nanoscale therapeutics based on synthetic 
 polymer scaffolds offers new routes to improved cancer drug delivery , with water - soluble polymers aiding in drug solubility , increasing 
 the therapeutic window through long circulation half - life ( t1/2 ) , and increasing uptake into tumors by the 
 enhanced permeability and retention ( epr ) effect . for polymer - based prodrugs , recent interest centers on 
 triggered release of therapeutic moieties from the polymer scaffold , for example by exploiting intratumoral or intracellular 
 environmental triggers such as ph . this approach is intended 
 to reduce nonspecific , off - target toxicity often associated with systemic 
 delivery . we reported the preparation of poly(2-methacryloyloxyethyl 
 phosphorylcholine)doxorubicin ( polympc  dox ) prodrugs , 
 in which dox was connected to the polymer backbone by hydrazone linkages , 
 which are ph sensitive . these prodrugs 
 were prepared by copolymerization of mpc with methacrylates containing 
 pendent acyl hydrazides and then incorporating dox by hydrazone formation , 
 to give tunable dox loading that can reach or exceed 30 wt % . these 
 polympc  dox conjugates displayed ph sensitive release profiles , 
 with half - life ( t1/2 ) values ranging from 
 2 to 40 h at ph 5.0 , while only 2 to 20% of dox was released in 48 
 h at ph 7.4 . in cell culture , the half - maximal inhibitory concentration 
 ( ic50 ) values for polympc dox ranged from 1.5 to 
 16 m for human breast cancer ( mcf-7 and mda - mb-231 ) and colorectal 
 ( colo 205 ) adenocarcinoma cell lines . , we report in vivo pharmacokinetic , 
 biodistribution , and treatment efficacy data for polympc the 4t1 mammary carcinoma 
 was selected as an extremely aggressive breast cancer model that is 
 highly tumorigenic and metastatic , and thus can be considered as a 
 model for triple negative breast cancer . unlike many tumor models , 4t1 tumors can metastasize spontaneously 
 from the primary tumor to multiple distant sites including the lungs , 
 lymph nodes , liver , brain , and bone within weeks following injection . dox prodrugs , potentially enhancing the 
 utility of dox in late stage breast cancer . 4t1 cells can be introduced 
 orthotopically by direct injection into the mammary gland , such that 
 the primary tumor site is anatomically correct , and the syngeneic 
 nature of the cells allows for use of immunocompetent animals , and 
 thus examination of the effects of polympc  dox conjugates on 
 the immune system . the 4t1 breast cancer model has been used by others 
 to study polymer prodrugs in vivo , including paclitaxel , docetaxel , cisplatin , gemcitabine , and doxorubicin , with variable success with respect to slowing tumor growth and reducing 
 off - target toxicity . given the high level of water solubility and 
 degree of drug loading achievable with polympc dox prodrugs , 
 this study aimed to demonstrate their efficacy in 4t1 tumor - bearing 
 mice . methacryloyloxyethyl phosphorylcholine 
 ( mpc ) , ethyl bromoacetate , copper(i ) bromide , 2,2-bipyridine 
 ( bpy ) , ethyl 2-bromoisobutyrate ( ebib ) , hydrazine monohydrate , acetic 
 acid , magnesium sulfate , trifluoroacetic acid ( tfa ) , acetonitrile 
 ( anhydrous ) , methanol ( anhydrous ) , and dimethyl sulfoxide ( anhydrous ) 
 were purchased from aldrich . spectra / por 
 3 dialysis membrane ( mwco 1000 ) was purchased from spectrum laboratories , 
 inc . hanks balanced salt solution used for in vivo studies 
 was obtained from life technologies ( gibco ) . uv / vis 
 absorbance measurements were taken on a perkinelmer lambda 25 spectrometer . molecular weights and polydispersity indices ( pdis ) were estimated 
 by gel permeation chromatography ( gpc ) in sodium nitrate ( 0.1 m with 
 0.02 wt % of nan3 ) aqueous solution against poly(ethylene 
 oxide ) standards , operating at 1.0 ml / min with three waters ultrahydrogel 
 linear columns ( 300  7.8 mm ) equipped with ri and uv detectors . hplc was performed on a waters alliance system equipped with uv and 
 fluorescence detectors . a reverse phase c18 column ( 250  4.6 
