potentially kill cells by both pdt effects and released 
 ca4 . to prove this mechanistic difference , we tested the bystander 
 effects after the illumination .
after treating the cells in wells 
 of 24-well plates with the prodrug or pseudo - prodrug , one - half of 
 each well was exposed to light . at 48 h post - illumination ,
live cells 
 were stained with calcein am , and the center of each well was imaged 
 using a fluorescence microscope . because the lifetime of so 
 in aqueous medium / biological systems is very short ( 40 ns ) , so generated in the illuminated half of the 

so should decay 
 before reaching the other half of the well ( diffusion distance of 
 so = 20200 nm ) .
however , the released 
 ca4 can induce bystander effects because it can diffuse to the unilluminated 
 half of the well . as we expected , bystander effects were found in 
 the pc-(l - ca4)2-treated wells : cells in the nonilluminated 
 side were damaged as much as cells in the illuminated side ( figure 5b ) .
however , wells treated with pc-(ncl - ca4)2 had cell damage only in the illuminated halves of the wells 
 ( figure 5c ) .
this clearly supported our hypothesis 
 that illuminated pc-(l - ca4)2 releases ca4 that can induce 
 bystander effects , which is consistent with data from our previous 
 study with cmp - l - ca4 .
fluorescence live cell 
 images of the center of each well treated 
 with ( a ) vehicle ( diluting solution without a prodrug ) , ( b ) 25 nm 
 pc-(l - ca4)2 , and ( c ) 25 nm pc-(ncl - ca4)2 .
the 
 left half of each well was illuminated with a 690 nm diode laser ( 11 
 mw / cm for 15 min ) . at these concentrations
, pc-(l - ca4)2 and pc-(ncl - ca4)2 did not produce any significant 
 dark toxicity .
one of our 
 major goals was to make pc-(l - ca4)2 detectable in tissues 
 using fluorescence optical imaging .
we expected that the fluorescent 
 photosensitizer pc would provide sufficient fluorescence emission 
 for this goal .
pla polymer micelles of pc-(l - ca4)2 or pc-(ncl - ca4)2 . as a control formulation
, we also made 
 solutions of these prodrugs in 5% cremophor el , which does not produce 
 epr effects .
balb / c mice with sc tumors ( colon-26 cells , 46 
 mm in length ) were injected retro - orbitally with 2 mol / kg of 
 the prodrug .
then , the mice were imaged at various postinjection time 
 points ( figure 6 and figure 
 s4 ) .
as anticipated , we could clearly see the fluorescence 
 emissions from the two prodrugs in live mice .
first , the images from the mice that 
 received the polymer micelles of both prodrugs showed 
hot 
 spots  in tumors , with a peak at around 24 h postinjection , 
 presumably due to the epr effects of the nanosized polymer micelles . 

these hot spots were more evident in the mice who received injections 
 of pc-(l - ca4)2 .
resulting 
 from formulations with the polymer micelles persisted longer than 
 those  hot spots  resulting from formulations with cremophor 
 solutions .
the polymer micelles appeared to delay the clearance of 
 the prodrugs from the system . because the illumination for optical 
 imaging could theoretically 
 generate so and thus release ca4 from pc-(l - ca4)2 , we monitored 
 the body weight change and tumor growth pattern of the mice imaged 
 with pc-(l - ca4)2 .
we did not see any significant impact 
 on mouse body weight or tumor growth since the light dose used for 
 imaging was negligible [ 675 filter ( 660690 nm ) at 1.5 
 w / cm for 2 s ( 3.0  10 j / cm ) ] . the treatment used a ( 1.2  10)-fold higher light dose than the dose used for imaging .
fluorescence 
 optical images of the mice after retro - orbital injection 
 of 2 mol / kg of prodrug : ( a ) pc-(l - ca4)2 in polymer 
 micelles , ( b ) pc-(ncl - ca4)2 in polymer micelles , ( c ) pc-(l - ca4)2 in 5% cremophor solution , and ( d ) pc-(ncl - ca4)2 in 5% cremophor solution .
we expected that the new prodrug 
 pc-(l - ca4)2 would show better antitumor effects than our 
 previous prodrug cmp - l - ca4 , because pc has superior light absorption 
 properties and the prodrug releases two ca4 instead of one .
we used 
 balb / c mice with sc tumors to evaluate the antitumor effects of the 
 prodrugs with illumination .
