much of current cancer research is focused 
 on exploring novel targets 
 and discovering new anticancer drugs . while the availability of new 
 molecules certainly offers hope to patients who do not respond to 
 existing drugs , development of each new molecule
many highly active drug candidates 
 have been shelved because of poor physicochemical or pharmacokinetic 
 characteristics .
reformulation of such molecules can overcome their 
 unfavorable biological behavior and would offer a less expensive approach 
 to anticancer drug development .
tylocrebrine , a phenanthropiperidine alkaloid , is an example 
 of 
 a drug with potent anticancer activity and whose clinical trial was 
 discontinued following the discovery of severe central nervous system 
 ( cns ) toxicities .
the cns toxicities of the drug were likely caused 
 by its extensive penetration into the brain .
we 
 hypothesized that encapsulation of tylocrebrine in poly(lactide - co - glycolide ) ( plga)-based polymeric nanoparticles will 
 limit the distribution of the drug to the cns , potentially decreasing 
 its neurological toxicities .
additionally , nanoparticles are known 
 to passively accumulate in the tumor tissue through the enhanced permeability 
 and retention ( epr ) effect .
the combination of 
 these properties should result in significantly improved therapeutic 
 index . to further enhance the tumor cell uptake and retention of nanoparticles
, 
 we functionalized the nanoparticle surface with a peptide capable 
 of targeting the epidermal growth factor receptor ( egfr ) , which is overexpressed on the cell membrane of multiple solid tumors . using in vitro and in vivo models of egfr - overexpressing tumors
our studies show that this reformulation strategy significantly improved 
 the antitumor efficacy while reducing the brain penetration of tylocrebrine .
amine - terminated poly(ethylene glycol ) ( molecular 
 weight 3400 da ) was purchased from laysan bio inc .
plga 
 ( 50:50 molar ratio of lactide  glycolide , molecular weight 40 
 kda ) was purchased from lactel ( birmingham , al ) .
poly(vinyl alcohol ) 
 ( molecular weight 3070 kda ) and lactic acid were obtained 
 from sigma - aldrich co. ( st . louis , mo ) .
egfr - targeting peptide ( yhwygytpqnvi ) 
 and scrambled peptide ( hwpyahpthpsw ) were obtained from peptide 
 2.0 , inc .
radioimmunoprecipiation ( ripa ) buffer and 
 bicinchoninic acid assay kit were obtained from thermo scientific 
 ( rockford , il ) .
all other chemicals were obtained from sigma - aldrich 
 co. ( st . louis , mo ) .
a549 cells were cultured 
 in dulbecco s minimum essential media ( dmem ) , while a431 cells 
 were grown in roswell park memorial institute media ( rpmi-1640 ) .
both 
 media were supplemented with 10% v / v fetal bovine serum and 1% v / v 
 penicillin  streptomycin .
the cells were grown in a humidified 
 environment consisting of 5% co2/95% air and were maintained 
 at 37 c .
we determined the effect of extracellular 
 ph on the cell accumulation 
 of tylocrebrine . for low ph conditions ,
serum - free rpmi was acidified 
 with 9.1% v / v 0.1 m hcl .
the ph of acidified rpmi was maintained between 
 6.3 and 6.7 for 6 h when placed under routine cell culture conditions .
aliquots of 5 10 a431 cells were seeded in a 
 24-well plate and allowed to adhere overnight .
tylocrebrine was first 
 dissolved in 1 m hcl ( 1:1 molar ratio ) and then diluted in serum - free 
 rpmi to prepare a stock solution of 1 mg / ml . the stock solution was 
 then diluted to 5 g / ml in neutral or acidic serum - free rpmi 
 and added to the cells .
treatments were removed 1 h later , and the 
 cells were washed with cold 1x phosphate buffered saline ( pbs ) .
cells 
 were digested with ripa buffer ( 0.1 ml ) for 15 min , and the cell lysate 
 was divided into two parts .
one part ( 20 l ) was analyzed by 
 bicinchoninic acid assay ( bca ) to determine cell protein concentration 
 ( elx800 absorbance microplate reader , biotek inc . ,
winooski , vt ) . 
 the other part ( 80 l ) was extracted overnight with methanol 
 and tylocrebrine concentration in the methanol extract and was analyzed 
 using high - performance liquid chromatography ( hplc ) .
hplc was performed 
 on a beckman coulter hplc system equipped with a system gold 508 autosampler . 
 a beckman coulter c18 column (
4.6 mm  250 mm , 5 m ) was 
 used as the stationary phase . the mobile phase consisted of 80:20 
 mix of acetonitrile and 87 mm ammonium acetate ( ph 4.2 ) , run isocratically 
 at a flow rate of 1 ml / min .
tylocrebrine was analyzed by measuring 
 absorbance at 265 nm using a system gold uv detector .
a block copolymer of poly(lactide ) 
 ( pla ) and carboxyl - terminated poly(ethylene glycol ) ( peg - cooh ) was 
 synthesized in a two - step process . in the first step , lactic acid 

