the 
 combination of small - molecule drugs with therapeutic nucleic 
 acids has emerged as a promising strategy in cancer treatment . because of the heterogeneity of cancer and the 
 involvement of multiple genetic changes during tumorigenesis , such 
 combination treatments offer great advantages in targeting multiple 
 disease pathways and overcoming adaptive drug resistance .
however , 
 this potentially powerful combination modality is greatly hampered 
 by a lack of desirable delivery systems that could accommodate various 
 therapeutic payloads .
it is especially 
 challenging to design delivery vectors for drug  nucleic acid 
 combinations , owing to the physicochemical differences between the 
 two agents . among the available systems ,
biodegradable polycations 
 represent promising delivery platforms suitable for delivery of nucleic 
 acid / drug combinations .
natural polyamines spermine 
 ( spm ) , spermidine ( spd ) , and their 
 diamine precursor putrescine ( put ) are ubiquitous alkylamines that 
 are essential for cell growth , differentiation , and survival .
polyamines are present inside cells at millimolar concentration 
 levels , but the majority exist in the bound form .
most 
 polyamines are found in polyamine  rna complexes , thus influencing 
 protein synthesis .
intracellular levels 
 of natural polyamines are strictly regulated by the polyamine transport 
 system and metabolic enzymes ( scheme 1a ) .
the rate - limiting enzymes in the polyamine biosynthesis 
 pathway include ornithine decarboxylase ( odc ) and s - adenosylmethionine decarboxylase ( adometdc ) .
the catabolism of natural 
 polyamines is mediated by spermine / spermidine n - acetyltransferase ( ssat ) , polyamine oxidase ( apao ) , and spermine 
 oxidase ( smo ) .
ssat and smo are highly inducible enzymes whose expression 
 is regulated by a variety of stimuli , including changes in polyamine 
 levels , cellular stress , dna damage , or exposure to various drugs .
polyamine depletion leads to inhibition of cell growth and 
 acceleration of aging , whereas increased 
 levels of polyamines are associated with hyper - proliferative diseases 
 such as cancer . in cancer ,
dysregulation of the 
 enzymes involved in the polyamine pathway results in accumulation 
 of polyamines , which promotes tumorigenesis and tumor progression . 

elevation of polyamine levels enhances the malignant potential of 
 cancer cells and decreases antitumor immunity .
alteration in the polyamine pathway is also associated 
 with poor prognosis of certain cancers .
thus , the polyamine pathway represents a promising target in cancer 
 chemotherapy and chemoprevention .
( a ) benspm acts 
 in multiple 
 ways to regulate polyamine pathways and leads to the depletion of 
 all three natural polyamines and growth inhibition of cancer cells . 

( b ) dss - ben condenses dna into polyplexes via electrostatic interactions . 
 upon cell uptake ,
the polyplexes are subjected to intracellular reduction 
 by gsh , followed by disassembly of the polyplexes and release of the 
 dna for cancer gene therapy .
simultaneously , the intracellular degradation 
 of dss - ben leads to release of benspm and inhibition of cancer cell 
 growth due to the effect of benspm on polyamine metabolism .
initial efforts to develop anticancer agents based 
 on the inhibition 
 of polyamine biosynthesis enzymes yielded limited clinical success . 