twenty - four hours post - retro - orbital injection 
 of 1 or 2 mol / kg of the prodrug , the tumor was illuminated 
 by a 690 nm diode laser for 30 min at 100 mw / cm ( 180 j / cm ) or 200 mw / cm ( 360 j / cm ) .
six groups 
 were used to assess the antitumor effects of the prodrug and pseudo - prodrug : 
 g1 , negative control ; g2 , [ ca4 ( 1 mol / kg ) + no hv ] ; g3 , [ pc-(ncl - ca4)2 ( 1 mol / kg ) + hv ( 100 mw / cm ) ] ; g4 , [ pc-(l - ca4)2 ( 1 mol / kg ) + hv ( 100 mw / cm ) ] ; g5 , [ pc-(l - ca4)2 ( 2 mol / kg ) + hv ( 100 mw / cm ) ] ; and g6 , 
 [ pc-(l - ca4)2 ( 2 mol / kg ) + hv ( 200 mw / cm ) ] .
[ the tumor growth curves of g5 and g6 
 were nearly identical to g4 until day 15 .
so , these curves were omitted 
 from figure 7a for clarity ( figure s2 ) .
] we found outstanding antitumor effects 
 in the mice treated with pc-(l - ca4)2 , g4g6 .
after 
 24 h illumination , all tumors shrank to a nonmeasurable size and remained 
 so for almost 15 days ( figure 7a ) .
mice in 
 group g4 experienced tumor growth only after day 16 ( figure s2 ) .
all mice in g4g6 lived until day 30 , while 
 tumor size of all 4 mice in the control group ( g1 ) reached > 800 
 mm in 12 days ( figure 7b ) .
the 
 pdt effects 
 resulting from pc-(ncl - ca4)2 treatment ( g3 ) had a significant 
 impact on tumor size until day 3 ( p < 0.05 ) . however , 
 after day 3 , the tumors grew back at a rate similar to the tumors 
 in the control group g1 . throughout the observation period ,
pc-(l - ca4)2 treatment yielded significantly better antitumor effects 
 ( p < 0.01 ) , than pc-(ncl - ca4)2 treatment . 
 thus , it seemed that the pdt effects alone might not be sufficient 
 to produce such a robust antitumor effect as seen in group g4 .
we 
 hypothesized that our findings stemmed from the contribution of the 
 released ca4 in addition to pdt effects .
interestingly , no mice in 
 g1g6 experienced a significant decrease in body weight ( figure s3 ) .
the histological data were 
 consistent with the antitumor effects 
 ( figure 7c ) .
after 24 h of treatment , tumors 
 were collected and stained with h&e to visualize the tissue damage . 
 while mice treated with pc-(ncl - ca4)2 experienced tissue 
 damage only on the skin , mice receiving pc-(l - ca4)2 also 
 experienced direct tumor damage .
in fact , the volume of tumors treated 
 with pc-(l - ca4)2 shrank to about 1/8 of the volume of the 
 tumors in the control group .
( a ) tumor growth curves , 
 drug iv administration : 
 once a day on day 1 , illumination 24 h postdrug administration 
 [ hv : 100 mw / cm for 30 min ( 180 j / cm ) or 200 mw / cm for 30 min ( 360 j / cm ) ] , 5 
 mice per group except the control group ( 4 mice ) .
error bars represent 
 se . in inset : the order of tumor size was g1 > g3 > g4 during 
 day 
 1 to day 4 [ * * p < 0.01 ( g1 vs g3 , from day 1 
 to day 4 ) and # # p < 0.01 ( g3 vs g4 , from day 
 1 to day 4 ) ] .
( c ) h&e staining of tumors 24 h post - illumination from ( i ) control 
 mice or mice treated with ( ii ) pc-(ncl - ca4)2 , or ( iii ) 
 pc-(l - ca4)2 .
prodrug administration and illumination conditions 
 were same as for those in ( a ) .
vt , viable tumor ; dt , damaged tumor ; 
 and ds , damaged skin .
we successfully demonstrated a multifunctional 
 photounclickable 
 prodrug that can be visualized by optical imaging , and ablates tumors 
 with a combination of pdt and local chemotherapy .
the prodrug pc-(l - ca4)2 and its pseudo - prodrug pc-(ncl - ca4)2 were prepared 
 in high yields through a facile and flexible scheme .
the cytotoxicity 
 of these prodrugs was lower than that of the parent drug ca4 , but 
 both prodrugs showed enhanced cytotoxicity upon illumination .
the mechanisms of cell damage of pc-(l - ca4)2 and pc-(ncl - ca4)2 combined with illumination should be different . while the 
 use of pc-(ncl - ca4)2 and illumination killed cancer cells 
 through pdt effects , the use of pc-(l - ca4)2 and illumination 
 combined these pdt effects with local chemotherapy through the released 
 ca4 .
this was supported by the bystander effects demonstrated in vitro . through the use of optical imaging
, we found that 
 both prodrugs were detected at the therapeutic dose within tumors . 