was reacted with amine - terminated peg to generate an amine - terminated 
 block copolymer ( pla  peg - nh2 ) . in the second step ,
the terminal amino group was reacted 
 with succinic anhydride to produce pla  peg - cooh .
all glassware was rinsed with toluene 
 and dried overnight at 100 c prior to both reactions . for step 
 one , amine - terminated peg ( 400500 mg ) was dissolved in 80 
 ml of dichloromethane and added to a round - bottom flask .
lactic 
 acid ( 2 g ) was added to this mixture and stirred for 10 min .
about 
 20 l of 1,8-diazabicycloundec-7-ene was 
 added as a catalyst , and the reaction was allowed to proceed for 1 
 h. after 1 h , the solvent was reduced to 20 ml using a rotovap . 

the solution was added dropwise to chilled diethyl ether to precipitate 
 the amine terminated block copolymer .
the suspension was filtered , 
 and the solid was dried at 25 c in a vacuum oven .
the product 
 was dissolved in deuterated chloroform and characterized using h nmr ( varian 400 mhz ) . for step two ,
the amine terminated 
 block copolymer ( 1 g ) was dissolved 
 in 50 ml of tetrahydrofuran and 5 ml of triethyl amine in a round - bottom 
 flask .
succinic anhydride ( molar ratio of succinic anhydride to block copolymer 
 was 1.1:1 ) was added to this solution .
the reaction was allowed to 
 proceed at 50 c for 2 h. the reaction mixture was then concentrated 
 using a rotavap and added to cold diethyl ether to precipitate the 
 polymer .
plga nanoparticles loaded with tylocrebrine 
 and surface functionalized with carboxyl - terminated peg were synthesized 
 by the interfacial activity assisted surface functionalization technique 
 developed by our lab .
briefly , plga ( 3035 
 mg ) and tylocrebrine ( 5 mg ) were dissolved in 1 ml of chloroform . 

an aqueous solution of 2% w / v poly(vinyl alcohol ) in pbs ( 0.15 mm , 
 ph 7.4 ; henceforth referred to as 1x pbs ) was prepared .
drug 
 mixture was added to the aqueous surfactant to form an o / w emulsion . 

the emulsion was probe sonicated at an output of 1821 w for 
 5 min over an ice bath ( sonicator xl , misonix , ny ) and then stirred 
 at 650 rpm on a magnetic stir plate .
peg - cooh ( 8 mg ) dissolved 
 in 0.2 ml of chloroform was added dropwise to the emulsion .
the organic 
 solvent was evaporated overnight under ambient conditions and then 
 for 2 h under vacuum .
the nanoparticle dispersion was washed twice 
 with 30 ml of 1x pbs by ultracentrifugation ( 35  000 rpm , 35 
 min , 4 c ) ( beckman , palo alto , ca ) .
after the second wash , nanoparticles 
 were dispersed in 1.5 ml of 1x pbs . to this dispersion , 0.7 mg of n - hydroxysuccinimide ( nhs )
, 1.14 mg of 1-ethyl-3-[3-(dimethylamino)propyl ] 
 carbodiimide hydrochloride ( edc ) , and 1.8 mg of targeting or control 
 peptide were added , and the reaction was allowed to proceed for 3 , 
 5 , or 8 h. following the conjugation reaction , nanoparticles were 
 dispersed in 30 ml of deionized water and centrifuged ( 35  000 
 rpm , 35 min , 4 c ) to remove unconjugated peptide .
the nanoparticle 
 pellet was redispersed in 10 ml of deionized water , frozen 
 below 50 c for 2 h , and lyophilized ( labconco freezone 
 4.5 , kansas city , mo ) .
the lyophilized formulation was stored at 20 
 c . to determine particle size and zeta potential ,
a dispersion 
 of 
 nanoparticles ( 1 mg / ml ) was analyzed by dynamic light scattering 
 ( delsa nano c , beckman coulter , fullerton , ca ) .
to determine 
 drug loading , nanoparticles were dispersed in a mixture 
 of methanol and acetic acid ( 95:5 v / v ) .
nanoparticles were separated from the extract 
 by centrifugation ( 14  000 rpm , 15 min , 4 c ) .
the amount of peptide on the surface of 
 nanoparticles was determined 
 using bca .
nanoparticles were dispersed in deionized water and incubated 
 with the bca reagent for 20 min at 37 c .
the nanoparticle dispersion 
 and standards were then centrifuged ( 14  000 rpm , 15 min , 25 
 c ) , and the absorbance of the supernatant was measured at 562 
 nm ( elx800 absorbance microplate reader , biotek inc . ,
nanoparticles 
 were dispersed in a sufficient volume of 1x pbs ( ph 7.4 or 6.5 ) to 
 maintain sink conditions .
the dispersions were kept on a shaker at 
 37 c and 100 rpm [ c24 incubator shaker , new brunswick scientific 
 ( now eppendorf inc .
the released 
 drug was separated from nanoparticles using a microkros filter module 
 ( spectrum laboratories , rancho dominguez , ca ) .
drug solution was lyophilized , 
 dissolved in a mixture of methanol and acetic acid ( 95:5 v / v ) , and 
 analyzed by hplc .
we first determined whether cellular uptake 
 of egfr - targeted nanoparticles 
 was higher than nontargeted nanoparticles and if their uptake was 
 mediated by egfr .
a549 cells were seeded in a 24-well plate at a seeding 
 density of 5  10 cells / well and allowed to adhere 
 overnight .
on the next day , nanoparticles loaded with a fluorescent 
 dye ( coumarin 6 ) , in the presence or absence of excess free targeting 
 peptide , were added to the cells .
cells were incubated with the treatments 
 at 4 c for 1 h , washed twice with 1x pbs , and incubated with 
 fresh media at 37 c .
after 1 h , the media was aspirated , cells 
 were washed with 1x pbs , and lysed with ripa buffer .
one part of the 
 lysed cells was extracted with methanol , and coumarin 6 content was 
 determined using hplc .
the other part 
 was analyzed by bca assay to determine the amount of cell protein .
we also compared the cell uptake of targeted and nontargeted nanoparticles 
 loaded with tylocrebrine .
a431 cells were seeded in a 24-well plate 
 as described previously . on the day of the experiment , tylocrebrine - loaded 
 nanoparticles , dispersed in serum - free media ,
after 1 h , the media was aspirated , 
 and the cells were washed thrice with cold 1x pbs .
the cells were 
 then lysed with 0.1 ml of ripa buffer for 15 min .
one part ( 20 l ) was analyzed by 
 bca assay to determine the amount of cell protein .
the other part 
 ( 80 l ) was extracted with a mixture of methanol and acetic 
 acid ( 95:5 v / v ) .
we compared the tumor cell kill efficacy of 
 free drug , nontargeted , and targeted nanoparticles in both neutral 
 and acidic media .
a549 cells or a431 cells were seeded in a 96-well 
 plate ( 8  10 cells / well ) and allowed to attach overnight . 

