hereinafter , the present invention will be described in greater detail with reference to the following examples . the examples are given only for illustration of the present invention and not to be limiting the present invention . as shown in fig2 , ligands a and b were synthesized by using poly ( ethylene glycol )- poly ( β - benzyl - l - aspartate ) ( peg - pbla ) as a template . in order to prepare peg - pbla , β - benzyl - l - aspartate n - carboxy anhydride ( bla - nca ) ( 3 g , 12 mmol ) was polymerized in a mixture of dmf ( 20 ml ) and ch 2 cl 2 ( 50 ml ) at 40 ° c . by initiation from the terminal primary amino group of α - methoxy - ω - amino - poly ( ethylene glycol ) ( mw = 2 , 000 da , 240 mg , 120 μmol ). peg - pbla was purified by precipitation in ether ( 3 l ) three times . in order to synthesize peg - pbla - ce6 ( platform ligand ), ce6 was attached to the amine groups of peg - pbla via the conventional carbodiimide reaction . the peg - pbla ( 0 . 5 g , 32 . 5 μmol ,) and a mixture of ce6 ( 23 . 4 mg , 39 . 0 μmol ), dicyclohexylcarbodiimide ( 9 . 6 mg , 46 . 8 μmol ), and n - hydroxysuccinimide ( 5 . 4 mg , 46 . 8 μmol ) were dissolved separately in dmso ( 5 ml ) and the solutions were stirred thoroughly for 3 h prior to the condensation reaction . the two reactant solutions were then mixed and stirred at room temperature . after 24 h , the reaction mixture was filtered to remove the insoluble by - products ( e . g ., dicyclohexylurea ) and dialyzed against deionized water for 2 days ( spectra / por ; molecular weight cutoff size , mwco : 1 , 000 da ). the final solution was lyophilized to obtain the platform ligand . the hydrogen nuclear resonance spectroscopy (“ h - nmr ”) analysis of the platform ligand ( peg - pbla - ce6 ) at 300 mhz with dmso - d 6 is illustrated in fig1 , where δ = 9 . 7 ppm ( 1h , s , — ch ═ of ce6 ), 8 . 1 ppm ( 1h , s , — nh —), 7 . 3 ppm ( 5h , s , — ch 2 c 6 h 5 —), 5 . 0 ppm ( 2h , — c h 2 c 6 h 5 —), 4 . 6 ppm ( 1h , m , — nhchc ═ o —), 3 . 5 ppm ( 182h , s , — ch 2 ch 2 o — of mpeg ), 3 . 2 ppm ( 3h , s , ch 3 — of mpeg ) and 2 . 9 - 2 . 5 ppm ( 2h , m , chch 2 c ═ o ). ligand a was synthesized via aminolysis of the platform ligand with 1 -( 3 - aminopropyl ) imidazole ( api ) and dopamine . peg - pbla - ce6 ( 0 . 5 g , 18 . 5 μmol ) was dissolved in dmso ( 5 ml ), followed by the reaction with dopamine ( 0 . 1 g , 0 7 mmol ) under nitrogen atmosphere at 25 ° c . for 1 h . then , api ( 0 . 5 g , 3 . 9 mmol ) was added under nitrogen at 25 ° c . and stirred for 4 h . the reaction mixture was added dropwise into a cooled aqueous solution of 0 . 1 n hcl ( 20 ml ) and dialyzed against a 0 . 01 n hcl solution three times ( spectra / por ; mwco : 1 , 000 da ). the final solution was lyophilized to obtain ligand a . the h - nmr analysis of ligand a ( peg - p ( api & amp ; dopa - l - asp )- ce6 ) at 300 mhz with dmso - d 6 is illustrated in fig2 , where δ = 7 . 8 ppm ( 1h , s , — nch ═ n — of imidazole ring ), 7 . 7 ppm ( 1h , s , — nch ═ ch — of imidazole ring ), 7 . 3 ppm ( 1h , s , — chch ═ n — of imidazole ring ), 6 . 6 - 6 . 5 ppm ( 1h , m , — ch 2 ═ chch — and — ch 2 ch ═ ch — of dopa ), 6 . 4 ppm ( 1h , m , ═ chco — of dopa ), 4 . 5 ppm ( 1h , m , — nhchc ═ o —), 4 . 2 ppm ( 2h , m , ═ nch 2 ch 2 ), 3 . 5 ppm ( 182h , s , — ch 2 ch 2 o — of mpeg chain ), 3 . 0 ppm ( 2h , m , — nhch 2 ch 2 —), and 2 . 8 - 2 . 5 ppm ( 2h , m , chch 2 c ═ o ). ligand b was synthesized via aminolysis of the platform ligand with api and 3 - phenyl - 1 - propylamine ( ppa ). peg - pbla - ce6 ( 0 . 5 g , 18 . 5 μmol ) was dissolved in dmso ( 5 ml ), followed by reacting with ppa ( 0 . 1 g , 0 . 9 mmol ) under nitrogen at 25 ° c . for 1 h . then , api ( 0 . 5 g , 3 . 9 mmol ) was added under nitrogen at 25 ° c . and stirred for 4 h . after the reaction , the mixture was added dropwise into a cooled 0 . 1 n hcl solution ( 20 ml ) and dialyzed against an aqueous solution of 0 . 01 n hcl three times ( mwco : 1 , 000 da ). the final solution was lyophilized to obtain ligand b . the h - nmr analysis of ligand b ( peg - p ( api & amp ; ppa - l - asp )- ce6 ) 300 mhz with dmso - d 6 is illustrated in fig3 , where δ = 7 . 8 ppm ( 1h , s , — nch ═ n — of imidazole ring ), 7 . 7 ppm ( 1h , s , — nch ═ ch — of imidazole ring ), 7 . 3 ppm ( 1h , s , — chch ═ n — of imidazole ring ), 7 . 3 - 7 . 