optical imaging also provided the information about the pk profiles 
 of the prodrugs so that we could find the optimal time point for illumination . 

as expected , the antitumor effects in mice treated with pc-(l - ca4)2 were dramatically better than in mice treated with pc-(ncl - ca4)2 .
treatment with either pc-(ncl - ca4)2 or ca4 produced 
 minimal antitumor effects , suggesting that the outstanding antitumor 
 effects of pc-(l - ca4)2 may have been a result of the synergistic 
 effects of pdt and chemotherapy .
in addition to confirming that 
 our current so - activatable prodrug 
 strategy provides improved antitumor efficacy , we demonstrated the 
 innovative use of a fluorescent photosensitizer within the prodrug . 
 using optical imaging
, we were able to noninvasively generate pk information 
 about the prodrug without causing any observable acute toxicity to 
 mice .
our multifunctional prodrug strategy includes ( 1 ) activation 
 by the clinical translatable far - red light ( or nir ) , ( 2 ) the unique 
 combination of pdt and local chemotherapy , and ( 3 ) the dual function 
 of optical imaging and treatment with one prodrug .
we anticipate that 
 this strategy will be applicable to various drug delivery forms , clinically 
 approved drugs , and advanced drug delivery systems targeted at tumors .
ca4 , compound 1 , and compound 2(10 ) were synthesized 
 as reported previously . 

the purity of the biologically evaluated compounds pc-(l - ca4)2 and pc-(ncl - ca4)2 was confirmed to be > 95% 
 by 
 high - performance liquid chromatography ( hplc ) ( figures s9 and s10 ) .
1-(2-hydroxyethyl)-piperazine 
 ( 1.91 g , 14.70 mmol ) and pyridine ( 2.5 ml ) were added to a solution 
 of silicon(iv ) phthalocyanine dichloride ( pc - cl2 , 1 g , 
 1.63 mmol ) in 50 ml toluene .
the reaction mixture was refluxed for 
 12 h. the solvent was then removed under reduced pressure .
the solvent of the combined organic layers was removed by evaporation , 
 and the crude was recrystallized with chcl3/n - hexane ( 1:4 v / v ) to give a blue solid compound 3 ( 1.22 
 g , 94% ) .
h nmr ( 300 mhz , cd2cl2 ) 
  1.19 ( t , j = 5.8 hz , 4h ) , 0.82 
 ( t , j = 5.8 hz , 1h ) , 0.28 ( m , 8h ) , 1.75 ( m , 8h ) , 
 8.308.40 ( m , 8h , pc - h ) , 9.559.69 
 ( m , 8h , pc - h ) ; c nmr ( 300 mhz , cd2cl2 )  123.4 , 131.0 , 135.9 , 149.2 ; hrms esi 
 ( m / z ) : [ m + h ] calculated 
 for c44h43n12o2si , 799.3401 ; 
 found , 799.3395 .
compound 1 ( 46 
 mg , 0.13 mmol ) and compound 3 ( 50 mg , 0.06 mmol ) were 
 dissolved in 20 ml dry thf , and the solution was stirred at room temperature 
 for 15 min .
the solvent was removed under reduced pressure to yield 
 the crude product , which was then recrystallized from chcl3/n - hexane ( 1:5 v / v ) to give pc-(l - ca4)2 ( 83 mg , 87% ) .
