polymerdrug conjugates are established as one of the first - generation nanomedicines for the treatment of cancer .
their use facilitates the uptake and transport of therapeutic agents and creates a dose - differentiation between the treatment target and the rest of the body , due to the fact that macromolecules can accumulate passively in solid tumor tissue by the phenomenon called 
enhanced permeability and retention ( epr ) effect.(1 ) the enhanced drug accumulation in tumor tissue increases the therapeutic effect while reducing nonspecific side - effects . moreover ,
the intracellular trafficking mechanism of polymerdrug conjugates renders drug efflux pumps ineffective.(5 ) to obtain such multifunctional capabilities , polymerdrug conjugates have been designed to consist of ( a ) a nonimmunogenic and biocompatible water - soluble polymer backbone such as poly(ethylene glycol ) or n-(2-hydroxypropyl)methacrylamide ( hpma ) copolymers,(6 ) ( b ) therapeutically active molecules , ( c ) linkers between the polymer backbone and the active molecule that are stable in blood circulation and release the free drug at target sites , and ( d ) a targeting moiety to mediate biomolecular recognition .
the main advantages of hpma copolymerdrug conjugates over their low molecular weight drugs ( reviewed in kopeek,(6 ) putnam and kopeek,(8 ) kopeek et al.,(5 ) duncan,(9 ) cuchelkar and kopeek,(10 ) pan and kopeek(11 ) ) include the following : ( 1 ) enhanced water solubility of poorly soluble or insoluble drugs with concomitant improvement of drug bioavailability ; ( 2 ) long - lasting circulation in the bloodstream ; ( 3 ) decreased nonspecific toxicity of the conjugated drug and immunogenicity of the targeting moiety;(14 ) ( 4 ) increased passive and/or active accumulation of the drug at the site of its action by the epr effect and/or by targeting , respectively ; ( 5 ) active uptake by fluid - phase pinocytosis ( nontargeted polymer - bound drug ) or receptor - mediated endocytosis ( targeted polymer bound drug ) ; ( 6 ) the potential to overcome efflux pump - mediated mechanism of drug resistance ; and ( 7 ) ability to deliver several active components with different properties to the same target site that enhance the specific activity of the main drug . the incorporation of targeting moieties , such as monoclonal antibody ( mab ) or antibody fragment , into hpma copolymerdrug conjugates results in specific delivery and enhancement of the amount of the polymeric conjugate being internalized by receptor - mediated endocytosis.(23 ) consequently , the intracellular concentration of the polymeric conjugates is enhanced with concomitant increase in antitumor activity.(17 ) moreover , the polymer modification of mab or antibody fragments reduces their immunogenicity and extends their circulating half - lives .
the use of antibody fragments provides a better control of the structure of hpma copolymer conjugates compared to full - length mab .
the unique sulfhydryl group near the c terminus of fab fragments has provided a convenient way for coupling to hpma copolymers containing maleimide groups and allow the antigen - binding site to be more approachable . to improve the therapeutic outcome and reduce the toxicity of anticancer agents , a novel concept of combining chemotherapy and photodynamic therapy ( pdt ) , using hpma copolymer bound drugs , was developed.(27 ) the in vivo studies on two cancer models , neuro 2a neuroblastoma induced in a / j mice(28 ) and human ovarian carcinoma heterotransplanted in nude mice , demonstrated that combination therapy with hpma copolymer - bound dox ( doxorubicin ) and hpma copolymer - bound mce6 ( mesochlorin e6 monoethylenediamine ) produced tumor cures which could not be obtained with either chemotherapy or pdt alone .
furthermore , significantly lower nonspecific toxicities were observed when compared to low molecular weight drugs .
previously , in vitro studies of the binary combination of free and hpma copolymer - bound sos [ 2,5-bis(5-hydroxymethyl-2-thienyl)furan , nsc 652287 ] , dox , and mce6 in the treatment of human a498 renal carcinoma cells using the median - effect method showed that these combinations displayed synergistic - to - additive effects , depending on the cytotoxic mechanisms of each agent.(31 ) in the present study , fab-targeted and nontargeted hpma copolymerdrug ( sos and mce6 ) conjugates for combination chemotherapy and pdt against human ovarian ovcar-3 carcinoma cells were synthesized .
its mechanism of action consists of disrupting the p53-hdm-2 ( human double minute-2 ) interaction , resulting in an increased p53 accumulation , thereby inducing cell cycle arrest and apoptosis . for pdt the second - generation synthetic photosensitizer , mce6 , was used .
photosensitizer molecules can be activated by specific wavelength of light and interact with molecular oxygen to produce reactive singlet oxygen , causing irreversible photodamage to cells resulting in cell death.(36 ) the antibody fab fragment was prepared from ov - tl16 antibody , which recognizes the oa-3 surface antigen , also known as cd47 or iap ( integrin - associated protein ) , overexpressed on most human ovarian carcinoma cells .
it was hypothesized that a combination of these agents may produce synergistic effects and has higher efficiency than each agent alone .
accordingly , the efficiency of free , nontargeted , and fab fragment - targeted hpma copolymer - bound sos and mce6 against ovcar-3 cells as single agents and in combination was evaluated .