 mm ) eluting with 40% acetonitrile in water + 1% tfa at a flow rate 
 of 1 ml / min was used to analyze biological samples obtained from the 
 pharmacokinetic and biodistribution studies . sodium methacrylate ( 9.7 g , 90 mmol ) 
 and 10.02 g of ethyl bromoacetate ( 60 mmol ) were added to 55 ml of 
 dry acetonitrile . to this suspension the salt was removed by filtration , and solvent was removed by evaporation 
 under reduced pressure . the organic phase was dried over 
 mgso4 , and concentration under vacuum gave the desired 
 monomer as a pale yellow oil ( 9.8 g , 95% ) . h nmr ( cdcl3 , 300 mhz ) :  6.21 ( s , 1h ) , 5.64 ( m , 1h ) , 4.66 ( s , 2h ) , 
 4.22 ( q , 2h ) , 1.97 ( s , 3h ) , 1.27 ( t , 3h ) ppm . c nmr ( cdcl3 , 75 mhz ) :  167.9 , 166.7 , 135.4 , 126.8 , 61.4 , 60.9 , 
 18.2 , 14.1 ppm . ebib ( 5.9 mg , 0.03 mmol ) , 
 mpc , and etoema were combined in a 10 ml two - neck round - bottom flask , 
 and three cycles of vacuum  nitrogen were employed . dmso and 
 meoh ( previously degassed with n2(g ) ) were injected with 
 a degassed syringe . cu(i)br ( 8.6 mg , 0.06 mmol ) and bipyridine ( 18.7 mg , 0.12 
 mmol ) were added quickly as solids under nitrogen atmosphere . the 
 mixture was then purged with nitrogen gas for an additional 20 min 
 and then left under nitrogen atmosphere . the mixture was stirred at 
 room temperature , and the polymerization conversion was monitored 
 by h nmr spectroscopy . the crude product was purified by column chromatography 
 on silica gel , eluting with methanol to give the poly(mpc - etoema ) 3 random copolymer as a white solid . the monomer ratio in 
 the copolymer was characterized by h nmr spectroscopy , 
 integrating signals at 3.58 ppm ( ch2n in mpc ) and at 1.27 ppm ( ( ch3 ) in etoema ) . poly(mpc - etoema ) was dissolved in methanol at 
 a concentration of 100200 mg / ml . hydrazine monohydrate was 
 added to the polymer solution to a final concentration of 25% . the 
 reaction mixture was stirred at room temperature , with monitoring 
 by h nmr spectroscopy . upon completion , the reaction mixture 
 was diluted with water and purified by dialysis against water using 
 a mwco 1000 membrane for 2 days and passing through a 0.45 m 
 membrane . the typical yield was over 80% , and the loading 
 of the hydrazine group was calculated by integration of signals at 
 3.58 ppm ( ch2n in mpc ) 
 and 4.64 ppm ( ch2conhnh2 ) in the h nmr spectrum . h nmr ( meod , 
 300 mhz ) :  = 0.91.2 ( br , 3h ) , 1.82.3 ( br , 2h ) , 
 3.3 ( s , 9h ) , 3.75 ( br , 2h ) , 4.16 ( br , 2h ) , 4.3 ( br , 2h ) , 4.38 ( br , 
 2h ) , 4.64 ( br , 2h ) . c nmr ( meod , 100 mhz ) :  = 
 16.8 , 18.5 , 44.7 , 45.0 , 53.4 , 59.3 , 62.2 , 62.9 , 64.8 , 66.1 , 167.1 , 
 176.9 , 178.1 . gpc ( 0.1 m nano3 + 0.02 wt % nan3 , peo standards ) : mn , 25,000 ; pdi 1.4 . mpc copolymer 4 ( 200 mg , 0.165 mmol 
 of nhnh2 ) and dox ( 58 mg , 0.1 mmol ) were dissolved 
 in anhydrous methanol ( 5 ml ) . to this solution were added 60 l 
 of acetic acid and 200 mg of anhydrous magnesium sulfate . the reaction 
 mixture was stirred in the dark at room temperature for 2 days . the 
 resulting conjugate was purified by passage over a sephadex lh-20 
 column eluting with methanol . dox 
 conjugate were concentrated by rotary evaporation , redissolved in 
 water , and further purified by sephadex g-25 column eluting with pure 
 water . dox conjugate 5 was obtained as 
 a dark red powder after lyophilization ( 230 mg , 88% ) . doxhcl was dissolved in pure water at 
 0.01 mg / ml , and polympc  dox conjugate 5 was dissolved 
 in pure water at a concentration of 0.1 mg / ml . the dox loading of 
 the conjugate was calculated on the basis of uv / vis absorbance at 
 490 nm , using the molar absorptivity of doxhcl at 490 nm . all experiments 
 were performed in accordance with protocols approved by the baystate 
 institutional animal care and use committee . four week old balb / c 
 female mice were injected subcutaneously in the right flank with 5 
  10 4t1 murine breast cancer cells suspended in 
 100 l of hanks balanced salt solution ( hbss ) . once tumors reached 