one of the most well - known inhibitors of polyamine biosynthesis is 
 difluoromethylornithine , an irreversible inhibitor of odc .
alternative research focused on developing polyamine 
 analogues that are capable of competing with natural polyamines for 
 transport , biosynthesis , and catabolism .
this line of research has 
 proven to be more successful , as illustrated in the example of benspm 
 ( scheme 1a ) .
it has shown promising antitumor activity against 
 a wide range of cancers , including melanoma , ovarian , breast , and 
 pancreatic cancers .
benspm induces ssat , downregulates odc and adometdc , and ultimately causes cell growth inhibition 
 and apoptosis .
although benspm did not 
 show a satisfactory clinical outcome as a single agent , recent studies demonstrated its potential when used in combination 
 with other chemotherapy drugs such as 5-fluorouracil and paclitaxel . in this study , we report the synthesis of biodegradable polycation 
 prodrugs based on benspm ( dss - ben ) .
the synthesized prodrugs utilize 
 bis(2-hydroxyethyl ) disulfide as a self - immolative linker to facilitate rapid intracellular degradation 
 and release of benspm from the polycations .
we report results of the 
 intracellular benspm release and the effect of dss - ben on polyamine 
 metabolism in cancer cells .
finally , we explore the ability of the 
 synthesized polycations to deliver dna and demonstrate a promising 
 enhancement in the anticancer activity of tnf-encoding plasmid 
 dna by the action of benspm from dss - ben .
1,1-carbonyldiimidazole 
 ( cdi ) , bis(2-hydroxyethyl ) disulfide ( bhed ) , spermidine ( spm ) trihydrochloride , 
 putrescine ( put ) dihydrochloride , and branched polyethylenimine ( pei , 
 25 kda ) were purchased from sigma - aldrich ( st . louis , mo ) .
dichloromethane 
 ( dcm ) ( 99.9% , extra dry , acroseal ) , tetrahydrofuran ( thf , 99.85% , 
 extra dry , acroseal ) , 5-sulfosalicylic acid dihydrate ( ssa ) , 1,7-diaminoheptane 
 ( dah ) , dansyl chloride ( 5-dimethylamino-1-naphthalenesulfonyl chloride , 
 98% ) , and 1,6-hexanediol were from acros organics ( fair lawn , nj ) .
l - proline was from alfa aesar ( ward hill , ma ) . spermine ( spm ) 
 was from mp biomedicals ( santa ana , ca ) .
plasmid dna containing a luciferase reporter gene ( gwiz - luc ) was 
 from aldevron ( fargo , nd ) , and human tumor necrosis factor ( tnf ) 
 plasmid dna was obtained from invivogen ( san diego , ca ) .
dulbecco s 
 modified eagle s medium ( dmem ) , eagle s minimum essential 
 medium ( emem ) , dulbecco s phosphate - buffered saline ( pbs ) , 
 fetal bovine serum ( fbs ) , l - glutamine , and penicillin 
all other reagents and chemicals were 
 obtained from fisher scientific or vwr international unless otherwise 
 stated .
all reactions were performed under anhydrous 
 conditions under nitrogen . to synthesize dss - ben , 403.2 mg of bhed 
 ( 2.6 mmol )
was dissolved in a mixture of 3.3 ml of dcm and 0.7 ml 
 of thf on ice .
then , a solution of cdi ( 887.5 mg , 5.5 mmol ) in 3.5 
 ml of dcm was added dropwise , and the reaction was kept on ice for 
 1 h. a solution of benspm ( 2.6 mmol , 640 mg ) in dcm was then added , 
 and the reaction was left to proceed for 18 h at 45 c .
the reaction 
 mixture was then allowed to cool to room temperature , and the final 
 product was precipitated in 25 ml of diethyl ether , followed by two 
 washes with 20 ml of diethyl ether .
the product was then dried under 
 vacuum and redissolved in 0.1 mm hcl , followed by extensive dialysis 
 ( mwco 3.5 kda ) against 0.1 mm hcl and then pure water before lyophilization . 

a total of 372 mg of dss - ben hydrochloride was obtained . to synthesize 
 dcc - ben ,
a solution of cdi ( 1.260 g , 7.77 mmol ) in 5 ml of dcm was 
 added dropwise into a solution of 1,6-hexanediol ( 437.3 mg , 3.7 mmol ) 
 in 15 ml of dcm and 1 ml of thf .
the reaction was kept on ice for 
 1 h , and 10 ml of benspm ( 904 mg , 3.7 mmol ) in dcm was added .
the 
 reaction mixture was then refluxed for 96 h before precipitation in 
 diethyl ether , extensive dialysis , and lyophilization following the 
 same procedure as that described for dss - ben .
h nmr spectra 
 of the polymers were recorded on 400 mhz bruker nmr spectrometer , 
 and chemical shifts (  ) are expressed in ppm .
the composition 
 of the polymers was determined by elemental analysis from n , s , h , 
 and cl content ( atlantic microlab , inc . , norcross , ga ) .
weight - average 
 molecular weight ( mw ) of the polymers 
 was determined by size - exclusion chromatography using an agilent technologies 
 1260 infinity equipped with an isocratic pump , degasser , variable 
 wavelength detector , thermostated column compartment , and autosampler 
 from agilent technologies , inc .
a wyatt minidwan 
 treos multiangle light scattering detector and optilab t - rex differential 
 refractometer ( wyatt technology , santa barbara , ca ) were used as detectors , 
 and astra 6.1 software was used for chromatographic data processing . 
 the refractive index increment ( dn / dc )
was determined experimentally as 0.1693 g / ml using a serial dilution 
 of dss - ben .
the analysis was conducted using single - pore aquagel columns 
 paa-202 and paa-203 from polyanalytik ( london , on , canada ) .
sodium 
 acetate buffer ( 0.3 m , ph 5.0 ) was used as the mobile phase at a flow 
 rate of 0.3 ml / min .
degradation 
 of dss - ben and release of benspm were evaluated using h nmr .
briefly , dss - ben or dcc - ben ( 10 mg ) was dissolved in a 0.9 
 ml mixture of 0.1 m phosphate buffered d2o and acetone - d6 ( 3:2 v / v ) , and the solution was purged with 
 argon for 10 min .
dithiothreitol ( dtt ) ( 15 mg , 0.097 mmol ) was then 
 added to the solution immediately before the start of nmr acquisition 
 at 25 c .
polymer degradation was determined from the relative 
 decrease of the integral intensity of the benspm methylene protons 
 next to the carbamate bond in the polymer ( 3.353.55 ppm in 
 dss - ben and 3.233.50 ppm in dcc - ben ) relative to the d2o solvent peak at 4.8 ppm .
human osteosarcoma 
 u2os cells were from thermo scientific ( waltham , ma ) and cultured 
 in dmem supplemented with 2 mm l - glutamine , 10% fbs , 1% pen  strep , 
 and 0.5 mg / ml g418 .
all cells were maintained in a 37 c cell 
 culture incubator with 5% co2 .
b16f10 and u2os cells were treated 
 with 2.5 g / ml benspm , 5.7 
 g / ml dss - ben , or 5.7 g / ml dcc - ben for 48 or 72 h. the 
 cells were harvested by trypsinization , and the amount of benspm and 
 natural polyamines ( spm , spd , put ) was determined as described previously .
briefly , harvested cells were homogenized in 
 5% ssa and centrifuged for 5 min at 12  000 g .
the supernatant containing the polyamines was functionalized with 
 dansyl chloride and purified with a bond - elut c18 column ( agilent 
 technologies , santa clara , ca ) .
the samples were then injected onto 
 an eclipse plus c18 column ( 4.6  150 mm , agilent technologies ) 
 using dah as the internal standard .
a 1260 infinity quaternary lc 
 system ( agilent technologies ) was used for the analysis using a gradient 
 of acetonitrile and phosphate buffer ( 10 mm , ph 4.4 ) as the eluent 
 system .
data were collected and analyzed using openlab cds chemstation 
 edition software ( agilent technologies ) .
the amount of benspm , spm , 
 spd , and put in the samples was quantified using standard curves constructed 
 with the corresponding standard compound .
relative polyamine depletion 
 ( % ) was calculated from polyamine concentrations found in treated 
 vs untreated cells .
expression of polyamine catabolic enzymes 
 smo and ssat in b16f10 and u2os cells was quantified using rt - pcr . 