the combination index ( ci ) analysis was used to quantify the synergism , antagonism , and additive effects of drug combinations .
sos was kindly supplied by the drug synthesis and chemistry branch , developmental therapeutics program , division of cancer treatment and diagnosis , national cancer institute .
all other chemicals were purchased from sigma chemical co. ( st . louis , mo ) .
cells were cultured in rpmi 1640 medium ( sigma ) containing 10 g / ml insulin ( sigma ) supplemented with 10% fetal bovine serum ( hyclone laboratories , logan , ut ) , at 37 c in a humidified atmosphere of 5% co2 ( v / v ) .
the ov - tl16 antibody was produced as described previously.(44 ) briefly , the ov - tl16 antibody was produced by in vitro cartridge bioreactor ( cellulosic - mps , spectrum laboratories , rancho dominguez , ca ) culture of ov - tl16 hybridoma cells with serum free hybridoma medium ( gibco life sciences , carlsbad , ca ) .
the antibody was purified by applying the supernatant of cell suspension harvested from bioreactor to a protein g sepharose 4 fast flow column ( pharmacia , piscataway , nj ) , equilibrated with binding buffer ( 0.01 m na2hpo4 , 0.15 m nacl , 0.01 m edta ph 7.2 ) .
the antibody was dialyzed ( mol wt cutoff 68 kda ) against phosphate buffered saline ( pbs ) overnight at 4 c .
the antibody fab fragment was prepared freshly as described previously . the ov - tl16 antibody in 0.1 m citric buffer ph 4.0 was digested by 10% ( w / w ) pepsin ( sigma ) for 2.5 h at 37 c to give f(ab)2 .
the f(ab)2 was reduced to fab with 20 mm cysteine ( sigma ) in 20 mm tris buffer ph 8.5 for 1 h at 37 c .
p - gflg - mce6 was prepared as described previously.(28 ) briefly , the conjugate was synthesized using a polymer analogous reaction in two steps .
first , the polymer precursor ( p - gflg - onp ) was prepared by radical precipitation copolymerization of hpma and n - methacryloylglycylphenylalanylleucylglycine p - nitrophenyl ester ( ma - gflg - onp ) .
second , mce6 was bound to p - gflg - onp by aminolysis of reactive onp groups in n , n - dimethylformamide ( dmf ) .
the reaction solution was precipitated into a mixture of acetone : ether ( 3:2 ( v / v ) ) .
the dried precipitate was dissolved in methanol and purified on a sephadex lh-20 column with methanol/0.5% acetic acid as the elution solvent .
the product was dissolved in deionized ( di ) water , dialyzed overnight and lyophilized .
the structure and characterization of nontargeted polymer conjugates are summarized in scheme 1a and table 1 . determined by uv spectrophotometry in methanol : 395 = 158000 m cm for mce6 , and 358 = 33000 m cm for sos .
apparent molecular weight ( mw ) of polymers was estimated by size exclusion chromatography using akta / fplc ( pharmacia ) system equipped with a superose 6 column , calibrated with polyhpma fractions .
pbs buffer ph 7.3 + 30% ( v ) acetonitrile and 0.1 m acetate buffer ph 5.5 + 30% ( v ) acetonitrile were used for polymer conjugates containing mce6 and polymer conjugates containing sos , respectively .
determined by ninhydrin assay . determined by 5-((2-(and-3)-s-(acetylmercapto)succinoyl)amino)fluorescein assay ( samsa assay , molecular probes ) . determined by spectrophotometric determination of fitc ( 497 = 73 000 m cm in 0.1 m sodium borate buffer ) .
fluorescently labeled targeted conjugates were prepared by reacting p-(gflg - mce6)-fab and p- ( gflg - sos)-fab ( table 2 ) with 5-sfx .
p - gflg - sos ( scheme 1a ) was synthesized by binding of sos to the p - gflg - onp polymer precursor via an ester linkage.(31 ) briefly , p - gflg - onp was dissolved in dmf and mixed with dmf solution of sos .
4-dimethylaminopyridine ( dmap ) was added , and the reaction was allowed to proceed for 72 h in the dark at room temperature .
the product was isolated , after reduction of volume , by precipitation into a mixture of acetone : ether ( 3:1 ( v / v ) ) .
the precipitate was dissolved in methanol and applied to a sephadex lh-20 column with methanol as the mobile phase .
first , a polymerizable derivative of mce6 , n - methacryloylglycylphenylalanylleucylglycine mce6 ( ma - gflg - mce6),(25 ) was synthesized by reacting ma - gflg - onp ( 60 mg , 0.103 mmol ) with mce6 ( 63.5 mg , 0.093 mmol ) in dmf ( 2 ml ) .
the reaction solution was stirred at room temperature in the dark for 2 h. n , n-diisopropylethylamine ( dipea ; 18 l , 0.103 mmol ) was added and stirring continued overnight . 1-amino-2-propanol
( 8 l , 0.103 mmol ) and a small amount of tert - octylpyrocatechol were added and dmf was removed under reduced pressure .
the residue was isolated using a sephadex lh-20 column with acetone : methanol : acetic acid ( 2:1:0.1 ) as the mobile phase .
the product fraction was evaporated to dryness , washed with ether , collected by filtration , and dried under vacuum .
