multidrug resistance 
 ( mdr ) of cancer constitutes a significant 
 challenge to clinical oncology and serves as one of the major reasons 
 for tumor recurrence .
cancer stem cells ( cscs ) were discovered in 
 various types of cancer , including breast , colon , colorectal , lung , glioma , and pancreatic cancers .
recently , 
 cscs have been linked to the mdr since cscs are often enriched in 
 tumors after conventional chemotherapies ; and cscs were found more resistant to conventional 
 chemotherapeutics than differentiated tumor cells .
mechanisms 
 that cscs utilize to achieve drug resistance include entering a quiescent 
 cell cycle status , possessing a high 
 dna repair capacity , avoiding apoptosis , upregulating atp - binding cassette transporter , and overexpressing of detoxification enzymes such as aldehyde dehydrogenase 
 1 .
the distinct susceptibility 
 to conventional chemotherapeutics between 
 cscs and differentiated tumors leads to enrichment of the csc populations 
 in some tumors treated by conventional chemotherapeutics .
what aggravates this alarming 
 situation is cscs ability to recapitulate heterogeneity and 
 hierarchy .
therefore , cscs not only are 
 one important reason for mdr but also play a critical role in progression 
 and metastasis of cancer .
consequently , 
 a failure of eradicating cscs in tumors could have a huge adverse 
 impact on the prognosis of cancer . due to this rationale ,
an effective 
 way to elimination of cscs becomes critical to prevent mdr development 
 and results in a favored prognosis during cancer treatment by conventional 
 chemotherapeutics .
because of the role of cscs in tumorigenesis 
 and the mdr , there 
 is an intensive , ongoing effort to discover new compounds that have 
 selective toxicity to cscs .
proposed mechanisms 
 behind the selective toxicity of sali include p38 mapk activation , accumulation of reactive oxygen species , inhibition of wnt signaling pathways , and inhibition of p - glycoprotein activity by 
 sali .
consistent with its selective toxicity 
 to cscs and the role of cscs in causing mdr , sali can sensitize a 
 wide spectrum of tumor cell lines that would be otherwise resistant 
 to conventional cancer chemotherapeutics such as paclitaxel ( ptx ) . while using sali to eradicate cscs
may 
 have immediate clinical 
 benefits , such an application is currently hindered by poor solubility 
 of sali , as well as its toxicities to healthy tissues and cells in 
 mammals . on the other hand
, nanocarriers have in general been 
 shown to increase tumor accumulation of small molecule drugs , redistribute 
 their accumulation in organs , and/or resolve their solubility issues .
therefore , a nano delivery system is highly desired that can help 
 sali overcome the solubility issue and increase its accumulation in 
 tumors while reducing its distribution in off - target organs .
recently , 
 a hyaluronic acid based nanogel was shown in vitro to facilitate the uptake of sali to cscs , which are cd44 positive . 

the increased uptake compared to free sali was attributed to interactions 
 between hyaluronic acid and cd44 . meanwhile , 
 another group used peg - b - pcl polymeric micelles to 
 deliver sali as encapsulated drugs and found that the micelles improved 
 csc - specific toxicity of sali in vivo using a mouse 
 subcutaneous , xenograph model of human breast cancer . however , the 
 improvement was marginal as the overall therapeutic outcomes resulted 
 from micelle - delivered sali and free sali treatments were indifferent 
 statistically .
we speculated that 
 one reason for the aforementioned suboptimal 
 therapeutic outcomes of sali may be that the micellar carrier failed 
 to accumulate more sali in tumors than the free form of sali , and 
 hence did not result in a stronger inhibition to tumor growth .
the 
 speculation can not be answered as the report did not include pharmacokinetics 
 and tumor accumulation evaluations . here 

we seek to develop a novel and biocompatible nanoparticle ( np ) carrier 
 to deliver sali to clinically and biologically relevant orthotopic 
 breast tumors .
we hypothesize that the np using sali as its hydrophobic , 
 together with our new encapsulation method , can deliver satisfactory 
 pharmacokinetics and tumor accumulation of sali , which in turn lead 
 to a better therapeutic outcome of sali . to this end
, we first obtained 
 nps self - assembled from the conjugates between sali and itep , a biodegradable 
 recombinant polypeptide we invented recently ( m. chen , personal communication ) ; 
 the immune tolerance nature of itep may prove an advantage when the 
 carrier is administered repeatedly and tested in clinical trials , 
 in which cases the immunogenicity of therapeutics becomes a focal 
 point of consideration .
sali nps 
 to encapsulate free sali , which is a novel strategy for sali delivery . 