nanoparticles or free drug were dispersed in serum - free rpmi media 
 to form a stock solution .
this stock solution was then diluted with 
 either neutral or acidified serum - free media and added to the cells 
 at various dilutions .
the treatments were removed 6 h later , and the 
 cells were washed with cold 1x pbs .
the cells were then incubated 
 with serum containing media for further 90 h. cell viability was measured 
 using celltiter 96 aqueous one solution cell proliferation 
 assay kit ( promega , madison , wi ) according to the supplier s 
 protocol .
all the animal studies described here were 
 approved by the university of minnesota s institutional animal 
 care and use committee .
a431 cells ( 12  
 10 ) were dispersed in 0.05 ml of 1x pbs and injected subcutaneously 
 in female athymic nude mice ( 46 weeks old , taconic biosciences , 
 hudson , ny ) .
mice were lightly anesthetized using isofluorane and 
 treated with various formulations of tylocrebrine ( dose , 12 mg / kg ; 
 dosing volume , 10 ml / kg ) via retroorbital injection .
the free 
 drug formulation was prepared by dissolving the drug in 
 1 m hcl ( 1:1 molar ratio ) and diluting the solution with saline .
excess 
 acid was neutralized with 1 m naoh to obtain a final drug concentration 
 of 1.2 mg / ml . to prepare nanoparticle treatments , nanoparticles were 
 dispersed in saline and probe sonicated at an output of 1821 
 w thrice for 30 s each on an ice bath .
the nanoparticle dispersion 
 was centrifuged ( 1000 rpm , 5 min ) to remove any large aggregates , 
 and the supernatant was used . at various time points ,
cohorts 
 of mice were sacrificed , and blood 
 was collected by cardiac stick .
the dry organs were then extracted overnight with a mixture of methanol 
 and acetic acid ( 95:5 ) .
the dried residue was redispersed 
 in a mixture of acetonitrile and acetic acid ( 95:5 ) .
the resulting 
 suspension was centrifuged ( 14  000 rpm , 15 min , 4 c ) , 
 and the supernatant was used for lc 
ms / ms 
 was performed using an acquity ultra - performance 
 liquid chromatography ( uplc ) system equipped with a waters / micromass 
 quattro ultima mass spectrometer .
lc was performed using agilent xdb - c18 
 column ( 4.6 mm  50 mm , 1.8 m ) as the stationary phase . 