the molecular weight ( mw ) of ma - gflg - mce6 was 1083.6 da as determined by electrospray ionization mass spectrometry ( esi - ms ) .
second , the polymeric precursor p-(gflg - mce6)-nh2 was prepared by radical copolymerization of hpma(47 ) ( 107.6 mg , 0.751 mmol ) , n-(3-aminopropyl)methacrylamide hydrochloride ( apma , 16.27 mg , 0.091 mmol ; polysciences , warrington , pa ) , and ma - gflg - mce6 ( 76.9 mg , 0.068 mmol ) in methanol ( 1.8 ml ) at 50 c for 48 h , using 2,2-azobisisobutyronitrile ( aibn ; 19.27 mg ) as the initiator .
the polymerization mixture contained 12.5 wt % of monomers and 1.2 wt % of aibn .
the molar ratio of hpma : apma : ma - gflg - mce6 was 82.5:10:7.5 .
the residue was precipitated in a mixture of acetone : ether ( 1:2 ( v / v ) ) .
the precipitate was dissolved in water , dialyzed ( mol wt cutoff 68 kda ) against di water and lyophilized .
third , polymeric precursor p-(gflg - mce6)-mal was prepared by reacting p-(gflg - mce6)-nh2 ( 55 mg , 0.026 mmol nh2 group ) with succinimidyl trans-4-(maleimidomethyl)cyclohexane-1-carboxylate ( smcc ; 17.36 mg , 0.052 mmol ) ( soltec ventures , beverly , ma ) and dipea ( 13 l , 0.078 mmol ) . the mole ratio of nh2:smcc : dipea was 1:2:3 .
p-(gflg - mce6)-nh2 was dissolved in 0.3 ml of dmf and smcc in 0.9 ml of dmf .
the residue was dissolved in a small volume of methanol and purified on a sephadex lh-20 column eluted with a mixture of methanol and 0.1% acetic acid .
the copolymer band was collected , evaporated and precipitated into a mixture of acetone : ether ( 1:1 ) .
first , ma - gflg - onp ( 278.6 mg , 0.480 mmol ) and sos ( 200 mg , 0.680 mmol ) were dissolved in tetrahydrofuran ( thf ; 15 ml ) .
the reaction mixture was stirred at room temperature in the dark for 72 h. the mixture was concentrated under reduced pressure .
the excess free drug was recovered by elution with acetone : hexane ( 2:1 ) , and the fractions were collected by eluting with acetone and then with methanol .
the mw of ma - gflg - sos was 734.2 da as determined by esi - ms .
second , polymeric precursor , p-(gflg - sos)-nh2 , was synthesized by copolymerization of hpma ( 141.3 mg , 0.987 mmol ) , apma ( 14.2 mg , 0.079 mmol ) , and ma - gflg - sos ( 50 mg , 0.068 mmol ) in dmf ( 1 ml ) and acetone ( 0.5 ml ) at 50 c for 48 h , using aibn ( 19.7 mg ) as the initiator . the molar ratio of hpma : apma : ma - gflg - sos was 87:7:6 .
third , polymeric precursor p-(gflg - sos)-mal was prepared by using the same procedure as for p-(gflg - mce6)-mal .
p-(gflg - sos)-nh2 ( 145 mg , 0.040 mmol nh2 groups ) and smcc ( 26.41 mg , 0.079 mmol ) were dissolved separately in dmf ( 2.5 ml and 0.5 ml , respectively ) .
the product was applied on a sephadex lh-20 column and eluted with methanol without acetic acid .
the product was precipitated and filtered off , with a yield of 130 mg ( 76% ) .
the targeted conjugates were prepared by dissolving p-(gflg - mce6)-mal or p-(gflg - sos)-mal precursor in 20 mm mes buffer ph 6.5 and reacting with freshly prepared fab fragment ( polymer : fab weight ratio = 1:2 ) overnight in the dark at 4 c .
the product was purified on a deae sepharose fast flow ion exchange column ( pharmacia ) , eluted using 20 mm bis - tris buffer ph 6.5 with a gradient nacl concentration of 00.5 m. the fraction corresponding to conjugate was confirmed by size exclusion chromatography using superose 6 ( hr 10/30 ) column .
the structure and composition of polymer conjugates are summarized in scheme 1b and table 2 .
mce6 and sos contents determined by uv spectrophotometry in methanol : 395 = 158000 m cm for mce6 , 358 = 33000 m cm for sos .
calculated from the composition of polymer ( molecular ratio of drug , polymer , and fab ) .