2 ( 5h , m , — ch 2 c 6 h 5 — of ppa ), 4 . 5 ppm ( 1h , m , — nhchc ═ o —), 4 . 2 ppm ( 2h , m , ═ nch 2 ch2 ), 3 . 5 ppm ( 182h , s , — ch 2 ch 2 o — of mpeg chain ), 3 . 0 ppm ( 2h , m , — nhch 2 ch 2 —), and δ2 . 8 - 2 . 5 ppm ( 2h , m , chch 2 c ═ o . in the gpc measurements ( fig4 ), all polymeric ligands showed unimodal molecular weight distribution indicating no homopolymer residues in the copolymer products . the ce6 content of the ligand was examined by the fluorescence spectroscopy ( λ ex = 650 nm and λ em = 675 nm , table 1 ). esions were synthesized via thermal decomposition of iron - oleate complex in the presence of oleyl alcohol using the previously reported method ( kim , b . h . ; lee , n . ; kim , h . ; an , k . ; park , y . i . ; choi , y . ; shin , k . ; lee , y . ; kwon , s . g . ; na , h . b . ; park , j . g . ; ahn , t . y . ; kim , y . w . ; moon , w . k . ; choi , s . h . ; hyeon , t . j . am . chem . soc . 2011 , 133 , 12624 ). briefly , 1 . 8 g of iron - oleate complex ( 2 mmol ), 0 . 57 g of oleic acid ( 2 mmol ), and 1 . 61 g of oleyl alcohol ( 6 mmol ) were dissolved in 10 g of diphenyl ether at room temperature . the mixture was heated to 250 ° c . at a constant heating rate of 10 ° c ./ min and then kept at this temperature for 30 min under inert atmosphere . as the reaction proceeded , the initial brown transparent solution turned black . after the reaction , the mixture containing nanoparticles was removed from the heater and allowed to cool to room temperature and , then , 50 ml of acetone was added to precipitate the nanoparticles . the nanoparticles were pelleted by centrifuging at 40 , 000 rpm for 4 hours , the supernatant decanted and the nanoparticles redispersed in n - hexane or chloroform . for self - assembly , a solution prepared by mixing 15 mg of ligand a and 15 mg of ligand b in 3 ml of dmso was added slowly to 5 ml of colloidal esions ( 0 . 4 mg fe / ml ) in chloroform . the mixture was incubated on a shaker at room temperature for 30 min . chloroform was then completely removed by evaporation under vacuum and deionized water was added to the colloidal suspension in dmso to reach a total volume of 5 ml . the dmso was completely substituted with deionized water using a dialysis membrane ( spectra / por ; mwco : 12 , 000 da ). excess ligands were removed by centrifugation and washed 3 - 5 times with spin filter ( millipore , mwco : 100 , 000 da , 10 , 000 × g , for 10 min ) the resulting nanoparticles were re - dispersed in water . a mixed solution of 15 mg of ligand a and 15 mg of ligand b in 3 ml of dmso was added slowly to 5 ml of chloroform . the mixture was incubated on a shaker at room temperature for 30 min . then , chloroform was removed completely by evaporation under vacuum . thereafter , deionized water was added to the colloidal solution in dmso to reach a total volume of 5 ml . dmso was completely substituted with deionized water using a dialysis membrane ( spectra / por ; mwco , 12 , 000 da ). excess ligands were removed by centrifugation or washed with spin filter ( millipore , mwco : 100 , 000 da , 10 , 000 × g , for 10 min ) 3 - 5 times . the resulting nanoparticles were re - dispersed in water . a solution prepared by mixing 30 mg of platform ligand in 3 ml dmso was added slowly to 5 ml of a colloidal esions ( 0 . 4 mg fe / ml ) suspension in chloroform . the mixture was incubated on a shaker at room temperature for 30 min . then , chloroform was removed completely by evaporation under vacuum . thereafter , deionized water was added to the colloidal solution in dmso to reach a total volume of 5 ml . dmso was completely substituted with deionized water using a dialysis membrane ( spectra / por ; mwco , 12 , 000 da ). excess ligands were removed by centrifugation or washed with spin filter ( millipore , mw , 100 , 000 da , 10000 × g , for 10 min ) 3 - 5 times . the resulting nanoparticles were re - dispersed in water . tem measurements were performed on a jeol em - 2010 microscope operated at 200 kv . the powder x - ray diffraction patterns were obtained with a rigaku d / max - 3c diffractometer equipped with a rotating anode and a cu kα radiation source ( λ = 0 . 