 a size of 100300 mm ( calculated by length  
 width  /6 ) , mice were injected through the 
 lateral tail vein with 100 l of hbss , free doxorubicin ( 6 mg / kg ) , 
 or polympc  dox ( 6 mg / kg , dox equivalent ) ( n = 8/treatment ) . blood samples ( 3050 l ) were taken 
 from the submandibular vein prior to injection , and then 30 min , 2 
 h , 6 h , 12 h , 1 day , 2 days , 3 days , and 5 days postinjection . blood 
 samples were clotted on ice and centrifuged at 1500 g for 15 min at 4 c . serum was collected and stored at 80 
 c until hplc analysis to determine doxorubicin concentration . 
 on day 3 and day 5 postinjection blood was collected by cardiac puncture ( 800 l ) , and 
 a complete blood count ( cbc ) was performed on 500 l using the 
 vetscan hm5 . the remaining samples were allowed to clot on ice , and 
 centrifuged at 1500 g for 15 min at 4 c . serum 
 was collected and stored at 80 c for elisa of cytokine 
 response . tumor , heart , liver , lung , kidney , and spleen samples were 
 collected , weighed , and frozen in liquid nitrogen . livers and spleens 
 were divided , and half of each tissue was additionally fixed in 10% 
 buffered formalin overnight at 4 c , transferred to 70% etoh 
 at 4 c , and paraffin embedded for histological analysis . frozen 
 tissues were homogenized at maximum speed in acidified isopropanol 
 ( 90% isopropanol containing 0.6 ml of concentrated hcl ) . samples were 
 then centrifuged at 1500 g for 15 min at 4 c , 
 and the upper aqueous phase was collected and stored at 80 
 c until hplc analysis of doxorubicin concentration . four week old balb / c female 
 mice were injected into lower right mammary fat pad with 5  
 10 4t1 murine breast cancer cells suspended in 100 l 
 of hanks balanced salt solution ( hbss ) . once tumors reached a size 
 of 42132 mm ( calculated by length  width  /6 ) , mice were injected through the lateral 
 tail vein with free doxorubicin ( 3 mg / kg ) , polympc  dox ( 15 
 mg / kg dox equivalent ) , or hbss ( control ) ( n = 15/treatment ) . a second injection of the same concentration was given on day 7 , and 
 mice treated with polympc animals were monitored for signs of distress , and body weights 
 and tumor measurements were collected every 2 days . upon completion , 
 tumor , heart , liver , lung , kidney , and spleen samples were collected , 
 weighed , and fixed in 10% buffered formalin overnight at 4 c , 
 transferred to 70% etoh at 4 c , and paraffin embedded for histological 
 analysis . polympc  dox 
 prodrugs were prepared by copolymerization of mpc with 2-ethoxy-2-oxoethyl 
 methacrylate ( etoema ) by atom transfer radical polymerization ( atrp ) 
 using copper bromide and bipyridine as the catalyst and ligand , respectively . the ethyl esters of copolymer 3 are 
 converted to acylhydrazines by substitution with hydrazine to give 
 polympc - hydrazine , shown as polymer 4 in figure 1 . polymer 4 was characterized by aqueous 
 gel permeation chromatography ( gpc ) against linear peo standards , 
 and by h nmr spectroscopy to determine the mole percent 
 of hydrazine monomer units within the polymer . under these conditions , hydrazine substitution was preferred at the ketone of the ethyl ester , 
 as confirmed by nmr spectroscopy noting loss of the ethyl ester methyl 
 group at 1.25 ppm , and retention of the methylene ( ch2conhnh2 ) at 4.64 ppm . the 
 mole percent incorporation of comonomer remained constant through 
 the transition from copolymer 3 to polympc - hydrazine 4 . polymer 4 was conjugated to dox by hydrazone 
 formation in methanol , in the presence of magnesium sulfate and acetic 
 acid , to give polympc dox 5 was purified by preparative size exclusion chromatography 
 and lyophilized to give a bright red powder , which proved stable for 
 months when stored as a dry solid at 20 c . for the efficacy 
 study described here , we used polympc  dox with an estimated 
 molecular weight of 25  000 g / mol , and dox loading of 22 wt 
 % . synthesis of poly(mpc - co - etoema ) ( 3 ) , followed by acylhydrazine formation ( 4 ) and conjugation 