6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester ( 5-sfx ; 0.1 mg , 0.170 mol ) was dissolved in dimethylsulfoxide ( 25 l ) and di water ( 100 l ) .
the polymer precursor p-(gflg - mce6)-nh2 or p-(gflg - sos)-nh2 ( 3 mg , 1 mol of nh2 ) was dissolved in di water ( 300 l ) .
the mixture was stirred at room temperature in the dark for 1 h. saturated na2hpo4 ( 20 l ) was added to stop the reaction .
sos was dissolved in pbs containing cyclodextrin ( 5% ( w / v ) cyclodextrin in pbs/1 mg of sos ) to enhance the solubility of sos.(48 ) p - gflg - sos , p-(gflg - mce6)-fab , and p-(gflg - sos)-fab were prepared in pbs .
other samples ( mce6 and p - gflg - mce6 ) were prepared in di water .
all stock solutions were freshly prepared and gradually diluted with rpmi 1640 culture medium before use .
fifty thousand ovcar-3 cells were subcultured into an eight - chamber slide and incubated for 2 days at 37 c in a humidified atmosphere of 5% co2 .
the cells in each chamber were exposed to fluorescein - labeled copolymer conjugates ( at 20 m fitc equivalent ) at 37 c for 1 h in the dark .
cells were fixed with 2% paraformaldehyde for 20 min at room temperature and washed twice with pbs .
the chamber slide was covered with a cover slide utilizing antifade reagent ( molecular probes ) .
the cell internalization of fluorescently labeled hpma copolymer conjugates was imaged using a zeiss ( thornwood , ny ) lsm 510 confocal imaging system .
ovcar-3 cells ( 75,000 cells / well ) were seeded into a 24-well plate and incubated for 24 h at 37 c in a humidified atmosphere of 5% co2 .
cells in each well were exposed to the fluorescein - labeled copolymer conjugates ( at 20 m fitc equivalent ) at 37 c for 1 h in the dark .
the cell monolayer was rinsed twice with ice - cold pbs and detached from the well surface by incubation with tryple express ( gibco ) for 2 min .
the cells were suspended with ice - cold pbs containing 0.2% fbs , maintained in suspension on ice in the dark and processed for flow cytometry utilizing facscan instrument ( becton dickinson ) .
control cells were not exposed to the sample to assess the endogenous fluorescence of the cells .
additional experiments were performed at 0 c ( on ice ) to demonstrate the surface binding of targeted conjugates .
ovcar-3 cells , growth medium and the fluorescein - labeled conjugates ( at 10 m fitc equivalent ) were precooled on ice before experiments .
the cells were exposed to conjugates for 2 h , then the medium containing conjugates was discarded , and cells were washed extensively with cold pbs and processed for flow cytometry as described above . the drug concentration that inhibited cell growth by 50% compared with control cells ( ic50 ) was determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay.(49 ) cells were seeded in 96-well flat bottom microplates at a density of 10,000 cells / well in 200 l of rpmi 1640 medium and allowed to grow for 30 h. the cells were then exposed to various concentrations of each free drug alone ( mce6 and sos ) , each nontargeted copolymer conjugate ( p - gflg - mce6 and p - gflg - sos ) , each targeted copolymer conjugate [ p-(gflg - mce6)-fab and p-(gflg - sos)-fab ] , or their sequential combinations ( n = 6 in single experiment ) .
the different treatment protocols are shown in scheme 3 . during the cells exposure to drug(s ) , they were incubated at 37 c in a humidified atmosphere of 5% co2 and in the dark condition . after 4 days of exposure for sos and its conjugates or 1 day of exposure for mce6 and its conjugates ,
the drugs were removed , cells were washed with warm pbs and the medium ( 300 l ) was replaced . for cell growth inhibition studies using mce6 , p - gflg - mce6 , or p-(gflg - mce6)-fab ( alone or in combinations ) ,
the cells were irradiated with three tungsten halogen lamps through a 650 nm band - pass filter at 3.0 mw / cm for 30 min . after an additional 1 day or 4 days in culture for sos and its conjugates or for mce6 and its conjugates , respectively , medium was removed and replaced with 100 l of fresh medium and 10 l of sterile - filtered mtt solution ( 5 mg / ml in pbs ) . after incubating for 24 h , 150 l of 20%
( w / v ) sodium dodecyl sulfate in water was added to each well and incubated overnight .
the following day , the absorbance of each well was read at 570 nm with a reference wavelength at 630 nm .
untreated cells served as a 100% cell viability control , and the media served as background reference .
growth inhibition was expressed as the growth of drug - treated cells related to that of untreated control cells .
( a ) cells were treated with single agents : different concentrations of mce6 , sos , or their conjugates .
( b ) cells were treated with sequential combinations of sos or hpma copolymersos conjugates , followed by treatment with mce6 or hpma copolymermce6 conjugates . in combination treatment studies ,
ovcar-3 cells were treated with a dose range of sos for 4 days followed by a dose range of mce6 for 1 day , and irradiated for 30 min ( n = 6 in single experiment ) , as shown in scheme 3 .
after each step the drug was removed and the cells were washed with warm pbs . drug interaction and ci were determined using median - effect analysis according to the method of chou and talalay .
the median - effect equation describes doseeffect relationships , which is expressed by fa / fu = ( d / dm ) or log(fa / fu ) = m log(d )  m log(dm ) where fa and fu are the fraction affected [ 1  ( absorbance of treatment well  average of absorbance of blanks)/(average of absorbance of untreated cell wells  average of absorbance of blanks ) ] and unaffected ( fu = 1  fa ) by the dose or concentration d , dm is the median - effect dose ( ic50 ) that inhibits the cell growth by 50% , and m is the coefficient signifying the shape of the doseeffect relationship .
based on the logarithmic conversion , the plot of x = log(d ) versus y = log(fa / fu ) is called the median - effect plot and dm is calculated from the antilog of the x - intercept .
the ci describes the interaction between two drugs and quantitates the synergism , antagonism or additive effects .
the ci is determined by the equation ci = [ ( d)1/(dx)1 ] + [ ( d)2/(dx)2 ] where ( dx)1 and ( dx)2 are the doses of drug 1 alone and drug 2 alone that inhibit the cell growth x% , respectively .