then , we found that encapsulated sali was released in a controlled 
 manner and maintained csc - selective toxicity of sali .
we also discovered 
 that coencapsulation of sali with two additives , -tocopherol 
 and dmha , noticeably improved the loading efficiency and drug release 
 profile of the sali .
another benefit of the resulting nps is that 
 they significantly retarded in vivo release of the 
 sali and therefore led to better pharmacokinetics and tumor accumulation 
 profiles of the sali . through tumor regression study , we demonstrated 
 that the encapsulated sali was able to inhibit the growth of bulk 
 tumors because of these desired properties conferred by the nps .
lastly 
 and most importantly , the np - delivered sali depleted cscs in breast 
 tumors more efficiently than free sali .
all chemicals , unless otherwise described , 
 were purchased from thermo fisher scientific inc .
organic solvents including acetonitrile ( acn ) , 
 dichloromethane ( dcm ) , dimethylformamide ( dmf ) , isopropanol , and methanol 
 were purchased from thermo fisher scientific inc .
the cell 
 culture media and supplements including rpmi-1640 ( with 2 mm l - glutamine ) , media 199 , and fetal bovine serum ( fbs ) were purchased 
 from life technologies , inc .
all antibodies 
 were purchased from biolegend ( san diego , ca , usa ) including pe anti - mouse 
 cd24 antibody ( cat . # 101808 ) , apc anti - human cd44 antibody ( cat . 

4t1 , a highly metastatic murine cell line derived 
 from a spontaneous 
 syngeneic breast cancer of balb / c mice , was purchased from american 
 type culture collection ( rockville , md , usa ) .
4t1 cells were maintained 
 in monolayer cultures within an rpmi 1640 medium supplemented with 
 10% fbs .
female balb / c mice that were 2428 
 days old ( 1819 
 g ) were purchased from charles river laboratories international , inc . 

all the animal experiment protocols were approved by the institutional 
 animal care and use committee at the university of utah .
iteps or itep  sali conjugates were 
 measured using the malvern 
 zetasizer nano ( malvern , chester county , pa , usa ) at 25 m in 
 pbs at 37 c using a low - volume disposable sizing cuvette or 
 a clear disposable zeta cell . before size measurements ,
all samples 
 were reduced overnight in a 100 mm tcep solution to reduce possible 
 disulfide bonds .
the instrument 
 settings include the following : material ri = 1.59 , material absorption 
 = 0.010 , water dispersant ri = 1.330 , viscosity = 0.6864 cp .
the instrument 
 was allowed to automatically optimize the count rate , duration , and 
 attenuator .
specifically , for zeta potential determination , the dispersant 
 dielectric constant was set as 74.4 .
5.0 mg of 
 itep  sali nps and 3.0 mg of salinomycin were codissolved in 
 125 l of dmf and mixed well . in some cases , 13.34 mg of dmha 
 ( sigma - aldrich , st .
louis , mo , usa ) and 0.87 mg of -tocopherol 
 ( sigma - aldrich , st .
the ratio of the above components was based on our independent trial 
 experiments and previous reports .
the mixture 
 was then supplemented with 750 l of milli - q water in a dropwise 
 manner .
the mixture was stirred for 0.5 h at room temperature before 
 being dialyzed against 3
the 
 complete removal of dmf was critical and was monitored using a characteristic 
 absorption of dmf at 210 nm .
the product solution was filtered through 
 0.45 m acrodisc syringe filters with supor membrane ( pall corporation , 
 port washington , ny , usa ) and concentrated by amicon ultra-15 centrifugal 
 filter units ( mw cutoff = 3000 da ) .
the loading efficiency was defined 
 as in the following equation : in 
 vitro release profile of sali from itep 
sali nps 
 was measured by a previously described dialysis method with minor 
 modification .
sali nps with 
 or without encapsulated sali were diluted in 0.5 ml of milli - q water 
 containing 4% bsa and kept in dialysis bags ( spectrum laboratories , 
 inc . ,
rancho dominguez , ca , usa , mw cutoff = 8000 da ) . the bags were 
 then immersed into 100 ml of pbs solution ( ph = 7.4 ) in a beaker , 
 respectively .
the entire dialysis systems were shaken at 100 rpm at 
 37 c . at predetermined time points ,
the 0.5 ml sample solutions 
 in the bags were mixed and 10 l of sample was collected from 
 each sample .
sali inside the bags was presumed and measured as unreleased 
 drug in the nps .
the relationship of 
 the percentage of sali release ( f ) with time ( t ) was fitted using the following equation and graphpad 
 v5.0.k is the release rate constant : pharmacokinetics 
 studies were carried out according a protocol modified from a previous 
 report .
sali nps or sali 
 were injected in a 100 l solution into balb / c mice via the 
 tail vein at 5 mg / kg free sali or encapsulated sali dose .
the sali 
 solution used for the injection contained 1% dmso to dissolve sali 
 ( sigma - aldrich , st .
about 50 l blood samples were collected by submandibular bleeding 
 into tubes containing 150 l of pbs with heparin at 1,000 u / ml . 
 blood plasma was collected from blood samples after samples were centrifuged 
 to pellet blood cells .
next , 100 l of isopropanol was added 
 to each plasma sample to precipitate proteins .
the supernatants were collected ; concentrations 
 of sali in the supernatant were determined by the precolumn derivitization 
 with dnph .
the changes of plasma sali concentrations 
 over time were fitted to a two - compartment model using graphpad v5.0 . 