a mixture of 10 mm ammonium acetate with 0.06% v / v acetic acid and 
 acetonitrile ( 55:45 v / v ) was used as the mobile phase .
the flow rate 
 was 0.4 ml / min , and the run time was set to 6 min .
the mass spectrometer conditions 
 were as follows : cone voltage , 50 v ; collision voltage , 20 v ; dwell 
 time , 0.4 s. tylocrebrine was detected by monitoring the m / z transition of 394  324.9 .
concentration 
 of tylocrebrine was normalized to tissue weight and 
 injected dose and was represented as % injected dose ( id)/g of organ 
 weight .
drug exposure in each tissue was determined by calculating 
 the area under the concentration curve ( auc ) .
the relative benefit of using the nanoparticulate 
 formulation over the free drug was determined using the drug targeting 
 index ( dti ) .
the dti was calculated as 
 shown below : the therapeutic efficacy of 
 various formulations of tylocrebrine was determined in a mouse model 
 of epidermoid cancer .
about 1  10 a431 cells , suspended 
 in 50 l of 1x pbs , were injected subcutaneously in female nude 
 mice .
when tumor 
 volume reached 75 mm , animals were treated with 
 saline , free drug , nontargeted nanoparticles , or egfr - targeted nanoparticles 
 ( three doses , every 96 h , 12 mg / kg ) .
the treatments were prepared 
 and administered as described in the biodistribution study . at the end of the study , animals were sacrificed , and the tumors 
 were excised .
the tumors were fixed using 5% formalin solution in 
 1x pbs for 24 h. after the initial fixation , tumors were preserved 
 in 70% ethanol .
microtome sectioning was performed on the fixed and 
 mounted tumor samples , and the sections were stained for cleaved caspase 
 3 ( to determine apoptosis ) and ki67 ( as a marker of proliferation ) . 

staining of tumor sections was quantified using imagej 1.48v software . 
 to determine apoptotic and proliferative indices , the fraction of 
 the total cellular area that stained positive for the individual markers 
 was determined and presented as apoptotic and proliferative indices .
all statistical analyses were 
 performed using one - way analysis of variance ( anova ) and posthoc tukey 
 test . for the efficacy study ,
slopes of the tumor growth profile were analyzed at 
 each time point for each animal .
one - way anova and posthoc tukey test 
 were performed to determine if the differences in various treatments 
 were statistically significant .
tumors are often characterized 
 by increased production of lactic 
 acid due to the warburg effect .
impaired 
 drainage of the acid from the tumor microenvironment leads to low 
 extracellular ph in the tumor .
ionization 
 of the drug molecule can decrease its diffusion across the cell membrane 
 and hence lead to decreased intracellular drug availability .
we measured the effect of extracellular ph on 
 drug accumulation in a431 cells ( figure 1 ) . 

decreasing the ph of the media from 7.4 ( physiologic ) to 6.36.7 
 ( to mimic tumor microenvironment ) resulted in reduced drug uptake 
 ( 60% reduction ) .
this is likely due to the protonation of 
 the indolizidine nitrogen , leading to a positive charge on the molecule .
tylocrebrine ( as free drug ) was incubated with a431 cells at an extracellular 
 ph of 7.4 ( physiological ) or 6.5 ( acidic ) .
data represented as mean  sd , n = 
 6 ,  indicates p < 0.005 . to overcome this unfavorable ionization of tylocrebrine 
 and reduced 
 availability inside the cells , we formulated the drug in egfr - targeted 
 polymeric nanoparticles .
nanoparticles loaded with tylocrebrine and 
 surface functionalized with peg - cooh were first fabricated using the 
 interfacial activity assisted surface functionalization ( iaasf ) technique .
these nanoparticles were then conjugated to either the 
 egfr - targeting peptide or a control , nontargeting peptide .
peg - cooh 
 block copolymer was used to incorporate carboxyl groups on the surface 
 of plga nanoparticles .
peg - cooh through 
 a two - step reaction involving the generation of pla  peg - nh2 from peg - nh2 followed by the reaction of the terminal 
 amino group with succinic anhydride .
the nmr spectra of peg - nh2 , pla  peg - nh2 , and pla  peg - cooh are 
 shown in figure 2 .
conjugation of lactide to 
 peg was confirmed by the appearance of peaks at  = 5.3 ppm 
 and  = 1.6 ppm .
conjugation of 
 succinate moiety to pla  peg - nh2 was confirmed by 
 the appearance of a peak at  = 2.6 ppm . on the basis of the auc of the peaks ,
block 
 copolymer of pla and carboxyl - terminated peg were synthesized by a 
 two - step procedure . the starting material ( peg ) , intermediate product 
 ( pla  peg - nh2 ) , and final product ( pla  peg - cooh ) 