( d)1 and ( d)2 are for doses in combination that also inhibit x% . the ci values were calculated for different values of fa and plotting the ci values as a function of fa values , using compusyn software ( combosyn inc . ,
ci < 1 , = 1 , and > 1 indicate synergism , additivity , and antagonism , respectively .
all mean values are presented as means  standard deviation ( n = 6 in a single experiment ) .
the structures of hpma copolymer conjugates , p - gflg - mce6 , p - gflg - sos , p-(gflg - mce6)-fab , and p-(gflg - sos)-fab , are shown in scheme 1 .
the drugs , mce6 and sos , were bound to the hpma copolymer backbone via a gflg spacer , stable in the bloodstream , but susceptible to enzymatically catalyzed hydrolysis in the lysosomal compartment of the cells . for fab attachment , the amino groups of apma monomer units in hpma copolymer precursors were first converted to maleimido groups by reaction with a heterobifunctional agent , smcc ( scheme 2 ) , followed by attachment of fab via thioether bonds . for some experiments ,
the characteristics of hpma copolymer precursors , nontargeted hpma copolymer conjugates , fab-targeted hpma copolymer conjugates , and fluorescently labeled conjugates are summarized in tables 1 and 2 .
p - gflg - mce6 , p - gflg - sos , p-(gflg - mce6)-fab , and p-(gflg - sos)-fab conjugates contained 2.9 , 3.4 , 2.0 , and 5.0 drug molecules per macromolecule , respectively .
p-(gflg - mce6)-fab and p-(gflg - sos)-fab had drug : polymer : fab molecular ratios of approximately 2:1:1 and 5:1:1 , respectively .
the mw of fab-targeted copolymer conjugates were 2.2 to 2.8 times higher than those of nontargeted conjugates . unlike low molecular weight drugs that enter cells by diffusion through the plasma membrane
several basic internalization mechanisms , clathrin - mediated endocytosis , caveolae - mediated endocytosis , clathrin- and caveolin - independent endocytosis , and macropinocytosis have been identified . to a greater or lesser extent ,
two or more distinct mechanisms coexist when a single cell type internalizes macromoleculedrug conjugates.(55 ) studies on the subcellular fate of hpma copolymerdrug conjugates demonstrated that the conjugates are lysosomotropic and will accumulate in the lysosomal compartment of the cell .
tijerina et al . , using subcellular fractionation , determined the localization of a considerable fraction of hpma copolymermce6 conjugates in the lysosomal compartment of human ovarian carcinoma a2780 cells.(56 ) omelyanenko et al .
used ph dependent fluorescence of fitc to display the lysosomotropism of fitc - labeled hpma copolymers containing n - acylated galactosamine in hepg2 hepatocarcinoma cells.(57 ) recently , the uptake mechanism of hpma copolymerdox conjugate in human ovarian carcinoma ovcar-3 cells was studied by confocal fluorescence microscopy and by colocalization experiments with substrates specific for a particular internalization mechanism .
the results suggested that the hpma copolymerdox conjugate is internalized via both clathrin- and caveolae - mediated endocytosis.(55 ) the biorecognition and cellular uptake of p-(gflg - mce6)-(fab-fitc ) and p-(gflg - sos)-(fab-fitc ) was studied using confocal microscopy ( figure 1 ) and flow cytometry ( figures 2 and 3 ) .
after a 1 h exposure of ovcar-3 cells to hpma copolymer conjugates at 37 c , the intracellular concentrations of targeted polymer conjugates containing mce6 and sos were significantly higher when compared to nontargeted conjugates . both confocal microscopy images and flow cytometry profiles displayed very similar results .
confocal image of fixed ovcar-3 cells incubated with fluorescein - labeled hpma copolymer conjugates in rpmi 1640 culture medium for 1 h in the dark .
( a ) p-(gflg - mce6)-fitc , ( b ) p-(gflg - sos)-fitc , ( c ) p-(gflg - mce6)-(fab-fitc ) , and ( d ) p-(gflg - sos)-(fab-fitc ) .
flow cytometry profiles of ovcar-3 cells incubated with fluorescein - labeled hpma copolymer conjugates in rpmi 1640 culture medium for 1 h in the dark .
( a ) control cells , ( b ) p-(gflg - mce6)-fitc , ( c ) p-(gflg - sos)-fitc , ( d ) p-(gflg - sos)-(fab-fitc ) , ( e ) p-(gflg - mce6)-(fab-fitc ) .
flow cytometry profiles of ovcar-3 cells incubated with fluorescein - labeled hpma copolymer conjugates in rpmi 1640 culture medium for 2 h in the dark at 0 c .