cells were treated with 2.5 g / ml benspm , 5.7 g / ml dss - ben , 
 or 5.7 g / ml dcc - ben for 48 h. total rna ( 500 ng ) was isolated 
 using rneasy mini kit ( qiagen , valencia , ca ) and reverse - transcribed 
 to complementary dna ( cdna ) using quantitect reverse transcription 
 kit ( qiagen ) , and the relative amount of mrna was determined by rt - pcr 
 ( icycler iq real time pcr detection system , biorad , hercules , ca ) . 

gapdh primer assay and quantifast sybr green pcr kit ( qiagen ) were 
 used following the manufacturer s protocol .
the following primers 
 were used : murine smo ( forward 5-cacgtgattgtgaccgtttc ; 
 reverse 5-tgggtaggtgagggtacagtc ) ; murine ssat 
 ( forward 5-cgtccagccactgcctctg ; reverse 5-gcaagtactctttgtcaatcttg ) ; 
 human smo ( forward 5-cgcagacttacttccccggc ; reverse , 
 5-cgctcaattcctcaaccacg ) ; and human ssat ( forward , 
 5-atctaagccaggttgcaatga ; reverse , 5-gcactcctcactcctctgttg )
. relative expression of the mrna 
 of the enzymes was calculated from the threshold values ( ct ) of the target genes and the housekeeping gene gapdh .
the ability of dss - ben and dcc - ben to condense 
 plasmid dna was determined by an ethidium bromide ( etbr ) exclusion 
 assay by measuring the changes in etbr / dna fluorescence .
a total of 
 1 ml of dna solution ( 20 g / ml ) in 10 mm hepes buffer ( ph 7.4 ) 
 was mixed with etbr ( 1 g / ml ) , and the raw fluorescence was 
 set to 100% using a quantech fluorometer ( ex 540 nm / em 590 nm ) from 
 thermo scientific ( waltham , ma ) .
relative fluorescence ( % ) readings 
 were then recorded following the stepwise addition of a polycation 
 solution . the condensation curve for each polycation
dss - ben / dna and dcc - ben / dna polyplexes were formed by adding predetermined 
 volume of dss - ben or dcc - ben to a dna solution ( 20 g / ml in 
 10 mm hepes ph 7.4 ) to achieve the desired polymer / dna w / w ratio , 
 and the samples were mixed by vigorous vortexing for 10 s. polyplexes 
 were incubated at room temperature for 30 min prior to use .
hydrodynamic 
 diameter and zeta potential of the polyplexes were determined by dynamic 
 light scattering ( dls ) using a zen3600 zetasizer nano - zs ( malvern 
 instruments ltd . ,
the polyplexes were prepared at a w / w ratio 
 of 8 and incubated with increasing concentrations of heparin in the 
 presence or absence of 20 mm glutathione ( gsh ) for 30 min at 37 c . 