15418 nm ). the present inventors performed elemental analysis by inductively coupled plasma atomic emission spectroscopy ( icp - aes ) using an icps - 7500 spectrometer ( shimadzu ) and inductively coupled plasma - optical emission spectrometer ( icp - oes ) ( perkin - elmer optima 4300 dv ). uv / visible absorption spectra were collected on uv - 2450 spectrophotometer ( shimadzu , japan ). particle size and zeta potential were measured with zetasizer nano zs ( malvern instruments , malvern , uk ). for the analysis of size , the following parameters , material refractive index ( 2 . 42 ), material absorbance ( 0 . 2 ), dispersant refractive index ( 1 . 33 ), and dispersant viscosity ( 0 . 8872 ) were used . the magnetic properties were investigated using a superconducting quantum interface device ( squid ) magnetometer ( quantum design mpms xl ). hydrodynamic sizes were obtained using dynamic light scattering ( dls ) ( zetasizer nano zs ( malvern instruments , malvern , uk )) instrument at 25 ° c . a stock solution of hoechst 33342 ( 1 . 4 × 10 − 3 m ) in double - distilled water was prepared and stored at 4 ° c . the hoechst 33342 solution was mixed with solutions containing the samples at the concentrations of 1 . 0 × 10 − 3 mg / ml to 1 . 0 mg / ml . the final concentration of hoechst 33342 in each sample solution was 7 . 0 × 10 − 4 m . the resultant fluorescence was measured on a rf - 5301pc ( shimadzu , japan ) with λ ex = 355 nm and λ em = 457 nm and the slit widths were ex = 3 nm and em = 3 nm , respectively . the light transmittance measurements of pmn solutions ( 2 mg / ml , without ce6 attachment ) were obtained using a uv - visible spectrophotometer at 500 nm while the ph value of solution was gradually decreased from 7 . 5 to 4 . 0 by adding 0 . 1 n hcl , and increased from 4 . 0 to 7 . 5 by adding 0 . 1 n naoh solution . the fluorescence intensity of pmns or self - assembled ligands ( 2 mg / ml , 650 nm excitation and 675 nm emission ) was measured using a fluorescence plate reader ( tecan genios , durham , n . c . ), while the ph of the solution was gradually decreased from 7 . 5 to 4 . 0 by adding 0 . 1 n hcl solution . in order to evaluate the sog , samples ( 2 mg / ml , 100 μl ) at different ph were mixed with singlet oxygen sensor green ( sosg , 2 . 0 μm , 100 μl ). sog was induced by irradiation with a 670 nm laser source ( institute of electronics ) at 5 mw / cm 2 intensity for 4 min . sosg fluorescence was detected ( λ ex = 494 nm and λ em = 534 nm ) after irradiation to determine the sog of the samples . sog was evaluated by sosg fluorescence enhancement compared to the background . hct116 ( human colon cancer , kclb no . 10247 ) cells , ct26 ( murine colorectal carcinoma cell , kclb no . 80009 ) and m2 - 10b4 ( murine bone marrow stromal cell , kclb no . 21972 ) were obtained from the korean cell line bank . hct116 cells were cultured in 10 ml of roswell park memorial institute ( rpmi - 1640 ) medium supplemented with 10 % fetal bovine serum ( fbs ) and 1 % penicillin / streptomycin . ct26 cells were cultured in dulbecco &# 39 ; s modified eagle medium ( dmem ) supplemented with 10 % fbs and 1 % penicillin / streptomycin . m2 - 10b4 was cultured in rpmi - 1640 medium supplemented with 10 % fbs and 1 % penicillin / streptomycin . culturing conditions for all cells were 37 ° c . in 100 % humidity and 5 % co 2 . drug resistant ct26 cells ( ct26 / mdr ) were developed by exposing ct26 cells to increasing doses ( 1 , 5 , 10 , 25 , 50 , 100 , 500 , 1 , 000 , and 5 , 000 ng / ml ) of dox for 24 h followed by 3 - 4 days of recovery before exposing to the next dose . ct26 / mdr was then frozen and stored in liquid nitrogen . fresh aliquots of ct26 / mdr were used in all experiments to ensure that they did not revert to a drug sensitive phenotype . hct116 cells were exposed to pmns , ins - nps and ce6 at different phs to verify their cellular uptake respectively . the cells were incubated for 2 h , washed , harvested , and re - suspended with dpbs . in vitro cellular uptake was quantified using a flow cytometer ( beckman , san jose , calif ., usa ). for each sample 10 , 000 cells ( gated events ) were counted , and free ce6 fluorescence was detected with logarithmic settings ( fl4 ; λ em = 670 nm ). cells were counted as positive if their fluorescence ( fl4 ) was higher than that of cells from an untreated cell suspension . each experiment was analyzed statistically using the cxp analysis program . confocal laser scanning microscope ( lsm 510 meta ; zeiss , germany ) was used to carry out fluorescence imaging . an optimal pinhole size of 120 μm was selected to exclude fluorescent light emitted from out - of - focus planes above and below the focusing plane . the excitation / emission wavelengths were 340 / 488 nm for dapi , 490 / 520 nm for fitc , 555 / 578 nm for ritc , and 650 / 670 nm for ce6 . fluorescence images were analyzed using lsm image browser software ( zeiss ). in order to label the cells with pmns , hct116 cells were seeded onto culture dishes in 10 ml of media and grown overnight . subsequently , pmns of 0 , 12 . 