the details are described below : using the graphpad , we obtained the 
 initial concentration ( cp(0 ) ) , the distribution 
 rate constant ( a ) , and elimination rate constant 
 ( b ) directly .
other pk parameters were calculated 
 from the known parameters using following equations : for the 
 biodistribution study , balb / c mice were inoculated subcutaneously 
 with 10 4t1 cells in 50 l of pbs at # 4 mammary 
 fat pad on the right side of mouse abdomen .
sali 
 np3s or sali was injected intravenously into the mice at 5 mg / kg sali 
 or an equivalent sali dose , respectively . at 12 h postinjection , hearts , 
 livers , spleen , lungs , kidneys , and tumors
water 
 solution on ice for 3 min using a brinkmann polytron homogenizer ( brinkmann 
 instruments , inc . ,
next , 3 ml of the above abstract 
 solution was used for liver tissues while 1 ml of the solution was 
 used for heart , spleen , lungs , kidneys , and tumor tissues .
after sali 
 was extracted from the homogenized tissues into the solution , the 
 samples were centrifuged at 14,000 rpm for 10 min at 4 c .
supernatants 
 containing sali were collected , and sali quantity was determined by 
 precolumn derivitization with dnph . the final tissue distribution 
 was normalized to percentage of initial dose averaged by weight of 
 each organ ( % id / gram ) .
10 4t1 mammosphere 
 cells or 10 regular 4t1 cells were seeded in wells of 
 96-well plates in 100 l of rpmi-1640 medium , which contained 
 serially diluted sali , paclitaxel ( ptx ) , or the nps in tests for different 
 studies . after the incubation with these drugs , live cells were quantified 
 using the celltiter 96 aqueous one solution cell proliferation assay 
 ( promega , madison , wi , usa ) .
the plates were read with a spectramax 
 m2 plate reader ( molecular devices , inc . , sunnyvale ,
quantities 
 of live cells in each well were represented by the absorbance values 
 at 490 nm of that well .
consequently , cell viabilities in each treated 
 well were expressed as the absorbance values in percentage after the 
 values were normalized with the mean absorbance value of the wells 
 containing untreated cells .
the viability data was fitted into a sigmoidal 
 dose  response curve using the graphpad prism 5.0 ( graphpad 
 software , inc .
the ic50 and 95% confidence 
 index ( ci ) were obtained from the fitting .
balb / c mice were 
 inoculated subcutaneously 
 with 4t1 cells as described earlier . at the seventh day after inoculation 
 and when tumors all reached to or above 100 mm ,
the mice 
 were randomly assigned into three groups and treated with the control 
 ( 1% dmso pbs treatment ) , free sali ( 5 mg / kg in 1% dmso pbs ) , or itep 
the length and width dimensions of tumors 
 were measured by a caliper every other day .
tumor volumes were estimated 
 using the following formula : tumor volume = ( length  width/2 ) .
the mice were sacrificed 
 the day after the fifth treatment . the tumors were harvest to determine 
 their weight and csc populations .
single cell suspensions of cells 
 were prepared from 4t1 tumor tissues 
 that we collected from the tumor growth study according to a previously 
 published protocol .
an approximately 
 300 mg of tumor mass was incubated with 3 ml of medium 199 ( life technologies , 
 inc . ) with 250 u / ml ultrapure collagenase iii ( worthington biochemical , 
 inc . , lakewood , nj , usa ) .
the tissue digestion lasted for 2 h at 37 
 c while shaken at 100 rpm .
after digestion , 3 ml of serum - containing 
 m-199 medium was added to inhibit the activity of collagenase , and 
 cells were filtered through a 45 m nylon mesh and washed with 
 rpmi-1640 containing 10% fbs .
cells were counted , transferred to a 
 5 ml tube , and washed twice with dpbs with a 0.1% heat - inactivated 
 fbs at 4 c for 5 min at 1,000 rpm .
antibodies were then incubated 
 with the cells for 30 min on ice at a 1:80 dilution as recommended 
 by the manufacturer of the antibodies . after the incubations ,
cells 
 were washed twice with the above washing medium and then were resuspended 
 in 0.4 ml of dpbs/0.1% fbs containing 1 g / ml dapi per million 
 cells .
flow cytometry analyses of the cells were performed on a cytek 
 dxp ( cytek development , inc . ,
the gating for cscs 
 was set up based on the cd44 and cd24 expression profile of the cells 
 collected from 4t1 mammospheres . to study mammosphere - forming 
 ability of tumors
, the above - mentioned single tumor cells were seeded 
 in wells of 6-well cell culture plate at a density of 500 cells / well . 