the polyplex samples were then loaded onto an 0.8% agarose gel containing 
 0.5 g / ml etbr and run for 60 min at 120 v in 0.5 tris / borate / edta 
 running buffer .
the gels were visualized under uv illumination on 
 a kodak gel logic 100 imaging system .
cytotoxicity 
 of dss - ben and dcc - ben in b16f10 , u2os , and hepg2 cells was evaluated 
 by mts assay ( celltiter 96 aqueous one solution cell proliferation 
 assay , promega , madison , wi ) .
cells ( 8000 , u2os ; 10  000 , b16f10 
 and hepg2 ) were seeded in 96-well microplates 1 day before polymer 
 treatment .
culture medium was replaced with 200 l of increasing 
 concentrations of dss - ben or dcc - ben in serum - supplemented medium , 
 and the cells were then incubated for 24 h. to measure cell viability , 
 medium was aspirated and replaced with a mixture of 100 l of 
 serum - free medium and 20 l of mts reagent . after 1.5 h incubation , 
 the absorbance was measured using spectramax m5 multi - mode 
 microplate reader ( molecular devices , ca ) at  = 490 nm . the 
 relative cell viability ( % )
the ic50 values were calculated as the polymer concentration 
 that inhibits growth of 50% of cells relative to growth of untreated 
 cells using graphpad prism , version 5.0c .
cells 
 were seeded in 48-well plates at a density of 40  000 cells / well 
 for b16f10 or 20  000 cells / well for u2os 24 h prior to transfection . 

on the day of transfection , cells were incubated with the polyplexes 
 containing luciferase dna ( pluc ) ( 0.4 g dna / well ) in 170 l 
 of medium ( with or without 10% fbs ) . after 4 h incubation
, polyplexes 
 were completely removed , and the cells were incubated in medium with 
 10% fbs for another 24 h. to measure luciferase expression , the medium 
 was discarded , and the cells were lysed in 100 l of 0.5 
 cell culture lysis reagent buffer ( promega , madison , wi ) for 30 min . 

one hundred microliters of 0.5 mm luciferin solution was then automatically 
 injected into each well containing 20 l of cell lysate , and 
 the luminescence was integrated over 10 s using glomax 96 microplate 
 luminometer ( promega , madison , wi ) .
total cellular protein in the 
 cell lysate was determined by the bicinchoninic acid protein assay 
 using a calibration curve constructed with standard bovine serum albumin 
 solutions .
transfection activity was expressed as relative light units 
 ( rlu)/mg cellular protein  sd ( n = 4 ) .
transfections 
 of u2os cells with tnf plasmid dna ( ptnf ) were conducted 
 in 96-well plates .
the cells were seeded at a density of 3000 cells / well 
 24 h prior to polyplex addition .
transfection was performed as above 
 using a dna dose of 0.2 g / well .
after 4 h incubation , 115 l 
 of fresh medium with 10% fbs was added , and cells were incubated for 
 another 48 h prior to measuring cell viability using mts assay .
the 
 relative cell killing mediated by tnf expression was normalized 
 to the viability of untreated cells and expressed as the mean  
 sd of triplicate samples .
the cationic character of benspm recently prompted 
 us to explore 
 its potential for use in dual - function delivery vectors suitable for 
 combination drug / nucleic acid therapies . in proof - of - principle studies , 

we synthesized lipid prodrug based on benspm and established its utility 
 for use in combination drug / nucleic acid delivery .
herein , we extend the concept of dual - functioning delivery vectors 
 that target polyamine metabolism to benspm - based polycations .
we previously 
 found that benspm has to be released from the vector in its unmodified 
 form to exert the desired effect on polyamine metabolism . because of the presence of four secondary amines , 
 the easiest approach to synthesize benspm polycations would be to 
 polymerize benspm using amide or carbamate chemistry .
unfortunately , 
 benspm amides and carbamates were too stable in a biological environment 
 for the intended application .
as a solution , 
 we employed self - immolative linker chemistry to synthesize rapidly 
 degradable polycations that release unmodified benspm as the main 
 degradation product .
self - immolative linkers represent an alternative 
 strategy to traditional simple linkers used in prodrug design in situations 
 where the linker is sterically inaccessible or , as in our case , the 
 rates of the linker cleavage are too slow for the intended use ( scheme 2a ) . in the self - immolative approach ,
the linker 
 uses a readily cleavable bond with an additional protecting group 
 and a stable bond to the desired drug ( i.e. , benspm ) .
the stable bond 
 becomes labile when the cleavable bond is broken , and the process 
 leads to the rapid release of a drug from the conjugate .
multiple 
 types of self - immolative linkers have been explored in prodrug and 
 polymer synthesis . in this study , we selected bhed as the self - immolative 
 linker to 
 synthesize the benspm - based polycation prodrug dss - ben
. dss - ben was 
 synthesized as shown in scheme 2b by reacting 
 an equimolar amount of benspm and the bhed linker activated with cdi . 