( a ) control cells , ( b ) p-(gflg - mce6)-fitc , ( c ) p-(gflg - sos)-fitc , ( d ) p-(gflg - sos)-(fab-fitc ) , ( e ) p-(gflg - mce6)-(fab-fitc ) . however , at 37 c two processes , biorecognition at surface and internalization by endocytosis , are operative . to clearly demonstrate the biorecognition of targeted conjugates by the cd47 antigen , ovcar-3 cells were exposed to conjugates at 0 c .
flow cytometry data at 0 c , at conditions that suppress endocytosis , demonstrated the biorecognition of p-(gflg - mce6)-fab and p-(gflg - sos)-fab conjugates by ovcar-3 cells ( figure 3 ) .
after incubation of ovcar-3 cells with targeted and nontargeted conjugates for 2 h , no increase in fluorescent intensity ( when compared to controls not exposed to any conjugates ) in cells incubated with p - gflg - mce6 and p - gflg - sos was observed .
in contrast , higher fluorescent intensities were detected in cells incubated with targeted ( fab containing ) conjugates p-(gflg - mce6)-fab and p-(gflg - sos)-fab. presumably , the fluorescent signals were derived from membrane associated conjugates as a result of fabcd47 interactions .
these results indicated the biorecognition of hpma copolymer conjugates containing the fab antibody fragment by ovcar-3 cells .
the results of confocal microscopy are consistent with the internalization of targeted hpma copolymer conjugates via receptor - mediated endocytosis , and of the nontargeted conjugates , containing hydrophobic drugs , by fluid - phase pinocytosis and adsorptive pinocytosis , concurrently .
the data are consistent with our previous results on the determination of binding constants of ov - tl16 antibody targeted hpma copolymers toward ovcar-3 cells.(58 ) the affinity constant , ka , of free antibody was 8  10 m , whereas the ka for the p-(gg - mce6)-fab was 3  10 m. the minor decrease in the affinity may be a result of chemical modification and/or steric hindrance of the polymer chain upon the formation of the antibodyantigen complex .
the growth inhibitory effects of mce6 , sos , p - gflg - mce6 , p - gflg - sos , p-(gflg - mce6)-fab , and p-(gflg - sos)-fab as single agents on ovcar-3 cells were evaluated after drug exposure using the mtt assay .
the ic50 values for the free drugs , nontargeted and targeted hpma copolymer conjugates are shown in table 3 . the 1-day mce6 exposure and 4-day sos exposure
have been selected based on preliminary experiments on the relationship between exposure time and cell inhibition effect ( data not shown ) .
after 1-day mce6 exposure and 4-day sos exposure , the cells were more susceptible to mce6 than sos about 2 times .
it is interesting to note the enormous difference in the activity of sos toward ovcar-3 cells and toward the human a498 renal cell line.(31 ) our previous study on human renal a498 cells demonstrated that free sos and p - gflg - sos conjugates were effective individually and in combination with free dox and p - gflg - dox or free mce6 and p - gflg- mce6 , respectively.(31 ) the ic50 values for mce6 toward both cell types ( ovcar-3 and a498(31 ) ) were similar .
however , sos was very effective toward a498 cells ( ic50 = 3 nm),(31 ) but its activity toward ovcar-3 was about 670 times lower .
these data are consistent with the results of the nci anticancer drug screen consisting of a panel of 60 human cancer cell lines .
rivera et al . showed that after a 2-day continuous exposure to sos , ovcar-3 cells were less sensitive to sos than the a498 cells.(35 ) these results reflect the different p53 status in these cell lines .
the inhibitory effect of sos is mediated through p53 ; the disruption of the p53-hdm-2 interactions results in increasing p53 accumulation in tumor cells.(33 ) it was demonstrated that the p53 status of a498 cells and ovcar-3 cells is wild - type and mutant , respectively.(59 ) however , the study of the p53 gene in human ovarian carcinoma cell lines by yaginuma and westphal showed that the wild - type p53 protein was detectable in ovcar-3 cells by immunoprecipitation analysis.(60 ) these reports indicated that ovcar-3 cells can be inhibited by higher concentrations of sos , but have a considerably lower sensitivity when compared to the a498 cell line .
ic50 and dm values are the means  sem ( n = 6 in single experiment ) .
the ic50 doses of nontargeted conjugates , p - gflg - mce6 and p - gflg - sos , were higher than those of free mce6 and sos , respectively .
these results reflect the different mechanisms of cell entry of free drugs vs copolymer conjugates.(61 ) in contrast , the targeted p-(gflg - mce6)-fab conjugate was 2 and 9 times more effective than mce6 and p - gflg - mce6 , respectively .
the cytotoxicity data with mce6 conjugates were in agreement with biorecognition data and internalization mechanisms ( figures 1 , 2 and 3 ) .
there was a discrepancy in data obtained for p-(gflg - sos)-fab. this conjugate showed faster internalization ( figure 1 ) and moderately better biorecognition ( figure 2 ) than p - gflg - sos ; however , it possessed a slightly weaker inhibitory effect than p - gflg - sos ( table 3 ) .
after long exposure times the intracellular drug content of targeted and nontargeted conjugates may be similar.(62 ) furthermore , the efficacies of sos and its conjugates might be limited by the problem of sos trafficking to the subcellular compartments where p53 is mainly located , such as nucleus and mitochondria.(63 ) subcellular targeting is very important for some active agents and macromolecular therapeutics that have to be transported to their assigned cell organelles .
once those molecules are delivered to the cytosol , various approaches to locate drugs in a particular subcellular organelle have been performed , such as the use of nuclear localization peptides , cell - penetrating peptides,(65 ) lipophilic cationic moieties,(2 ) and mitochondrial localization agents.(66 ) the oa-3 surface antigen ( cd47 or iap ) was chosen as delivery target in this study because it is overexpressed in about 90% of the ovarian tumors and only weakly expressed in normal tissue.(39 ) a panel of anti - cd47 mabs used in the study of mawby et al .