conjugation of lactide to peg was confirmed by the appearance 
 of peaks at 1.6 and 5.2 ppm .
conjugation of succinate to the terminal 
 amine group in peg was confirmed by appearance of a peak at 2.6 ppm .
we 
 initially determined the effect of reaction time on drug loading in 
 nanoparticles and efficiency of targeting peptide conjugation to the 
 surface of nanoparticles . increasing
the reaction time resulted in 
 an increase in peptide conjugation efficiency but resulted in decreased 
 drug loading ( figure 3 ) .
particle size and 
 zeta potential were unaffected by the duration of the reaction time . 
 in general ,
the particle size was found to be in the range of 300400 
 nm , and the particles had a net negative zeta potential ( table 1 ) .
we used an intermediate reaction time ( 5 h ) for 
 achieving optimum drug loading and conjugation efficiency .
the drug 
 and peptide loading for nanoparticles used in the rest of the studies 
 are summarized in table 1 .
egfr - targeting peptide was conjugated to the surface 
 of nanoparticles using nhs  edc chemistry .
the influence of 
 reaction time on ( a ) conjugation efficiency of the peptide and ( b ) 
 drug loading is shown .
the drug release profile from nanoparticles at physiological ph 
 is shown in figure 4 , panel a. both targeted 
 and nontargeted nanoparticle formulations showed a characteristic 
 initial burst release followed by sustained release over a period 
 of 48 h. drug release profiles were comparable for both targeted and 
 nontargeted nanoparticles .
nanoparticles 
 were dispersed in ( a ) 1x pbs ( ph 7.4 ) or ( b ) 1x pbs ( ph 6.5 ) and incubated 
 at 37 c and 100 rpm . drug release from nanoparticles was monitored 
 by analyzing tylocrebrine concentration in the release buffer using 
 hplc .
drug release from nanoparticles 
 was also determined at ph 6.5 ( figure 4b ) . 

however , the extent of burst release 
 in acidic ph was higher than that at physiologic ph ( 25% vs 
 50% ) . by using fluorescent dye labeled particles , we first determined 
 if 
 cellular uptake of targeted nanoparticles was greater than that of 
 nontargeted nanoparticles .
we found that targeted nanoparticles resulted 
 in 23-fold higher uptake than nontargeted nanoparticles . 

additionally , uptake of targeted nanoparticles was reduced ( not statistically 
 significant ) in the presence of excess free targeting peptide ( figure 5a ) .
this indicated that the enhanced uptake of targeted 
 nanoparticles could be mediated via egfr .
additional experiments investigating 
 uptake in egfr knockout cells are needed to confirm this finding .
( a ) 
 a549 cells were incubated with coumarin 6 loaded targeted nanoparticles 
 with or without excess peptide , or nontargeted nanoparticles for 1 
 h at 4 c .
treatments were removed , and the cells were incubated 
 in fresh media at 37 c for 1 h. intracellular levels of coumarin 
 6 were estimated using hplc .
data represented as mean  
 sd , n = 6 ,  indicates p < 
 0.05 . we determined the uptake of targeted 
 and nontargeted tylocrebrine 
 nanoparticles in a431 cells ( figure 5b ) .
we 
 found that encapsulation of tylocrebrine in targeted nanoparticles 
 increased the cellular drug uptake by about three - fold ( p < 0.05 ) .
we compared the in vitro efficacy 
 of various formulations of tylocrebrine in both a549 and a431 cells . 
 since our previous results showed that acidic ph led to a decreased 
 intracellular accumulation of tylocrebrine , we performed these studies 
 under both physiologic as well as acidic ph conditions .
the free drug 
 was found to be potent in both cell lines under neutral conditions , 
 with ic50 values of 210 and 37 nm ( figure 6a , c ) .
however , there was a dramatic decrease in the efficacy of tylocrebrine 
 under acidic conditions ( compare figure 6a , b 
 and 6c , d ) .
the ic50 values of tylocrebrine 
 increased from 210 nm and 37 nm to 432 nm and 361 nm , respectively . 
 at physiological ph ,
the efficacy of nontargeted tylocrebrine nanoparticles 
 was comparable to that of the free drug , while targeted tylocrebrine 
 nanoparticles were more effective than the free drug . with a decrease 
 in extracellular ph , the ic50 value of nanoparticle formulations remained 
 unchanged in a431 cells , while there was a small increase in ic50 
 for the nanoparticle formulations in a549 cells .
as a result , both 
 nontargeted and targeted tylocrebrine nanoparticles showed superior 
 efficacy than the free drug under acidic conditions .
the ic50 values 
 estimated from the efficacy studies are summarized in table 2 . in vitro cytotoxicity of tylocrebrine . 