control nondegradable polycation dcc - ben was synthesized by reacting 
 benspm with 1,6-hexanediol activated with cdi .
the polymers were purified 
 by dialysis and characterized by h nmr , elemental analysis , 
 and size - exclusion chromatography .
the molecular weights of the prepared 
 polymers were relatively low at mw = 3.8 
 kda for dss - ben and 2.8 kda for dcc - ben .
the low molecular weight 
 of dcc - ben partially explained the low yield ( 10% ) , as a significant 
 portion of the polymer was lost during dialysis using a membrane with 
 a molecular weight cutoff of 3.5 kda .
polymer loss during purification 
 also explains the low polydispersity index of the synthesized polymers , 
 which was typically in the range 1.11.3 . on the basis of the 
 molecular weight ,
dss - ben contained , on average , 8 benspm molecules , 
 whereas dcc - ben contained 7 benspm molecules .
attempts to increase 
 the molecular weight by exploring other solvents , temperatures , and 
 reaction times were unsuccessful .
it is likely that incomplete cdi 
 activation of bhed and 1,6-hexanediol resulted in deviation from the 
 assumed equimolarity and bifunctionality and thus in the low molecular 
 weight .
results of the elemental analysis suggested that benspm accounts 
 for 44 wt % in both the dss - ben and dcc - ben samples .
a similar 
 estimate was obtained from the analysis of the h nmr spectra . 

although the polymer structure in figure 1a suggests that only terminal benspm amines engaged in reaction 
 with bhed , the h nmr does not allow us to exclude the 
 possibility that the two internal secondary amines also participated 
 in the reaction .
( a ) ( top ) representative h nmr spectrum of 
 dss - ben 
 in d2o ( top ) .
( bottom ) h nmr spectrum of dss - ben 
 after 16 h degradation in dtt solution in phosphate - buffered d2o / acetone - d6 ( peaks highlighted 
 in the red box were used to determine polymer degradation ) .
( b ) degradation 
 kinetics and benspm release from dss - ben and dcc - ben in 100 mm dtt 
 in 100 mm phosphate - buffered d2o ( ph 7.4)/acetone ( 3:2 ) 
 at 25 c measured by h nmr .
( c ) intracellular release 
 of benspm from dss - ben after 48 h incubation with b16f10 cells and 
 72 h incubation with u2os cells determined by hplc analysis ( n = 3 ) .
the amount of intracellular benspm is expressed 
 as a percentage relative to intracellular benspm content observed 
 in cells treated with free benspm .
bhed contains a disulfide bond that can be readily cleaved by intracellular 
 thiol  disulfide exchange with free thiols such as glutathione 
 ( gsh ) . in dss - ben , the disulfide cleavage results in generation of 
 a free thiol , which then participates in intramolecular attack of 
 the carbamate carbonyl and cleavage of the otherwise stable carbamate 
 bond between benspm and bhed ( scheme 2c ) .
we 
 first confirmed the proposed mechanism of polymer degradation and 
 benspm release by investigating the degradation kinetics by h nmr spectroscopy using dtt as the reducing agent ( figure 1a ) . benspm release from the polymers was calculated 
 as the percentage of reduction in the integral intensity of the benspm 
 methylene peaks ( highlighted by a box in figure 1a ) relative to their integral intensity at time 0 ( i.e. , immediately 
 after dtt addition ) . plotting the results as the percent benspm release 
 vs degradation time ( figure 1b ) suggested that 
 dss - ben degradation followed first - order kinetics .
the estimated rate 
 constant was 3.5  10 min , and the corresponding half - life was 198 min .
these results are 
 in good agreement with previous reports on the degradation of several 
 self - immolative polymers containing similar cyclizing spacers .
in contrast , treatment with dtt had no effect on dcc - ben ( figure s1 ) , confirming the stability of the carbamate 
 bond and the benefits of the self - immolative linker in the degradation 
 of dss - ben and release of free benspm .
intracellular degradability 
 and release of benspm from the polymers were evaluated by incubating 
 b16f10 and u2os cells with 5.7 g / ml dss - ben or dcc - ben or with 
 control benspm ( 2.5 g / ml ) .
the polymer concentration was selected 
 such that the cells were exposed to 2.5 g / ml of benspm equivalent . 