5 , 25 , and 50 μg / ml were added . after 4 h , the cells were washed twice with pbs and detached by adding 1 ml of trypsin / edta . after centrifugation , cells were dispersed in culture media and transferred to a 1 . 5 ml test tube . cell pellets were prepared by centrifugation at 1 , 000 rpm for 5 min . t 1 - weighted mr images were acquired with a head coil on a 1 . 5 t mr scanner ( signa excite , ge healthcare ). for the plasma concentration - time experiment , the mice were injected with pmns and ins - nps respectively ( 2 mg fe / kg ) via the tail vein . blood was collected at 1 min , 30 min , 1 h , 3 h , 8 h and 20 h after the injection . plasma was isolated from red blood cells by centrifugation at 1 , 000 rpm for 10 min the plasma ( 100 μl ) for each blood sample was subsequently mixed with 70 % nitric acid ( 1 ml ) at room temperature for 12 h followed by centrifugation ( 10 , 000 rpm for 5 min ), and the supernatant was used for inductively coupled plasma optical emission spectrometer analysis ( icp - oes , optima 4300 dv , perkin - elmer ) after 5 - fold dilution with 2 % nitric acid . fe uptake in the tumor was measured 12 h after pmns or ins - nps injection ( 2 mg fe / kg ). dissected tumor tissues were weighed , homogenized , and treated with scintillation mixtures . a volume of 60 % nitric acid was added to each sample and tissue was incubated for 24 h at 60 ° c . after which the solutions were centrifuged at 13 , 000 rpm for 30 min and the supernatant was diluted 10 - fold with 2 % nitric acid . determination of fe uptake content in tumor was performed by icp - oes , and the nanoparticle uptake in tumor was calculated as gram of fe content per gram of tissue ( g fe / g tissue ). tumor tissues were fixed in 10 % neutral buffered formalin and frozen sectioned into 5 micron thick slices . sdf - 1 and p - gp were immunolabeled with anti - sdf - 1 antibody ( abcam ) and anti - p - glycoprotein antibody ( abeam ), then the samples were incubated for 60 min at ambient temperature with ritc ( λ ex / λ em , 555 / 578 nm ) or fitc ( λ ex / λ em , 490 / 520 nm ) conjugated secondary antibody for further confocal laser scanning microscope analysis . for histology analysis , tumor tissues from control and treated mice were fixed in 10 % neutral buffered formalin and frozen sectioned into 5 micron thick slices , stained with hematoxylin & amp ; eosin ( h & amp ; e ) and terminal deoxynucleotidyl transferase dutp nick - end labeling ( tunel ), and were examined by a digital microscope ( leica qwin ) and a confocal laser scanning microscope ( lsm 510 meta ; zeiss , germany ). healthy nude mice were intravenously injected with a suspension of pmns or normal saline ( control group ). after 2 weeks , the mice were sacrificed and major viscera collected . the heart , liver , spleen , kidney , and lung were stained with h & amp ; e . reagent kits of alt , ast , alp and d - ldh were employed to analyze the serum which was isolated from blood sampled by eyeball extirpation . the tumor cellular uptake of pmns was monitored at different ph conditions ( ph 7 . 4 and 6 . 8 ) using a kodak in vivo image station ( image station 4000 mm ; kodak , new haven , conn ., usa ) and a flow cytometer ( beckman , san jose , calif ., usa ). hct116 cells ( 1 × 10 5 cells / ml ) were incubated with each sample for 2 h in rpmi - 1640 medium ( ph 7 . 4 or 6 . 8 , with 1 % penicillin - streptomycin ) at 37 ° c . and then analyzed . to ameliorate photobleaching during microscope observation , a drop of anti - fade mounting solution ( 5 % n - propyl gallate , 47 . 5 % glycerol , and 47 . 5 % tris - hcl , ph 8 . 4 ) was added to the cells . all in vivo studies conformed to the guide for the care and use of laboratory animals published by the national institutes of health , usa ( nih publication no . 