a431 and 
 a549 cells were treated with different formulations of tylocrebrine 
 at physiological and acidic extracellular ph .
time profile of the drug in the tumor is shown 
 in figure 7 , panel a. accumulation of the drug 
 and targeted nanoparticles in the tumor was rapid and reached a peak 
 at 0.5 h. when encapsulated in nontargeted nanoparticles , tumor accumulation 
 was slower , reaching a peak at 2 h. at 0.5 , 2 , 4 , and 6 h 
 postdose , tumor tylocrebrine concentrations in targeted nanoparticle 
 treated group were 24-fold higher than free drug group .
tumor 
 concentration of tylocrebrine in animals treated with nontargeted 
 nanoparticles was two - fold higher than the free drug - treated animals 
 only at 2 and 4 h postdose ; the concentrations were similar to those 
 of the free drug treatment group at later time points .
aucs of the 
 free drug and nontargeted nanoparticles in the tumor were comparable . 

the auc of targeted nanoparticles was about three - fold higher than 
 that of the other formulations ( table 3 ) .
a431 tumor bearing mice were 
 treated intravenously with tylocrebrine in solution or encapsulated 
 in nanoparticles . at various time points , concentrations of tylocrebrine 
 in ( a ) tumor ,
data represented as mean  sem , n = 34 , 
  indicates p < 0.05 .
the 
 concentration  time profile of the drug in the brain 
 ( the major site of toxicity ) is shown in figure 7 , panel b. similar to that in the tumor , there was a rapid accumulation 
 of the drug in the brain . however , encapsulation in nanoparticles 
 resulted in a five - fold decrease in brain concentration of the drug 
 at the first time point .
overall , animals treated with free drug had a two - fold higher 
 drug exposure in the brain as compared to those treated with the nanoparticle 
 formulations ( table 3 ) .
targeted nanoparticles 
 had a dti value of 5 because of the reduced brain exposure 
 and enhanced tumor exposure . the concentration 
time profile 
 of the drug in the blood 
 is shown in figure 7 , panel c. nanoparticles 
 resulted in higher blood concentration of the drug relative to that 
 achieved after the administration of the free drug .
time 
 profiles of the drug in other key organs are summarized in supplemental 
 figure 1 of the supporting information .
we determined the antitumor 
 efficacy of the different formulations of tylocrebrine in a431 tumor 
 model ( figure 8) .
there was a reduction in 
 tumor growth rate in animals treated with tylocrebrine solution and 
 nontargeted nanoparticles relative to that in the saline - treated animals . 

treatment with nontargeted tylocrebrine nanoparticles and tylocrebrine 
 in solution resulted in comparable activities . in agreement with our 
 pharmacokinetic studies ,
treatment with egfr targeted tylocrebrine 
 nanoparticles resulted in greater tumor growth inhibition than that 
 with the other formulations .
antitumor efficacy 
 of various formulations of tylocrebrine was determined in mouse a431 
 tumor model .
tumor bearing mice were treated with three doses of tylocrebrine 
 ( 12 mg / kg ) administered at 96 h intervals .
 indicates p < 
 0.05 for saline versus targeted nanoparticles and nontargeted nanoparticles 
 versus targeted nanoparticles .
tumor samples were stained for ki67 and cleaved caspase 3 
 to determine 
 the proliferative and apoptotic indices , respectively .
saline - treated animals had significantly higher ki67 cells as compared to the other treatment groups .
there was 
 no significant difference in the staining profile of free drug and 
 nontargeted nanoparticle treated tumors .
an opposite profile was 
 observed for cleaved caspase 3 expression ( figure 10 ) .
cleaved caspase 3 staining was lowest in saline - treated 
 animals and highest in animals treated with targeted nanoparticles . 

taken together , these data suggest that treatment with tylocrebrine 
 inhibited tumor cell proliferation and induced apoptosis , with targeted 
 nanoparticles resulting in the greatest decrease in tumor cell viability .
tumors were excised at the end 
 of the efficacy study and stained for ki67 .
representative micrographs 
 of tumor from animals treated with ( a ) saline , ( b ) free drug , ( c ) 
 nontargeted nanoparticles , and ( d ) targeted nanoparticles are shown . 

data represented as mean  
 sem , n = 6 sections  3 images / section .  
 indicates p < 0.05 .
micrographs of cleaved 
 caspase 3 staining in tumors from animals treated with ( a ) saline , 
 ( b ) free drug , ( c ) nontargeted nanoparticles , and ( d ) targeted nanoparticles 
 are shown .
data represented as mean 
  sem , n = 6 sections  3 images / section . 

tylocrebrine was originally 
 isolated in 1962 from the north queensland vine , t. crebriflora . with accumulating evidence that this 
 family of phenanthropiperidine alkaloids had anticancer properties , 
 tylocrebrine was tested and found to be effective in several cancer 
 models .
unfortunately , there were unforeseen 
 problems with the drug , and clinical trials were terminated before 
 tylocrebrine s therapeutic efficacy could be established in 
 the clinic .
cns toxicity , as evidenced 
 by ataxia and disorientation , was considered the major reason for 
 abandoning clinical trials . in the decades that followed tylocrebrine
s 
 failure in the clinic , medical interest in tylocrebrine and other 
 members of its class waned .
recently , however , there has been a resurgence 
 of activity with regard to these natural products and their analogs . 