 of doxhcl to give polympc  dox prodrug ( 5 ) . we previously reported maximum 
 tolerated dose ( mtd ) data for polympc  dox in athymic nu / j mice , 
 where polympc dox was found to be well - tolerated at 30 mg / kg 
 over the course of 30 days ; mice which received doses of 50 mg / kg 
 showed only a 10% weight loss at 22 days . mg / kg ) , and are comparable to the liposomal 
 formulation doxil ( 2030 mg / kg ) and a pegylated polyester dendritic dox example ( 2040 mg / kg ) . to further extend our in vivo prodrug characterization , the pharmacokinetics of polympc animals were sorted into three groups 
 of eight , with a control group ( hbss ) , a free dox group , and a polympc dox 
 group ( 6 mg / kg dox equivalent doses ) , introducing drugs by a single 
 tail vein injection of 100 l total volume . blood serum levels 
 of dox were monitored over time , analyzing for the presence of drug 
 by hplc equipped with a fluorescence detector . as shown in figure 2 , in vivo free dox concentration 
 decreased rapidly , with a t1/2 of 15 min , 
 clearing to near - undetectable levels within 1 h. this is consistent 
 with reported values for dox . dox 
 displayed a significantly longer circulation half - life of 2 h. accordingly , 
 the area under the curve ( auc ) was dramatically higher for the polympc dox 
 prodrug ( 408 gh / ml ) compared to free dox ( 22 gh / ml ) . polymer conjugation 
 extends the circulation half - life from 15 min to 2 h and increases 
 the auc from 22 gh / ml to 408 gh / ml . error 
 bars represent  standard deviation . the biodistribution of dox was determined for both the free 
 drug and polympc  dox , 3 and 5 days postinjection from the 6 
 mg / kg dox equivalent doses administered to the tumor - bearing mice 
 used in the pk study ( figure 3 ) . dox was 700 ng / g of tissue 
 3 days after injection , and 390 ng / g 5 days after injection . this 
 represents a 2-fold increase over free dox at day 3 ( 350 ng / g ) and 
 a 3-fold increase over free dox at day 5 ( 130 ng / g ) . dox 
 conjugates displayed reduced accumulation in off - target organs , including 
 the spleen and especially the lungs , relative to free dox . significantly 
 higher drug accumulation was noted in the liver for the polympc dox 
 group compared to the free dox group , which we attribute to the prolonged 
 circulation times and the delayed clearance noted for the polymer 
 prodrug . while dox is known to be metabolized primarily by the liver , 
 the liposomal formulation doxil was found to have impaired hepatic 
 metabolism , suspected to be excluded from uptake based on the liposome 
 size . similarly , the increased size of 
 polympc  dox prodrugs relative to free dox may hamper hepatic 
 uptake , resulting in delayed accumulations . dox group is potentially 
 advantageous for reducing cardiotoxicity effects , a common dose - limiting 
 side effect associated with dox administration . the tumor to normal tissue distribution ratios are given 
 in table 1 , highlighting the preferential dox 
 uptake in tumor tissue relative to healthy tissue . the benefits of 
 passive tumor targeting of polymer prodrugs has been noted before , and the data presented here suggests that the polympc biodistribution analysis 
 of polympc  dox and free dox after ( a ) 3 days and ( b ) 5 days , 
 expressed as ng of dox / g of tissue . the significance was determined 
 using a two - tailed student t test [ * p = 0.05 to 0.01 ; * * p = 0.01 to 0.001 ; * * * p < 0.001 ] . error bars represent  the standard 
 error of the mean ( sem ) . at the conclusion of the pk and biodistribution study , the spleen , 
 liver , kidney , heart , lungs , and tumors were removed from the animals 
 and weighed , with livers and spleens fixed and paraffin - embedded for 
 histological analysis . as shown in figure 4 , histological analysis of tissue sections stained 
 with hematoxylin and eosin ( h&e ) suggested no significant off - target 
 toxicity at these high levels of dox , consistent with the use of polympc 
 as the carrier . despite the noted increase in drug accumulation in 
 the liver , h&e analysis revealed no sign of adverse effects or 