h nmr ( 300 mhz , cd2cl2 ) )  1.96 ( t , j = 5.6 hz , 4h ) , 0.55 
 ( t , j = 5.0 hz , 4h ) , 0.29 ( br s , 8h ) , 2.20 ( br s , 
 8h ) , 3.67 ( s , 12h ) , 3.72 ( s , 6h ) , 3.77 ( s , 6h ) , 4.34 ( d , j = 12.8 hz , 2h ) , 6.45 ( d , j = 4.9 hz , 4h ) , 6.62 
 ( s , 4h ) , 6.84 ( d , j = 8.1 hz , 2h ) , 6.96 ( s , 2h ) , 
 7.00 ( s , 2h ) , 7.09 ( d , j = 12.8 hz , 2h ) , 8.388.43 
 ( m , 8h , pc - h ) , 9.659.70 ( m , 8h , pc - h ) ; c nmr ( 300 mhz , cd2cl2 )  55.8 , 56.6 , 60.4 , 81.8 , 105.9 , 111.8 , 123.5 , 123.9 , 
 126.6 , 128.7 , 129.1 , 129.8 , 131.2 , 132.5 , 135.8 , 137.2 , 140.3 , 149.3 , 
 151.1 , 152.2 , 153.0 , 167.2 ; hrms esi ( m / z ) : [ m + h ] calculated for c86h83n12o14si , 1535.5921 ; found , 1535.5914 .
anhydrous k2co3 ( 68 mg , 0.500 
 mmol ) and compound 3 ( 200 mg , 
 0.25 mmol ) were added to a solution of compound 2 ( 218 
 mg , 0.50 mmol ) in 10 ml dry dmf .
the reaction mixture was stirred 
 at room temperature for 12 h. the k2co3 was 
 removed by suction filtration and the solvent was removed under reduced 
 pressure .
the crude product was then recrystallized from chcl3/n - hexane ( 1:5 v / v ) to give pc-(ncl - ca4)2 ( 302 mg , 80% ) .
h nmr ( 300 mhz , cd2cl2 )  1.96 ( t , j = 5.6 
 hz , 4h ) , 0.78 ( t , j = 5.8 hz , 4h ) , 0.41 ( br 
 s , 8h ) , 1.50 ( m , 2h ) , 1.90 ( m , 2h ) , 2.18 ( m , 2h ) , 2.49 ( m , 2h ) , 3.32 
 ( s , 4h ) , 3.60 ( br s , 8h ) , 3.65 ( s , 12h ) , 3.75 ( s , 6h ) , 3.80 ( s , 6h ) , 
 6.46 ( m , 4h ) , 6.51 ( m , 4h ) , 6.74 ( m , 2h ) , 6.81 ( m , 2h ) , 6.92 ( m , 2h ) , 
 8.38 ( m , 8h , pc - h ) , 9.66 ( m , 8h , pc - h ) ; c nmr ( 300 mhz , cd2cl2 )  
 55.8 , 60.4 , 105.1 , 111.4 , 113.6 , 115.2 , 117.5 , 123.4 , 126.7 , 126.8 , 
 126.9 , 130.9 , 132.8 , 135.9 , 149.1 ; hrms esi ( m / z ) : [ m + h ] calculated for c86h91n12o12si , 1511.6649 ; found , 1511.6612 .
briefly , 3 mg of pc-(l - ca4)2 or pc-(ncl - ca4)2 was dissolved in 1.3 ml of thf .
pla 
 ( cat # ak09 , vendor : polyscitech ) was dissolved in 1 ml of thf .
600 
 l of the 3 mg of pc-(l - ca4)2 or pc-(ncl - ca4)2 dissolved in 1.3 ml thf was added to the mpeg - pla - thf mixture . 

the volume of the resulting mixture was reduced to 300 l under 
 reduced pressure .
the 300 l of the mixture of pc-(l - ca4)2 or pc-(ncl - ca4)2 and mpeg  pla was added 
 to 3 ml of distilled water dropwise while stirring .
the mixture was 
 stirred for 3 h , after which the organic solvent was evaporated under 
 reduced pressure at 40 c .
the concentration of pc-(l - ca4)2 or pc-(ncl - ca4)2 micelles was determined by diluting 
 the micelles in thf : the absorbance was measured by absorbance of 
 pc group .
the concentration was calculated from the molar extinction 
 coefficient ( ec ) of pc-(l - ca4)2 or pc-(ncl - ca4)2 at 675 nm in thf ( ec of pc-(l - ca4)2 = 205,000 ; pc-(ncl - ca4)2 = 206,110 m cm ) using 
 the beer  lambert law .
the concentrations of pc-(l - ca4)2 or pc-(ncl - ca4)2 micelles were determined to be 
 211 and 210 m , respectively .
freshly prepared pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles were used for all the 
 experiments .