many of these alkaloids have been found to possess potent and broad 
 spectrum cytotoxicity .
several members of this class , including tylocrebrine , 
 were found to inhibit tumor cell growth with gi50 values in the low 
 nanomolar to subnanomolar range across the nci 60 cell line panel .
a major barrier to the clinical use of 
 tylocrebrine and other members 
 of its class is their neurological side effects . in a review , hitchcock and 
 pennington
show that the degree of passive diffusion across the blood  brain 
 barrier ( bbb ) correlates with a molecule s polar surface area 
 ( psa ) , number of h - bond donors ( hbd ) , clogp , clogd , and molecular 
 weight .
a calculation of these properties 
 for tylocrebrine reveals that each of these values is well within 
 the suggested parameters for cns penetrant drugs ( table 4 ) .
increasing the hydrophilicity of tylocrebrine can potentially 
 decrease its diffusion across the bbb . to this effect , lee and colleagues 
 recently reported water - soluble phenanthrene based tylophorine derivatives .
this effect is facilitated through binding of 
 the drug to the 40s and 80s ribosomal subunits .
tylocrebrine is a weak base and is mainly unionized at physiological 
 ph . because of its neutral charge and high hydrophobicity , tylocrebrine 
 can cross cell membranes efficiently under physiological conditions 
 ( for example , the bbb )
. however , under acidic conditions ( such as 
 those found in the tumor ) , tylocrebrine is ionized .
this is likely 
 to reduce its diffusion across the tumor cell membrane and its anticancer 
 activity . to address these twin problems of high cns penetration 
 and limited 
 tumor cell uptake , we encapsulated tylocrebrine in plga nanoparticles 
 surface functionalized with a peptide targeting the egfr .
plga nanoparticles , 
 likely due to their colloidal size range , do not cross the intact 
 bbb .
additionally , plga nanoparticles are rapidly taken up by cells 
 through endocytosis , a process augmented 
 by the presence of targeting ligands .
our in vitro studies showed that tylocrebrine 
 had potent activity with ic50 values in the nanomolar range .
encapsulation 
 in egfr - targeted nanoparticles further increased its potency . under 
 acidic conditions
, there was a dramatic decrease in the potency of 
 the free drug , while the activity of the nanoparticle formulations 
 showed a minor decrease .
this suggested that the issue of drug delivery 
 into the tumor cells could be addressed through nanoparticle encapsulation . 

the minor decrease in the efficacy of the nanoparticle formulations 
 can be attributed to the increase in the burst release observed in 
 acidic ph . upon iv
administration , we found that there was rapid 
 accumulation 
 of tylocrebrine in both the target tissue ( tumor ) and the site of 
 toxicity ( brain ) .
encapsulation of tylocrebrine in nontargeted nanoparticles 
 reduced the brain exposure but did not significantly improve the tumor 
 exposure . on the other hand , delivering tylocrebrine in targeted nanoparticles 
 resulted in both reduced brain exposure and improved tumor exposure . 

use of a targeting moiety 
 on the nanoparticle surface merely improves their accumulation at 
 the target site without affecting their accumulation at nontarget 
 sites .
usually , the target site has negligible 
 influence on the overall pharmacokinetics of the drug or drug carrier . 

there is still some debate in literature regarding whether targeting 
 ligands improve the tumor accumulation of nanoparticles or merely 
 increase their tumor cell uptake .
bartlett 
 et al . and choi et al . have shown that transferrin receptor targeted 
 nanoparticles have similar tumor accumulation to nontargeted nanoparticles . however , owing to improved tumor cell uptake , these nanoparticles 
 resulted in better efficacy . on the other hand , studies from our and 
 other laboratories have shown that nanoparticles targeting folic acid 
 receptor , biotin receptor , or egfr improved tumor accumulation of 
 encapsulated drug relative to that with nontargeted nanoparticles .
differences in tumor models , receptor expression levels , recycling 
 rates of different receptors , and affinities of targeting moieties 
 could contribute to these discrepancies .
other variables such as choice 
 of the drug , drug release kinetics and mechanisms , and plasma kinetics 
 of drug and drug carrier also make comparison between studies difficult . 

finally , current characterization techniques can not distinguish between 
 nanoparticle - encapsulated and free drug .
additional , in - depth characterization 
 studies are needed to draw strong mechanistic conclusions .
blood 
 concentrations of nanoparticle - encapsulated tylocrebrine 
 were significantly higher than the free drug ( about six - fold higher ) . 