the amount of intracellular benspm was then determined by hplc analysis 
 of the benspm in the cell lysate ( figure 1c ) . 
 in order to determine how efficiently dss - ben can generate intracellular 
 benspm when compared with treatments with free benspm , we expressed 
 the results as the percent of intracellular benspm content in cells 
 treated with dss - ben relative to the amount of benspm taken up by 
 cells after incubation with free benspm .
as shown in figure 1c , benspm was detected in both tested cell lines 
 treated with dss - ben .
in contrast , no detectable amount of benspm 
 was observed in cells treated with dcc - ben , suggesting the high stability 
 of the carbamate bond in the polymer and thus no intracellular degradation . 

we observed significant differences in the time course of intracellular 
 benspm between the two cell lines , with benspm levels peaking at 48 
 h in b16f10 and 72 h in u2os .
there were also substantial differences 
 in the achievable intracellular benspm concentrations between the 
 two cell lines .
in contrast to 67.5% benspm observed in u2os cells , 
 we found only 13.2% benspm in b16f10 cells relative to the intracellular 
 concentration found when incubating the cells with free benspm .
the 
 intracellular benspm content when delivered by dss - ben is a complex 
 function of the rate and extent of dss - ben uptake , intracellular trafficking , 
 rate of degradation , and extent of excretion from the cells .
we hypothesize 
 that the differences in the intracellular reducing capacity of the 
 two cell lines are among the most important factors behind the observed 
 results .
overall , our findings confirm the functionality of the self - immolative 
 linker in dss - ben in living cells .
benspm exhibits multiple functions 
 in the regulation of intracellular 
 polyamines . unlike selective inhibitors of the individual enzymes 
 in the polyamine pathway
benspm mimics the structure of spm , but 
 the alkylated terminal amines prevent its oxidation by oxidases such 
 as apao .
benspm is also a potent post - transcriptional inducer of the 
 activity of catabolic enzymes ssat and smo .
after 
 confirming that dss - ben is degraded into benspm in both tested cell 
 lines , we evaluated the ability of the released benspm to regulate 
 cellular polyamine metabolism . because benspm treatment leads to depletion 
 of all natural polyamines , we first focused on investigating the effect 
 of dss - ben treatment on changes in the expression of the key catabolic 
 enzymes ssat and smo using rt - pcr ( figure 2a ) .
as expected , treatment with free benspm upregulated the mrna 
 level of both ssat and smo .
overall , treatment with dss - ben resulted in a weaker upregulation 
 of the catabolic enzymes than that resulting from treatement with 
 free benspm .
for example , benspm increased expression of ssat mrna 
 3.9-fold in b16f10 , whereas dss - ben increased the expression by only 
 2-fold .
similarly , benspm upregulated smo mrna expression 21.8-fold 
 in u2os , whereas dss - ben achieved only a 12.1-fold increase .
treatment 
 with the nondegradable dcc - ben had the weakest effect on induction 
 of the two catabolic enzymes .
( a ) relative 
 changes in the expression of polyamine catabolic enzymes smo and ssat 
 mrna in b16f10 and u2os cells .
cells were incubated with 2.5 g / ml 
 benspm or 5.7 g / ml dss - ben or dcc - ben , and mrna levels were 
 measured by rt - pcr .
results are expressed as the fold induction of 
 specific mrna in treated cells relative to that in untreated control 
 ( n = 3 ) .
( b ) effect of benspm , dss - ben , and dcc - ben 
 on polyamine depletion in b16f10 and u2os cells determined by hplc . 