85 - 23 , 1985 , revised 1996 ), and mice were maintained under the guidelines of an approved protocol from the institutional animal care and use committee ( iacuc ) of the catholic university of korea ( republic of korea ). ce6 grafted poly ( ethylene glycol )- poly ( β - benzyl - l - aspartate ) ( peg - pbla - ce6 ) was synthesized to provide a platform ligand in which the flanking benzyl ester groups readily react with primary amines via nucleophilic attack . imidazole ( pka , ˜ 6 . 8 ) was then easily incorporated as an ionizable group to impart ph sensitivity to the tumor microenvironment . on the basis of this platform , the present inventors further engineered two ligand derivatives : ligands a and b . for ligand a , catechol groups were added to facilitate self - assembly as they can act as high - affinity anchors for iron oxide nanoparticles . in contrast , the hydrophobicity of ligand b was tuned using 3 - phenyl - 1 - propylamine to produce a critical phase transition of pmns that is activatable by tumor endo / lysosomal ph of ˜ 5 . 5 . the pmns were fabricated by coassembly of esions , ligand a and ligand b ( scheme 2 ) ( fig1 ). since esions ( about 3 nm ) were much smaller than the hydrodynamic dimensions of the peptide block of the ligands ( about 60 residues ; length of about 20 nm ), the catechol - anchored ligand a could wrap around the periphery of the esions . these functionalized esions can thus be considered polymer - metal analogues of conventional amphiphilic diblock copolymers since the functionalization permitted directed selfassembly of esions into colloidal magneto - core shell structures ( scheme 3 ) ( fig1 ). the hydrophobic core is then composed of the esions and hydrophobic blocks of the tumor - sensing polymeric ligands . transmission electron microscopy ( tem ) ( fig5 a ) revealed the particle size of pmns is ˜ 60 nm they were well dispersed in water , and the hydrodynamic diameter measured by dynamic light scattering ( dls ) was found to be 70 ± 5 nm ( fig5 a ), which is appropriate for the enhanced permeability and retention ( epr ) effect for passive tumor targeting . as a control , the present inventors fabricated ph - insensitive nanoparticle assemblies ( denoted as ins - nps ) by assembling esions and the platform ligand , which were similarly sized to pmns ( fig6 ). new ligand design of the present invention enabled a two - stage ph activation leading to surface charge reversal in the tumor periphery for increased cell adsorption and permeation as well as endo / lysosomal ph dependent theranostic activity of pmns . this ph - dependent structural transformation is schematically presented in fig5 b . first , pmns are slightly negatively charged at ph 7 . 4 . the drop in ph within the tumor environment causes increased imidazole ionization which reverses the polarity of the complex causing it to swell ( fig5 c ), thereby enhancing their payload delivery and cell internalization due to electrostatic interactions with the vicinal anionic cells . upon internalization , the particles further ionize as the endosomal ph decreases to 5 . 5 - 6 . 0 . here the hydrophobic interactions of the core of the pmns weaken , and the ionized unimers repel each other leading to complete dissociation as confirmed by both dls ( fig5 c ) and tem ( fig5 a ). acid - base titration ( fig7 a ) demonstrated a sharp drop in transmittance (% t ) at the critical ph range of 5 . 5 - 6 . 0 . when the ph was increased again , % t recovery was hysteretic , thereby demonstrating a reversible ph - dependent assembly / disassembly process . consequently , the photoactivity , i . e . singlet oxygen generation ( sog ) and fluorescence , of pmns was quenched at ph 7 . 4 due to fluorescence resonance energy transfer . at ph & lt ; 6 , the photoactivity was dramatically recovered upon disassembly , similar to that observed for the other self - assembled polymeric ligands ( fig7 b and 7 c ). interestingly , pmns showed a 2 - fold lower critical aggregation concentration (˜ 0 . 