, such as nbts / bric-125 ( bric-125 ) , nbts / bric-126 ( bric-126 ) and nbts / bric-154 ( bric-154 ) , showed an extremely broad tissue distribution , not only in ovarian carcinomas studied , but also in all hematopoietic cells , mesenchyme and epithelia at multiple sites.(38 ) on the contrary , the tissue distribution of i - labeled ov - tl16 mab in ovcar-3 bearing nude mice 48 h after iv injection studied by boerman et al .
demonstrated that i - labeled ov - tl16 mab possessed tumor / nontumor ratio of about 315.(39 ) furthermore , the mab ov - tl3 used by campbell et al . to define oa3 showed little or no reactivity with normal tissues but reacted with most ovarian carcinomas.(40 ) slobbe et al .
studied the structure of ov - tl3 and ov - tl16 antibodies and reported that both mabs are able to bind to same epitopic regions on the ovarian carcinoma membrane antigen oa3 , although structurally different in their vh regions.(67 ) the basis for the difference between mabs used in the study of mawby et al . and boerman et al . or campbell et al .
one possibility is that an unusual amino acid sequence in the oa3 isoforms is expressed in ovarian cancer cells , and that ov - tl3 and ov - tl16 recognize epitopes in this sequence.(38 ) the investigation of possible synergistic , additive , or antagonistic effects of sequential combinations of sos+mce6 , p - gflg - sos+p - gflg - mce6 , or p-(gflg - sos)-fab+p-(gflg - mce6)-fab against the ovarian carcinoma ovcar-3 cell line was performed in vitro by exposing cells to sos or its conjugates for 4 days , followed by exposure of cells to mce6 or its conjugates for 1 day , and finally , a 30 min irradiation .
this sequential combination was chosen because the optimal exposure times of sos / p - gflg - sos / p-(gflg - sos)-fab and mce6/p - gflg - mce6/p-(gflg - mce6)-fab were different ( 4 days for sos and 1 day for mce6 ) , as mentioned above .
the dose ratios of each combination ( table 4 ) were based on their respective ic50 concentrations from table 3 as a series of 2-fold dilutions from 4 to 0.03125 times ic50 .
figure 4 shows the composite doseresponse curves and median - effect plots of ovcar-3 cells , indicating the antiproliferative effects of single agents and their combinations .
the doseresponse curves for combined treatment were obtained by plotting % cell viability ( y ) vs the combined dose of two single agents ( x ) .
the median - effect plots of single agents and combinations were derived from the linear part of doseresponse curves .
the dose ratio and dm values of the combination treatments are shown in table 4 .
the ic50 dose of each drug in combinations was significantly lower than those of each drug as single agents ( compare tables 3 and 4 ) .
these results clearly indicate that all of the combination treatments were effective against ovcar-3 cells .
dm values are the means  sem ( n = 6 in single experiment ) .
doses of drug a and drug b were calculated approximately from the dm of each combination and dose ratio .
dose response curves and median - effect plots of ovcar-3 cells treated with mce6 , sos , p - gflg - mce6 , p - gflg - sos , p-(gflg - mce6)-fab , and p-(gflg - sos)-fab as single agents and sequential combinations at constant ratios of their respective ic50 concentrations .
the ci analysis was used to assess the drugdrug interaction of the sequential combinations of free drugs , nontargeted and targeted copolymer conjugates toward ovcar-3 cells in vitro . in the ci analysis ,
values of ci < 1 , ci = 1 , and ci > 1 indicate synergy , additivity , and antagonism , respectively .
figure 5 shows the combination index plots ( faci plots ) over all inhibition effect levels ( fa = 0.050.95 or 595% of inhibition effect ) in ovcar-3 cells .
the sequential combinations of sos+mce6 and p - gflg - sos+p - gflg - mce6 yield ci values lower than 1 over the entire range of cytotoxicity , indicating very strong synergistic to synergistic effects .
the p-(gflg - sos)-fab+p-(gflg - mce6)-fab combination also displayed a strong synergism for fa values up to about 0.85 , but showed synergistic effect and nearly additive effect at fa = 0.9 and 0.95 , respectively .
chemotherapeutic drugs and their hpma copolymer conjugates were gradually diluted at the ratio of their ic50 values as a series of 2-fold dilutions from 4 to 0.03125 times ic50 and ovcar-3 cells exposed to drugs
ci < 1 , = 1 and > 1 indicate synergism , additive effect , and antagonism , respectively .
the vertical bars indicate the 95% confidence intervals based on sequential deletion analysis ( sda ) and can be generated by using compusyn software .