2.0 mm stock 
 solutions of pc-(l - ca4)2 and pc-(ncl - ca4)2 prepared 
 in thf were further 
 diluted with 5% cremophor el in pbs to achieve appropriate concentrations .
the pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles in an aqueous solution were characterized by measuring 
 their hydrodynamic diameter and zeta potential via dynamic light scattering 
 ( dls ) .
the size measurement was carried out at a concentration of 
 1.0 mg / ml of pc-(l - ca4)2 or pc-(ncl - ca4)2 .
pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles were also imaged by 
 transmission electron microscopy ( tem ) at 20,000 operating at 
 80 kv .
tem samples were prepared by depositing 20 l of diluted 
 pc-(l - ca4)2 and pc-(ncl - ca4)2 micelle solution 
 on a 300 mesh copper tem grid with a carbon film .
a fluorescence - based tubulin 
 polymerization assay was conducted using a kit supplied by cytoskeleton , 
 inc .
the basic principle is that an increase in fluorescence 
 will occur as a fluorescence reporter is incorporated into microtubules 
 during the course of polymerization .
the assay was performed following 
 the experimental procedure described in version 2.1 of the tubulin 
 polymerization assay kit manual . briefly
, a drug in dmso stock solution 
 was added to a mixture of tubulin and gtp in a buffer solution , to 
 give a final concentration of 3 m .
fluorescence was monitored ( excitation = 
 360 nm and emission = 450 nm ) every 2 min for 1 h. pcx and ca4 were 
 included in the assay as positive controls , as well as a vehicle - only 
 negative control .
the cytotoxicity 
 of pc-(l - ca4)2 and pc-(ncl - ca4)2 was determined 
 with and without 
 illumination .
mcf-7 cells were maintained in minimum essential medium 
 ( -mem ) supplemented with 10% bovine growth serum , 2 mm l - glutamine , 50 units / ml penicillin g , 50 g / ml streptomycin , 
 and 1.0 g / ml fungizone .
mcf-7 cells ( 5000 cells / well ) were 
 seeded on 96-well plates in the medium and incubated for 24 h at 37 
 c in 5% co2 .
stock pc-(l - ca4)2 and pc-(ncl - ca4)2 micelle solutions ( 200 m ) were prepared in distilled 
 water .
the stock solutions were further diluted with medium to obtain 
 the necessary final concentrations . the diluted solution ( 10 l ) 

the plates were incubated 
 for 24 h and then removed from the incubator . for the phototoxicity 
 study : the uncovered plate was illuminated for 30 min using a diode 
 laser ( 690 nm , 5.6 mw / cm ) . to ensure uniformity of the 
 light during the illumination ,
each plate was shaken gently on an 
 orbital shaker ( lab - line , barnstead international ) .
for the dark toxicity 
 study : plates were kept in the dark for 30 min and then returned to 
 the incubator .
briefly , a 10 l solution of mtt ( 10 mg in 1 ml pbs 
 buffer ) was added to each well and the plate was incubated for 4 h 
 at room temperature or 37 c .
then , the mtt solution was removed 
 and the cells were dissolved in 200 l of dmso .
the absorbance 
 of each well was measured at 570 nm with background subtraction at 
 650 nm .
cell viability was quantified by measuring the absorbance 
 of the treated wells compared to that of the untreated control wells , 
 and expressed as a percentage .
colon-26 cells were seeded at 5000 
 cells / well on 24-well plates and incubated for 24 h. stock pc-(l - ca4)2 and pc-(ncl - ca4)2 micelle solutions ( 200 m ) 
 were prepared in distilled water .
200 m of the stock solutions 
 were added to wells ( 1 ml ) to obtain appropriate final concentrations 
 of both pc-(l - ca4)2 and pc-(ncl - ca4)2 at 25 
 nm .
after 24 h incubation , the plates were illuminated from the bottom 
 with a 690 nm diode laser at 11 mw / cm for 15 min . during 
 illumination ,
half of each well was blocked with black masking tape 
 ( cat # t743 - 1.0 , vendor : thorlabs ) .
the illuminated plates were incubated 
 for an additional 48 h. then , a calcein am live cell staining assay 
 ( cat # 4892 - 010-k , vendor : molecular , probes tervigen ) was performed . 