the culture medium was supplemented with b27 ( 1x , invitrogen , ca , 
 usa ) , 20 ng / ml egf , 20 ng / ml bfgf ( bd biosciences , franklin lakes , 
 nj , usa ) , 4 g / ml heparin ( sigma - aldrich , mo , usa ) , and 0.5% 
 methylcellulose ( stem cell technologies , vancouver , bc , v5z 1b3 , canada ) .
after 7 days of culture , number of mammospheres 
 in each well was counted and analyzed .
to design a nanocarrier to deliver sali to 
 solid tumors through the 
 enhanced permeability and retention ( epr ) effect , we took advantage of a self - assembling principle of amphiphiles 
 that amphiphlies with strong , spatially separated amphiphilicity are 
 able to assemble into micelle - like nps . since sali 
 is hydrophobic with a logd value of 3.24 at ph 7.4 , we paired it with 
 a hydrophilic itep that we recently invented ( chen , personnel communication ) . 

we expected that attaching sali to one terminus of the itep would 
 introduce sufficient , segmented amphiphilicity and the amphiphilicity 
 can drive the conjugates to form nps . to this end
, 32 cysteines were 
 appended to the c - terminal of the itep as conjugation sites ( figure 
 s1 in the supporting information ) .
the 
 resulting new itep ( figure s2 in the supporting 
 information ) maintains the hydrophilic nature of the parent 
 itep ( figure s3 in the supporting information ) .
to connect sali to the new itep , sali was first connected with 
 the mpbh linker ( figures s4 and s5 in the supporting 
 information ) ; then the formed mpbh 
sali conjugates were 
 connected with the new itep ( see the supporting 
 information ) .
after itep  sali conjugates were purified , 
 we found that the conjugates displayed a hydrodynamics diameter of 
 109.7  45.9 nm at 37 c according to dls measurements ( figure 1a ) .
the size of the conjugates was much larger than 
 that of the unconjugated itep , 7.9  1.49 nm , which suggests 
 a np structure of itep  sali conjugates .
sali 
 nps are narrowly dispersed with a low polydispersity index ( pdi ) value 
 of 0.217 .
the nps have a critical micelle concentration ( cmc ) of 15.32 
 m and a neutral charge ( figures s6 and s7 in the supporting information ) .
the nps were not toxic , 
 due presumably to the nonrelease of sali from the nps ( figure 1b ) .
the nonrelease result is reasonable since sali 
 is connected with mpbh through a stable amide bond and the mpbh , in 
 turn , is connected to the iteps through a stable thioester bond .
( a ) hydrodynamic 
 diameters of the unconjugated itep which has a 
 32-cysteine appendix ( blue filled area ) and itep  sali conjugate 
 ( black filled area ) .
, the unconjugated itep was reduced in 
 100 mm tcep solution overnight to cleave possible disulfide bonds . 