results are expressed as the percent polyamine depletion in treated 
 cells relative to that of the untreated control ( n = 3 ) .
having confirmed benspm release 
 from dss - ben and its effect on 
 the expression of catabolic enzymes , we evaluated if these findings 
 translate into the expected effect on polyamine depletion .
as above , 
 we treated the cells with benspm , dss - ben , and dcc - ben and measured 
 intracellular levels of put , spd , and spm by hplc ( figure 2b ) . in b16f10 cells , benspm treatment depleted 80% 
 of spd and 74% of spm ( we were unable to detect any put in this cell 
 line ) .
nondegradable dcc - ben had no observable effect on the polyamine pool 
 in b16f10 cells .
in u2os cells , benspm was highly effective at depleting 
 all three natural polyamines , as suggested by the fact that it depleted 
 100% of put , 88% of spd , and 95% of spm .
similarly , dss - ben was more 
 effective in u2os than in b16f10 , as it depleted 37% of spd and 65% 
 of spm .
the decreased ability of dss - ben to affect the cellular polyamine 
 pool in b16f10 cells is likely related to the lower overall intracellular 
 concentration of benspm in this cell line ( figure 1c ) .
surprisingly , only an 8% depletion of put was observed 
 in the case of dss - ben treatment of u2os cells .
we speculate that 
 this observation is related to the likely differences in the mechanism 
 of cellular uptake and trafficking between dss - ben and free benspm . to test the ability of dss - ben to deliver genes , 
 we first investigated dna condensation by the etbr exclusion assay 
 ( figure 3a ) .
dss - ben was able to fully condense 
 dna above a polymer / dna ( w / w ) ratio of 2.5 and displayed a typical 
 sigmoidal condensation curve .
despite having a comparable molecular 
 weight , dcc - ben exhibited a less efficient dna condensing ability 
 than that of dss - ben . as expected , free benspm showed poor dna condensing 
 ability , as indicated by the fact that less than a 30% decrease in 
 etbr fluorescence was observed even at the highest w / w ratios used .
( a ) dna condensation 
 by dss - ben and dcc - ben in 10 mm hepes buffer ( ph 7.4 ) by etbr exclusion 
 assay .
polyplexes were prepared at w / w 8 and incubated with increasing concentrations 
 of heparin either with or without 20 mm gsh for 1 h. ( c ) hydrodynamic 
 size and zeta - potential of dna polyplexes of dss - ben and dcc - ben .
we expected that the intracellular 
 cleavage of the disulfide bonds 
 in dss - ben will not only lead to release of free benspm but also facilitate 
 dna release from the polyplexes because of the inability of free benspm 
 to condense dna .
the reduction - triggered dna release was studied by 
 incubating dss - ben / dna and control dcc - ben / dna polyplexes with 20 
 mm gsh and increasing concentrations of heparin .
the stability of 
 the polyplexes was characterized by the minimum heparin concentration 
 required for polyplex disassembly and dna release using agarose gel 
 electrophoresis ( figure 3b ) . in the absence 
 of gsh , both dss - ben and control
the reducing activity of 20 mm gsh selectively 
 destabilized dss - ben polyplexes to a point that the dna was released 
 without the need for heparin .
these results suggest that the self - immolative 
 linker retains its sensitivity to reduction even when the polymer 
 is complexed with dna .
hydrodynamic 
 size and zeta - potential of the dss - ben and dcc - ben 
 polyplexes prepared at different w / w ratios were measured by light 
 scattering ( figure 3c ) .
the sizes of dss - ben 
 polyplexes ranged from 70 to 100 nm at all tested w / w ratios .
in contrast , 
 dcc - ben required w / w > 4 to achieve similar sized polyplexes . even 
 then , dcc - ben polyplexes were larger ( 120210 nm ) and 
 had a lower zeta - potential when compared with that of dss - ben polyplexes . 
 these size and zeta potential differences were observed despite the 
 structural and molecular weight similarity between dss - ben and dcc - ben . 

a possible explanation may rest in additional stabilization of the 
 dss - ben polyplexes by intrapolyplex disulfide 
toxicity associated with the use of 
 polycations remains a major hindrance for their use as gene delivery 
 systems .
one of the most effective strategies to reduce the toxicity 
 is to incorporate degradable moieties , such as the disulfide bonds 
 used in the present study , into the polymer backbone .
we evaluated the cytotoxicity of dss - ben and dcc - ben by 
 mts assay in three different cell lines : mouse melanoma b16f10 , human 
 osteosarcoma u2os , and human hepatocellular carcinoma hepg2 .
the goal 
 of this experiment was to separate the nonspecific polycation toxicity 
 from the toxic effects that originate specifically from the influence 
 of benspm on polyamine metabolism .
thus , we assessed only acute 24 
 h toxicity , which we believe can be mostly attributed to the polycation 
 character of the polymers .
the ic50 value of each polymer was calculated from the 
 concentration dependence of cell viability in the three cell lines 
 tested ( figure 4a ) .
as expected , bioreducibility 
 of dss - ben contributed to a significantly decreased acute toxicity 
 in all three cell lines when compared with those of control dcc - ben 
 and pei .
( a ) ic50 values of dss - ben , dcc - ben , and pei in b16f10 , 
 u2os , and hepg2 cells based on mts cell viability assay after 24 h 
 incubation with polymers ( n = 3 ) .
luciferase transfection 
 activity of dss - ben and dcc - ben in ( b ) b16f10 cells and ( c ) u2os cells . 