the cells were washed once with 1 ml calcein am wash buffer , then 
 250 l fresh wash buffer and 250 l working reagent were 
 added to the wells .
fluorescent 
 images were obtained with an olympus ix51 inverted microscope with 
 a green fluorescence channel to visualize live cells .
the concentrations 
 of pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles in 
 aqueous solution were determined by diluting 10 l of the formulation 
 stock in 1 ml of thf and measuring the absorbance of phthalocyanine . 

the concentration was calculated from the ec of pc-(l - ca4)2 at 672 nm in thf ( ec of pc-(l - ca4)2 = 205,000 ; 
 pc-(ncl - ca4)2 = 206,110 m cm ) using the beer  lambert law .
we used four- to six - week - old 
 balb / c mice to investigate the biodistribution and tumor targeting 
 ability of the polymeric micelles .
the mice were shaved before the 
 imaging experiments , and were imaged using the ivis imaging system . 

the mice were injected with pc-(l - ca4)2 and pc-(ncl - ca4)2 in the micelle formulation ( 2 mol / kg , i.v . ) . as a 
 comparison ,
fluorescence images were taken 0 , 3 , 6 , 12 , 24 , 48 , and 72 h after 
 retro - orbital injection . before taking the images ,
the mice were anesthetized 
 in an acrylic chamber with a 2.5% isoflurane / air mixture .
the following 
 parameters were used to acquire images with living image software : 
 fluorescence mode , exposure time : 2 s , binning : medium , f / stop : 2 , 
 excitation : 675 filter ( 660690 nm ) , and emission : 720 filter 
 ( 710730 nm ) . during post processing , image counts were adjusted 
 to 3  10 as minimum and 6.0  10 a.u . as maximum color scale .
four- to six - week - old balb / c 
 mice ( 1820 g ) were used for the murine tumor model .
the mice 
 were implanted sc with 2  10 colon 26 cells in pbs 
 ( 100 l ) on the lower back of the neck .
the longest axis of the tumor ( 1 ) and the 
 axis perpendicular to l ( w ) were used to calculate tumor volume ( lw/2 ) .
we used stock solutions of pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles in aqueous solution 
 and further dilutions to achieve final doses as follows : [ ca4 , ( 1 
 mol / kg each ) ] , [ pc-(ncl = ca4)2 , ( 1 mol / kg 
 each ) ] , [ pc-(l - ca4)2 , ( 1 mol / kg each ) ] , and [ pc-(l - ca4)2 , ( 2 mol / kg each ) ] .
to each mouse , 200 l of 
 sample was injected via iv once on day 1 .
twenty - four hours 
 later mice were anesthetized by ip injection of 80 mg / kg ketamine 
 and 6 mg / kg xylazine .
tumors were illuminated with a 690 nm diode 
 laser at 100 mw / cm or 200 mw / cm for 30 min 
 180 or 360 j / cm , respectively .
tumor size was 
 measured every day after the treatment . to evaluate antitumor 
 effect , mice from various groups
the specimens were fixed in 
 10% buffered formalin , embedded in paraffin , and 4 mm diameter 
 tissue sections were stained with hematoxylin and eosin following 
 a standard procedure at the tissue pathology core facility at ouhsc . 
 the sections were viewed and photographed by bright - field microscopy 
 at 4 magnification .
 we recently developed  photo - unclick 
 chemistry  , 
 a novel chemical tool involving the cleavage of aminoacrylate by singlet 
 oxygen , and demonstrated its application to visible light - activatable 
 prodrugs . in this study , we prepared an advanced multifunctional prodrug , 
 pc-(l - ca4)2 , composed of the fluorescent photosensitizer 
 phthalocyanine ( pc ) , an so - labile aminoacrylate linker ( l ) , and a 
 cytotoxic drug combretastatin a-4 ( ca4 ) . 
 pc-(l - ca4)2 had 
 reduced dark toxicity compared with ca4 . however , once illuminated , 
 it showed improved toxicity similar to ca4 and displayed bystander 
 effects in vitro . 
 we monitored the time - dependent 
 distribution of pc-(l - ca4)2 using optical imaging with 
 live mice . 
 we also effectively ablated tumors by the illumination 
 with far - red light to the mice , presumably through the combined effects 
 of photodynamic therapy ( pdt ) and released chemotherapy drug , without 
 any sign of acute systemic toxicity . 
Introduction
Results and Discussion
Conclusion
Experimental Section