the drug interactions may depend on the differences of drugs in the combination , such as physicochemical properties , the mechanisms of action , and the drug exposure schedules .
sos and mce6 are hydrophobic low molecular weight molecules , but both have different mechanisms and sites of action .
sos acts on p53 and dna while mce6 can cause damage to biological molecules by generation of reactive oxygen species . in our previous study , we compared the simultaneous combination of p - gflg - sos+p - gflg - mce6 to sos+mce6 against the human renal a498 carcinoma cell line .
after 16 h cell exposure to the combinations , both combinations displayed synergism for fa up to 0.8 , but showed slight antagonism and near additivity at fa = 0.95.(31 ) many researchers have studied the antitumor activities following different drug exposure schedules .
for example , the simultaneous and sequential exposures of irofulven with oxaliplatin or cisplatin against human breast , colon , and ovarian cancer cell lines showed that the sequence oxaliplatin followed by irofulven displayed better synergistic effect than the other schedules.(68 )
combination chemotherapy and pdt with free sos and mce6 , their nontargeted and fab-targeted hpma copolymer conjugates in human ovarian carcinoma ovcar-3 cells was evaluated .
sequential combinations of these therapeutics produced very strong synergism to nearly additivity in the treatment of ovcar-3 cells .
the synergistic effects ranked in the order p - gflg - sos+p - gflg - mce6 > sos+mce6 > p-(gflg - sos)-fab+p-(gflg - mce6)-fab. these data support continued in vivo investigations of sos and mce6 combinations to determine the antitumor activity for the treatment of ovarian cancer .
aibn , 2,2-azobisisobutyronitrile ; apma , n-(3-aminopropyl)methacrylamide hydrochloride ; ci , combination index ; dm , median - effect dose ; di , deionized ; dipea , n , n-diisopropylethylamine ; dmap , 4-dimethylaminopyridine ; dmf , n , n - dimethylformamide ; dox , doxorubicin ; epr , enhanced permeability and retention ; gflg , glycylphenylalanylleucylglycine ; hpma , n-(2-hydroxypropyl)methacrylamide ; ic50 , concentration that inhibited cell growth by 50% as compared with control cell growth ; ma , methacryloyl ; mab , monoclonal antibody ; mw , weight average molecular weight ; mce6 , mesochlorin e6 monoethylenediamine disodium salt ; mtt assay , modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay ; onp , p - nitrophenoxy ; p , hpma copolymer backbone ; pdt , photodynamic therapy ; p - gflg - mce6 , hpma copolymermce6 conjugate ; p-(gflg - mce6)-fab , antibody fab fragment targeted hpma copolymermce6 conjugate ; p - gflg - onp , hpma copolymer precursor containing reactive p - nitrophenyl ester groups at side chain termini ; p - gflg - sos , hpma copolymersos conjugate ; p-(gflg - sos)-fab , antibody fab fragment targeted hpma copolymersos conjugate ; 5-sfx , 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester ; smcc , succinimidyl trans-4-(maleimidomethyl)cyclohexane-1-carboxylate ; sos , 2,5-bis(5-hydroxymethyl-2-thienyl)furan ; thf , tetrahydrofuran .
 the biological activities of sequential combinations of anticancer drugs , sos thiophene ( sos ) and mesochlorin e6 monoethylenediamine ( mce6 ) , in the form of free drugs , nontargeted n-(2-hydroxypropyl)methacrylamide ( hpma ) copolymerdrug conjugates , p - gflg - mce6 and p - gflg - sos ( p is the hpma copolymer backbone and gflg is the glycylphenylalanylleucylglycine spacer ) , and fab-targeted hpma copolymerdrug conjugates , p-(gflg - mce6)-fab and p-(gflg - sos)-fab ( fab from ov - tl16 antibodies complementary to cd47 ) , were evaluated against human ovarian carcinoma ovcar-3 cells . 
 mce6 , sos , p - gflg - mce6 , p - gflg - sos , p-(gflg - mce6)-fab , and p-(gflg - sos)-fab , when used as single agents or in binary combination , exhibited cytotoxic activities against ovcar-3 cells , as determined using a modified mtt assay . 
 the binding and internalization of p-(gflg - mce6)-fab and p-(gflg - sos)-fab by ovcar-3 cells were visualized by confocal microscopy and flow cytometry . 
 the results confirmed an enhanced biorecognition by ovcar-3 cells of fab-targeted hpma copolymer conjugates over nontargeted conjugates . 
 the median - effect analysis and the determination of the combination index ( ci ) were used to describe the drug interaction and quantify the synergism , antagonism , or additivity in anticancer effects . 
 the sequential combinations of sos+mce6 and p - gflg - sos+p - gflg - mce6 displayed very strong synergism to synergism in the entire range of cell inhibition levels ( fa = 0.5  0.95 ) . 
 the p-(gflg - sos)-fab+p-(gflg - mce6)-fab exhibited a strong synergism for fa values up to about 0.85 , but showed synergistic effect and nearly additive effect at fa = 0.9 and 0.95 , respectively . 
 these observations support the continuation of in vivo investigations of these conjugates for the treatment of ovarian cancer . 
Introduction
Materials and Methods
Results and Discussion
Conclusion
Abbreviations Used