 off - target toxicity in the liver ( figure s1 in the supporting information ) . weights ( g ) of tissues collected at conclusion 
 of the study ( 5 days postinjection ) : liver , spleen , lung , kidneys , 
 heart , and tumor . error bars represent  sem ( * p = 0.50.1 ) . while our data , and other literature reports on polympc , 
 point to the safety of its use in vivo , we are not 
 aware of prior reports that examine potential in vivo immunogenicity arising from its presence in the bloodstream . thus , 
 in conjunction with these in vivo efficacy studies , 
 we sought to gauge whether there were innate or adaptive immune system 
 responses to polympc , accomplished through a complete blood count 
 ( cbc ) , and measurement of cytokine responses by an enzyme - linked immunosorbent 
 assay ( elisa ) ( figure 5 ) . analysis of serum 
 cytokine and white blood cell ( wbc ) levels indicated an initial increase 
 in total wbc count on day 3 with polympc  dox ( figure 5a ) , with no differences noted on day 5 ( figure 5b ) . the initial increase in white blood cell count , 
 suggestive of a foreign antigen response , was rectified by day 5 . red blood cell ( rbc ) counts indicated no differences across the treatment 
 groups . furthermore , we observed no significant differences between 
 polympc  dox and hbss in th1 versus th2 cytokines by elisa at 
 day 3 , with only a slight decrease in il12 and il10 noted at day 5 . however , this can not be attributed to the polympc carrier , since this 
 decrease is much more pronounced in the case of animals treated with 
 free dox ( figures 5c5h ) . these results suggest that polympc - bound dox does not 
 elicit significant adverse immunogenic effects that would produce 
 undesired anemia or inflammatory response in animals . analysis of immune response 
 across all treatment groups using complete blood count ( cbc ) and elisa 
 cytokine measurements 3 and 5 days after injection : ( a ) white blood 
 cell count ( wbc ) ( day 3 ) ; ( b ) wbc ( day 5 ) ; ( c ) interferon- 
 ( ifn- ) ( day 3 ) ; ( d ) ifn- ( day 5 ) ; ( e ) interleukin-12 
 ( il-12 ) ( day 3 ) ; ( f ) il-12 ( day 5 ) ; ( g ) interleukin-10 ( il-10 ) ( day 
 3 ) ; ( h ) il-10 ( day 5 ) . 4t1 tumor - bearing mice were used to evaluate the 
 therapeutic performance of polympc  dox conjugates for the treatment 
 of breast cancer . tumors were established by orthotopic injection 
 of murine 4t1 cells ( 5  10 4t1 cells in hanks balanced 
 salt solution , hbss ) into the mammary fat pad of the mice ( 17.5 
 g , 4 weeks old ) . the mice were randomized into three groups of 15 ; 
 at a tumor volume in the range of 42132 mm , mice 
 were administered either hbss , free dox ( 3 mg / kg , 1/2 mtd ) , 
 or polympc  dox ( 15 mg / kg dox equivalent , 1/2 mtd ) by 
 tail vein injection . subsequent doses were administered on days 7 
 and 17 ( polympc  dox group only ) . mice were examined and weighed 
 every 24 days , and tumor volume was determined by caliper 
 measurements ( calculated by 0.52  l  w ) over a period of 29 days . mice were removed 
 from the study when the tumor volume reached 1500 mm , 
 if weight loss exceeded 20% , or if the animal showed 
 signs of stress , such as a scruffy appearance or abnormal behavior . notably , mice receiving 
 the free dox treatment showed no improvement , essentially mirroring 
 results for the hbss group ; all these mice were removed from the study 
 by day 18 . in contrast , 80% of the mice receiving the polympc dox 
 treatment remained in the study at day 18 , with overall survival in 
 the polympc dox group extended almost 2-fold ( 29 days ) compared 
 to the other treatment groups . figure 6b shows 
 that tumor growth was greatly suppressed in the mice receiving polympc dox , 
 whereas the mice receiving free dox showed no difference relative 
 to the untreated mice . untreated and free dox treated mice surviving 
 to day 18 displayed tumors with average volumes ranging from 1600 
 to 1850 mm , requiring their removal from the study . polympc dox 