this indicates that the driving force for accumulation of tylocrebrine 
 in tumors is much higher when administered in the form of nanoparticles . 
 however , the increase in tumor levels of tylocrebrine was less dramatic 
 ( only about three - fold with targeted nanoparticles ) .
this may be attributed 
 to the favorable partition coefficient of the free drug into the tumor , 
 as illustrated by a high tumor to blood concentration ratio ( > 10 ) . 

thus , the advantage gained by nanoencapsulation strongly depends on 
 the drug being investigated .
additionally , a431 
 tumors were found to be fluid - filled ( not shown ) , suggesting that 
 the interstitial fluid pressure in these tumors is high , as previously 
 reported .
high interstitial fluid pressure ( ifp ) is likely to affect the intratumoral transport of nanoparticles 
 more significantly than that of free drug molecules .
the relatively large hydrodynamic particle size of the nanoparticles 
 used in our studies may also decrease their tumor penetration .
decreasing 
 the particle size can potentially increase the delivery of nanoparticles 
 into the tumor .
alternately , tumors with a low 
 ifp may prove to be better candidates for treatment with these formulations .
the in vivo tumor growth inhibition studies show 
 that free drug inhibited tumor growth even at the low dose used .
while 
 treatment efficacy did not improve with nontargeted nanoparticles , 
 targeted nanoparticles resulted in a considerably greater tumor growth 
 inhibition than other treatments .
this effect may be a manifestation 
 of both the higher tumor tissue accumulation and higher cytotoxicity 
 of targeted nanoparticles .
the dose of tylocrebrine used in 
 our studies was relatively low 
 ( 12 mg / kg ) .
this dose was effective only if the treatment was started 
 before the tumors reached a volume of 75 mm .
larger 
 tumors did not respond to this dose of tylocrebrine in any formulation 
 ( data not shown ) .
the overall dose of tylocrebrine that could be administered 
 was limited by its loading in nanoparticles .
additionally , burst release 
 of drug from nanoparticles may limit the overall efficiency of targeting 
 and chemotherapeutic efficacy of nanoparticles .
strategies that enable 
 higher drug loading and prolonged drug release will allow for larger 
 doses to be administered , and this could further enhance the antitumor 
 efficacy of the drug . using polymers that have greater interaction 
 with the drug can help achieve this goal . to this end , micelles formed 
 from polymers consisting of aromatic rings have been shown to improve 
 drug loading and stability as compared to micelles formed from aliphatic 
 polymers .
this has been attributed to the formation 
 of  stacks between the drug and polymer .
future 
 studies could investigate the use of such polymers for improving the 
 therapeutic index of tylocrebrine .
tylocrebrine 
 is a potent anticancer agent , but significant penetration 
 into the brain and low tumor cell uptake limit its use .
encapsulation 
 of the drug in plga nanoparticles significantly limited its cns penetration . 
 moreover , surface functionalizing nanoparticles with an egfr targeting 
 peptide led to enhanced tumor cell uptake , tumor tissue accumulation , 
 and in vivo antitumor efficacy .
we expect that the 
 reformulation approach presented here will enable further clinical 
 testing of a number of previously abandoned drug candidates while 
 potentially minimizing drug development costs .
 several 
 promising anticancer drug candidates have been sidelined 
 owing to their poor physicochemical properties or unfavorable pharmacokinetics , 
 resulting in high overall cost of drug discovery and development . 
 
 use of alternative formulation strategies that alleviate these issues 
 can help advance new molecules to the clinic at a significantly lower 
 cost . 
 tylocrebrine is a natural product with potent anticancer activity . 
 
 its clinical trial was discontinued following the discovery of severe 
 central nervous system toxicities . to improve the safety and potency 
 of tylocrebrine , we formulated the drug in polymeric nanoparticles 
 targeted to the epidermal growth factor receptor ( egfr ) overexpressed 
 on several types of tumors . through in vitro studies 
 in different cancer cell lines , we found that egfr targeted nanoparticles 
 were significantly more effective in killing tumor cells than the 
 free drug . in vivo 
 pharmacokinetic studies revealed 
 that encapsulation in nanoparticles resulted in lower brain penetration 
 and enhanced tumor accumulation of the drug . 
 further , targeted nanoparticles 
 were characterized by significantly enhanced tumor growth inhibitory 
 activity in a mouse xenograft model of epidermoid cancer . 
 these results 
 suggest that the therapeutic index of drugs that were previously considered 
 unusable could be significantly improved by reformulation . application 
 of novel formulation strategies to previously abandoned drugs provides 
 an opportunity to advance new molecules to the clinic at a lower cost . 
 
 this can significantly increase the repertoire of treatment options 
 available to cancer patients . 
Introduction
Materials
and Methods
Results
Discussion
Conclusion