01 mg / ml ) than the self - assembled polymeric ligands possibly due to polymer chain entanglement indicating their improved colloidal stability . the x - ray diffraction ( xrd ) pattern of pmns was similar to that of esions ( fig8 ), and the pmns show a weak magnetization because of the spin - canting effect , as illustrated in fig9 a and 9 b . the ph - dependent structural transformation of pmns in water is expected to affect proton relaxation . considering the large number of high - spin fe ( iii ) ions with five unpaired electrons ( s = 5 / 2 ) on the surface of esions , direct water coordination with fe 3 + species is a major contributor to the longitudinal relaxivity ( r1 ) of pmns . the intensity variation of the t 1 mr phantom of pmns at different ph values ( same fe concentration ) was well matched with the corresponding fluorescent imaging results ( fig7 d ). pmns showed an r1 of 3 . 30 mm − 1 · s − 1 with a transverse relaxivity ( r2 ) of 43 . 95 mm − 1 · s − 1 at ph 7 . 4 , and a concomitant r1 - increase and r2 - decrease was observed as the ph decreased from 7 . 4 to 5 . 5 ( fig7 e ). it is assumed as ph decreases , ligands become protonated and gain hydrophilicity . consequently , both the number of coordinated water molecules and the duration of their coordination with fe 3 + will increase . moreover , as ph - induced disassembly occurs , separated esions exhibit a lower r2 compared to initial pmns . although the efficiency of the contrast effect is evaluated in terms of relaxivity ( r1 ), the r2 / r1 ratio also plays an important role in positive t 1 imaging as an excessively high r2 may preclude their use as t 1 contrast agents . as the ph decreases , the r2 / r1 ratio of pmns significantly decreases along with r2 . finally , the pmns have a specific r1 value of 3 . 87 mm − 1 · s − 1 and a low r2 / r1 ratio of 5 . 8 at ph 5 . 5 , which results in a bright signal in t 1 - weighted imaging . therefore , the positive t 1 mr contrast of pmns was quenched at ph 7 . 4 but greatly recovered at ph 5 . 5 , which indicates pmns can be used for sensitive t 1 mr imaging of acidic tumor regions . in contrast , mr contrast of ins - nps was not dependent on ph ( fig1 ). to the best of the present inventors knowledge , this is the first demonstration of biocompatible iron oxide nanoparticles showing ph - sensitive t 1 contrast that can be used for tumor ph - sensitive t 1 mr imaging . although gd 3 + - based t 1 contrast agent can also be designed to respond to ph stimulus , they generally have short blood half - lives , preventing their accumulation in tumors for high - resolution tumor imaging . furthermore , gd 3 + - based contrast agents can be potentially toxic ; severe side effects were observed in patients with renal failure that received gadolinium - containing contrast agents , such that the u . s . fda recently released a warning regarding gd 3 + - based mr contrast agents and nephrogenic system fibrosis ( nsf ). while such side effects are quite rare , occurring in less than 5 % of patients , the present inventors believe iron oxide nanoparticles may provide a more biologically and metabolically compatible alternative . the use of biodegradable polymeric peptides for iron oxide - based pmn fabrication gives the final product a longer blood half - life and a greater biocompatibility for clinical translation . mri investigations were conducted using a 1 . 5 t mr scanner ( signa excite ; ge healthcare ) by using a litz coil ( diameter , 100 mm ; length , 85 mm ; doty scientific inc ., nc , usa ). spin - lattice and spin - spin relaxation times ( t 1 and t 2 ) were measured using fast spin echo ( fse ) sequence for different concentrations of pmns in media with different ph values at room temperature . for t 1 measurements , the field of view ( fov ) was set to 75 × 75 mm , slice thickness ( sl )= 3 mm , echo time ( te )= 9 . 3 ms and repetition time ( tr )= 505 . 2 , 525 . 0 , 545 . 0 , 565 . 0 , 585 . 0 , 605 . 0 , 625 . 0 , 645 . 0 , 665 . 0 , 685 . 0 , 705 . 0 , 730 . 0 , 755 . 0 , 805 . 0 , 855 . 0 , 905 . 0 , 955 . 0 , 1005 . 0 , 1055 . 0 , 1105 . 0 ms . for t 2 measurements , the following parameters were used : fov = 100 mm * 100 mm , sl = 3 mm , tr = 4 , 000 ms , te = 10 . 9 , 21 . 7 , 43 . 5 , 54 . 4 , 65 . 2 , 87 . 0 , 119 . 6 , 141 . 3 , 163 . 1 , 174 . 0 ms . the longitudinal ( r1 ) and transverse ( r2 ) relaxivities were calculated from r i =( 1 / t i − 1 / t i0 )/ c , where c is the fe concentration of pmns in mm , t i is the relaxation time at concentration c , t i0 is the relaxation time of water , and i = 1 and 2 for t 1 and t 2 . the cellular mr images were acquired using fse sequence ( fov = 100 mm * 100 mm , sl = 3 mm ) for t 1 measurement , tr = 400 ms and te = 10 ms were used , whereas for t 2 measurement , tr = 4 , 000 ms and te = 86 . 