 treated mice at day 18 had average tumor volumes of 1050 mm , and at the day 29 end point , average tumor volume was 1170 mm . the weights of the mice overall remained largely unchanged 
 over the course of the study , as shown in figure 6c . however , following the third dose of polympc  dox , 
 animal weights did not return to the normal range , 
 necessitating their removal from the study . at the conclusion of the 
 study , tissue samples ( liver , spleen , heart , kidney ) were collected 
 and analyzed to compare to the results obtained from the pk / biodistribution 
 study , with the mice from the efficacy study displaying comparable 
 tissue weights among the treatment groups , with the exception of the 
 lungs ( figure s2 in the supporting information ) . the significant weight increase in the lungs of the polympc dox 
 group is attributed to the numerous metastases in the lungs , likely 
 due to the prolonged survival of the mice in this group ( two times 
 that of the free dox and hbss mice ) . h&e analysis indicated no 
 apparent off - target toxicity in the liver even at the higher doses 
 of polympc dox used in the efficacy study , despite the previously 
 noted accumulation ( figure s3 in the supporting 
 information ) . dox 
 treatment were dosed below the previously determined mtd for these 
 conjugates , so that the cumulative dose received did not exceed the 
 mtd . dox is nearly cleared 
 within 48 h , future animal studies will examine a more frequent dosing 
 regimen , aiming toward complete tumor regression . nonetheless , this 
 experiment confirms the efficacy of polympc  dox prodrugs , even 
 when presented with aggressive , highly metastatic 4t1 cancer in live 
 animals . summary of efficacy data in 4t1 mouse model : ( a ) survival curve for 
 mice treated with hbss ( squares , solid line ) , polympc  dox ( triangles , 
 dashed line ) , and free dox ( inverted triangle , dotted line ) ; ( b ) tumor 
 growth over time for mice treated with hbss ( squares , solid line ) , 
 polympc  dox ( triangles , dashed line ) , and free dox ( inverted 
 triangle , dotted line ) ; ( c ) mouse weight for mice treated with hbss 
 ( squares , solid line ) , polympc  dox ( triangles , dashed line ) , 
 and free dox ( inverted triangle , dotted line ) arrows indicate days 
 on which treatments were administered : 0 , 7 , and 17 ( polympc  dox 
 only ) . the present study demonstrates the ability of 
 polympc  dox to prolong circulation half - life of dox from 15 min to 
 2 h , with more favorable drug accumulation in the tumor as opposed 
 to healthy tissue , and no significant innate or adaptive immunogenic 
 response . moreover , we demonstrate the efficacy of polympc  dox 
 in 4t1 tumor - bearing mice , increasing the overall survival 2-fold , 
 and significantly reducing tumor growth in mice . the aggressive 4t1 
 mouse model reveals the potential for polympc  dox in the treatment 
 of triple negative breast cancer , and ongoing studies include evaluating 
 the in vivo efficacy against a human breast cancer 
 cell line .
we 
 report the in vivo efficacy , in tumor - bearing mice , 
 of cancer prodrugs consisting of poly(methacryloyloxyethyl phosphorylcholine ) 
 ( polympc ) conjugated to doxorubicin ( dox ) . 
 our synthesis of polympc 
 dox 
 conjugates established prodrugs with tunable drug loading , ph sensitive 
 release kinetics , and a maximum tolerated dose in the range of 3050 
 mg / kg ( dox equivalent ) in healthy mice . here we show prolonged circulation 
 of polympc  dox , with a measured in vivo half - life 
 ( t1/2 ) 8 times greater than that of the 
 free drug . 
 we observed reduced drug uptake in healthy tissue , and 
 23 times enhanced drug accumulation in tumors for polympc 
 dox 
 prodrugs compared to free dox , using balb / c mice bearing 4t1 tumors . 
 prolonged survival and reduced tumor growth were observed in mice 
 receiving the polympc 
 dox prodrug treatment . 
 moreover , we evaluated 
 immunogenicity of polympc  dox prodrugs by examining complete 
 blood count ( cbc ) and characteristic cytokine responses , demonstrating 
 no apparent innate or adaptive immune system response .