results are expressed as rlu / mg protein  sd ( n = 4 ) .
after we determined the safe dosing 
 range of dss - ben , the gene 
 delivery efficiency was evaluated in b16f10 cells ( figure 4b ) and u2os cells ( figure 4c ) using a luciferase reporter plasmid .
the polyplexes were formulated 
 at varying polycation / pluc ( w / w ) ratios , and pei / pluc polyplexes ( w / w 
 1.2 ) were used as an additional control .
dss - ben polyplexes showed 
 considerably higher luciferase transfection compared to that of dcc - ben 
 polyplexes .
in addition , dss - ben polyplexes showed transfection that 
 was not only fully comparable with that of pei polyplexes but also 
 exhibited low sensitivity to w / w ratio and the presence of serum in 
 b16f10 cells .
inhibitors of polyamine 
 synthesis , including benspm , have been reported to sensitize various 
 human and mouse cancer cells to tnf - induced apoptosis . we hypothesized that using dss - ben as a delivery 
 vector for ptnf will result in improved anticancer activity 
 due to the combined effect of polyamine depletion by benspm released 
 from dss - ben and tnf cancer gene therapy ( scheme 1b ) . to study the dual - functionality of dss - ben in tnf 
 cancer gene therapy , we used dss - ben to deliver ptnf to u2os 
 cells and evaluated the combined anticancer activity in vitro ( figure 5 ) .
dss - ben / ptnf polyplexes 
 were prepared at different w / w ratios , and their cell killing activity 
 was evaluated . in order to dissect the contribution of benspm and 
 tnf to the overall cell killing activity , we used dss - ben polyplexes 
 with pluc and pei polyplexes with ptnf as controls .
as shown 
 in figure 5 , treatment with dss - ben / pluc polyplexes 
 resulted in 2329% cell death , which was attributed to the 
 effect of polyamine depletion by the benspm released from dss - ben . 
 when used to deliver ptnf
, dss - ben polyplexes exhibited the 
 highest combined cell killing activity ( 40% ) at w / w 4 . at this w / w 
 ratio ,
the cell killing activity was significantly higher than either 
 the activity of dss - ben / pluc polyplexes ( 29% ) or pei / ptnf polyplexes 
 ( 25% ) .
it is worth noting that under the used conditions pei showed 
 better transfection than dss - ben ( figure 4c ) . 

although the anticancer effect of tnf can not be fully captured in vitro due to the importance of the immune system for 
 tnf activity , the results nevertheless validate our hypothesis 
 and confirm dss - ben as dual - function delivery system that can deliver 
 a therapeutic gene and enhance its activity due to dss - ben intracellular 
 degradation to benspm and its beneficial effect via dysregulated polyamine 
 metabolism in cancer . enhanced anticancer activity of tnf gene delivered 
 by dss - ben 
 polyplexes .
u2os cells were treated with dss - ben / ptnf , dss - ben / pluc , 
 or pei / ptnf polyplexes prepared at different w / w ratios .
results are normalized to the viability 
 of untreated cells and shown as mean relative cell killing ( % )  
 sd ( n = 3 ) .
we have designed and developed self - immolative polycation dss - ben 
 and confirmed its dual functionality as ( i ) a prodrug of the anticancer 
 agent benspm that targets dysregulated polyamine metabolism in cancer 
 and ( ii ) a gene delivery vector . our results support the benefits 
 of self - immolative linkers in the design of biodegradable polymers 
 for use in gene delivery .
the dual functionality of dss - ben makes 
 it a promising delivery platform for combination anticancer therapy , 
 which can be further expanded to deliver a variety of other therapeutic 
 agents , including mirna , sirna , and proteins .
 polycations are explored as carriers 
 to deliver therapeutic nucleic 
 acids . 
 polycations are conventionally pharmacological inert with the 
 sole function of delivering therapeutic cargo . 
 this study reports 
 synthesis of a self - immolative polycation ( dss - ben ) based on a polyamine 
 analogue drug n1,n11-bisethylnorspermine ( benspm ) . 
 the polycation was designed 
 to function dually as a gene delivery carrier and a prodrug targeting 
 dysregulated polyamine metabolism in cancer . using a combination of 
 nmr and hplc 
 , we confirm that the self - immolative polycation undergoes 
 intracellular degradation into the parent drug benspm . 
 the released 
 benspm depletes cellular levels of spermidine and spermine and upregulates 
 polyamine catabolic enzymes spermine / spermidine n1-acetyltransferase ( ssat ) and spermine oxidase ( smo ) . 
 
 the synthesized polycations form polyplexes with dna and facilitate 
 efficient transfection . 
 taking advantage of the ability of benspm 
 to sensitize cancer cells to tnf-induced apoptosis , we show 
 that dss - ben enhances the cell killing activity of tnf gene 
 therapy . 
 the reported findings validate dss - ben as a dual - function 
 delivery system that can deliver a therapeutic gene and improve the 
 outcome of gene therapy as a result of the intracellular degradation 
 of dss - ben to benspm and the subsequent beneficial effect of benspm 
 on dysregulated polyamine metabolism in cancer . 
Introduction
Materials
and Methods
Results
and Discussion
Conclusions