72 ms were used . for the mr diagnosis of tumor in vivo , pmns were injected through the tail vein at a dose of 2 mg fe / kg body weight . mice were then placed in a 1 . 5 t mr scanner ( signa excite , ge healthcare ), and fse sequence was used with the followed parameters : fov = 90 mm × 90 mm , sl = 2 mm , flip angle ( fa )= 90 °, for t 1 mr imaging , tr = 400 ms , te = 10 ms , and for t 2 mr imaging , tr = 3 , 000 ms , te = 101 ms . the transverse section images were obtained and analyzed using onis dicom viewer ( digitalcore , tokyo , japan ). pmns showed higher cellular uptake at ph 6 . 8 than at ph 7 . 4 as evidenced by both fluorescence and flow cytometry results ( fig1 a ). in contrast , the cellular uptake of ce6 and ins - nps was not affected by changes in ph . for cell uptake , the present inventors followed the iron oxide nanoparticles by tem ( fig1 c ) which shows uptake in endosomes . the present inventors also followed the ce6 dye by confocal laser scanning microscopy ( clsm ) ( fig1 b ) which shows the fluorescence of pmns merges perfectly with that of lysotracker green . the existence of the ce6 signal suggests pmns indeed disassembled for fluorescence dequenching here in the endosome . consequently , pmns showed no cytotoxicity in the dark ( fig1 d ) but induced cell death much more efficiently than ce6 under illumination ( fig1 e ) at ph 6 . 8 . mr contrast and fluorescence of human colorectal carcinoma ( hct116 ) cells labeled with pmns ( fig1 f ) further confirmed their dual - modal imaging capability . the present inventors performed in vivo early stage tumor diagnosis with pmns . without conjugation of any tumor - targeting moiety and in contrast to ins - np injection , pmn injection resulted in significant t 1 enhancement of ultrasmall hct116 tumors of ˜ 3 mm in diameter ( fig1 a - 12 c ), thus confirming their successful tumor targeting and ph - dependent t 1 mr contrast effect . pharmacokinetic studies showed both pmns ( t 1 / 2 , pmn = 2 . 90 h ) and ins - nps ( t 1 / 2 , ins - np = 2 . 19 h ) had long blood circulation times ( fig1 d ). however , notably , pmn accumulation in tumors was & gt ; 2 - fold higher than that of ins - nps ( fig1 e ). moreover , pmns also enabled high - resolution fluorescent imaging of tumors in mice ( fig1 f ). macroscopic fluorescent imaging of excised organs demonstrated significant tumor accumulation of pmns ( fig1 ), and subcellular clsm confirmed uptake of pmns by tumor cells ( fig1 g ). these results indicate pmns are promising candidates for highly efficient early diagnosis of cancer . for in vitro pdt , hct116 cells ( 5 × 10 4 / well ) were incubated with the materials described above . photosensitization experiments were then performed by irradiating the cells with a 670 - nm laser source ( institute of electronics ) for 2 min the power at the level of the cells was fixed at 5 mw / cm 2 . cell viability was assessed using the mtt assay . absorbance intensity was measured at 595 nm using a microplate reader ( bio - tek , vt , usa ). a parallel set of control cells was also used in which cells incubated with the respective materials were not exposed to laser irradiation . for in vivo pdt , hct116 tumors were developed in the flanks of 6 - week - old male balb / c nude mice by subcutaneously implanting hct116 cells in 100 μl of a 1 : 1 mixture of matrigel ( bd biosciences , franklin lakes , n . j .) and serum - free medium . heterogeneous ct26 tumors were developed in the flanks of 6 - week - old male balb / c nude mice by subcutaneously implanting a mixture of ct26 ( 1 × 10 5 cells ), ct26 / mdr ( 1 × 10 5 cells ), and m2 - 10b4 cells ( 1 × 10 5 cells ) in 100 μl of serum - free medium . ten days after inoculating the tumor cells , pdt treatment was performed as follows : group 1 , saline only ; group 2 , free ce6 only ; group 3 , ins - nps ; and group 4 , pmns ( equivalent to 2 mg / kg body of ce6 ). nir images were obtained using kodak image station ( image station 4000 mm ; kodak , new haven , conn .) at 2 h post - injection . laser treatment was performed in groups 1 through 4 by irradiating the tumor region with a 670 - nm laser ( 250 mw / cm 2 , for 20 min ) at 12 h post - injection . the pdt treatment described above was repeated 5 days later . the tumor size was measured 3 times a week after the first pdt treatment by using a vernier caliper , and tumor volumes were calculated from width ( a ) and length ( b ) measurements ( v = a 2 × b / 2 , where a ≦ b ). all mice were killed when the tumor measured 1 , 000 mm 3 , at the first sign of suffering , or between 15 and 21 day post - transplantation . in order to explore the therapeutic effect of pmns , in vivo photodynamic therapy ( pdt ) was performed ( fig1 ). pdt is a clinically approved , minimally invasive therapeutic procedure for cancer treatment that relies on photosensitizers to produce cytotoxic singlet oxygen generation ( sog ) upon irradiation . however , currently available photosensitizers lack tumor selectivity which causes undesirable damage to normal tissues . in our pmn system , the activity of the photosensitizer can be self - quenched until it reaches the target tumor site where this suppression can be rapidly reversed by tumor ph stimulus , in particular intracellular ph stimulus , leading to a highly specific region of effect . hct116 tumor - bearing mice injected with pmns and then irradiated showed significant tumor regression relative to mice treated with ins - nps or free ce6 ( fig1 d and 14 e ). in particular , at 1 week post - injection , the tumors were almost completely destroyed with only scar tissue remaining . the enhanced antitumor effect was confirmed by hematoxylin and eosin staining ( h & amp ; e ) and terminal deoxynucleotidyl transferase dutp nick - end labeling ( tunel ) staining . these results clearly demonstrated that ph - targeted pdt using pmns was successful in homogeneous hct116 xenografts . in order to be a better match to clinical cancer treatment , the present inventors further demonstrated the therapeutic effect of pmns in tumors of more heterogeneous and drug - resistant nature . the present inventors hypothesized the ph - targeting approach of pmns would not be influenced by tumor heterogeneity , and since pdt kills by a nonspecific mechanism , drug - resistant cells are equally as susceptible as their naive counterparts . to test this hypothesis , the present inventors developed highly heterogeneous and drug - resistant ct26 tumors in mice . the heterogeneous ct26 tumors overexpress p - glycoprotein ( p - gp ), which is involved in the active efflux of anti - cancer agents , and stromal cell - derived factor - 1 ( sdf - 1 ), which induces angiogenesis ( cd31 ) ( fig1 c ). consequently , the ct26 tumors provided a physiologically relevant model of clinical tumors with a much faster growth rate than homogeneous hct116 tumors . notably , ins - nps produced some minor tumor growth inhibition in both the homogeneous hct116 and heterogeneous ct26 tumors ( fig1 f and 14 g ) compared to the untreated control . however , the heterogeneous model grows much more aggressively such that the positive effects observed for ins - nps are still far too weak . in contrast , pmns provided the same dramatic tumor destruction in both ct26 - tumor - bearing mice and homogeneous hct116 - tumor - bearing mice ( fig1 f and 14 g ). in the pmn - treated group , many cells in the tumor tissue and microvasculature as well as fibroblasts showed considerable destruction , whereas no obvious damage was observed in groups treated with ins - nps or ce6 ( fig1 f and 14 g ). therefore , ph targeting appears to play an important role in the improved anti - cancer therapeutic efficacy of pmns . consequently , for the first time , the present inventors successfully demonstrated treatment of drug - resistant heterogeneous tumor via ph - sensitive pdt , indicating pmns can be applicable to various tumor treatments including highly drug - resistant heterogeneous tumors . furthermore , no significant loss in body weight was observed throughout the pdt treatment ( fig1 ), and a series of in vivo biocompatibility tests including a histopathology examination and serum chemistry test ( fig1 ) demonstrated pmns are highly biocompatible .
the present invention relates to a nanoformulated self - assembled pharmaceutical composition for photodynamic therapy . more particularly , the present invention is directed to a self - assembled pharmaceutical composition for photodynamic therapy comprising a photosensitizer , a ligand a which is separated at a specific ph range , and a ligand b of which surface charge changes at a specific ph range and a method for manufacturing the same .