( b ) viability profiles of regular 4t1 cells after they were exposed 
 to different concentrations of sali or itep  sali nps for 48 
 h. the ic50 of free sali is 4.8 m .
the nps do not 
 show any toxicity up to 100 m . since 
 we have attained the itep 
sali nps , next we tried to use the 
 nps to deliver sali in an encapsulated form . using a modified cosolvent 
 method , we were able to load free sali into the nps .
sali np2s have a mean diameter 
 of 195.0  95.7 nm and a pdi of 0.288 ( figure 2a ) .
next , through an in vitro release study , 
 we found that the encapsulated sali was released from itep 
sali 
 np2 at a fairly rapid release rate with a half - life of 1.0 h ( figure 2b and table 1 ) .
( b ) the release profile for 
 sali from tep - sali np2 , itep 
the quantity of released sali was determined by 
 hplc in combination with a precolumn derivatization with dnph .
the 
 sali release data were fit to a release model described in the supporting information .
( c ) the loading efficiency 
 of sali by np2 , np2 plus dmha , or np3 .
the * indicates that the differences 
 between the np3 and np2 or np2 plus dmha are statistically significant 
 with a p < 0.0001 analyzed by one - way anova .
( d ) 
 viability profiles of 4t1 mammosphere cells after they were exposed 
 to different concentrations of sali or itep 
sali np3 for 48 
 h. the ic50 of free sali is 0.49 m with a 95% ci 
 = 0.400.60 m .
the ic50 of the np3 is 0.48 
 m with a 95% ci = 0.350.59 m . because sali has an ionizable 
 carboxyl group with a pka around 4.0 that 
 could confer negative charges to sali at a neutral ph , we suspected 
 the negative charges may have rendered instability to sali encapsulation 
 in hydrophobic cores of itep 
the instability , in 
 turn , may have led to the rapid release and low loading efficiency 
 of sali we observed . in order to test the conjecture and to attain 
 a carrier having a slower release rate and higher loading efficiency 
 of sali , we coencapsulated sali with dmha , a positively charged hydrocarbon 
 that has been used to neutralize encapsulated , negatively charged 
 chemicals inside micelles .
the results 
 from our in vitro release study showed that coencapsulation 
 of dmha and sali slowed down the release of sali from the np2s .
the 
 release half - life of sali from the np2 with dmha was 2.9 h , which 
 is significantly longer than that of the np2s without dmha ( figure 2b and table 1 ) .
the resulted loading efficiency 
 of 30.2  6.8% was same as that of the np2s without dmha ( figure 2c ) .
one early study pointed out that dmha was not 
 as effective as its more hydrophobic analogue , n , n - dimethyloctadecylamine ( dmoa ) , in stabilizing the encapsulation 
 of negatively charged chemicals .
dmoa , 
 however , was more toxic than both dmha and sali ( figure s8 in the supporting information ) , which excluded it from 
 serving as an additive for the sali coencapsulation .
we reasoned 
 that , to exploit the coencapsulation system of dmha and sali , we might 
 need to supplement more hydrophobicity to it .
therefore , we introduced 
 a second additive , -tocopherol ( vitamin e ) , to the system . 

the -tocopherol was able to double loading efficiency of rapamycin 
 due to its ability to increase the hydrophobic core of micelles .
we hypothesized that coencapsulation of -tocopherol 
 together with sali and dmha would not only provide a sufficiently 
 hydrophobic environment for sali but also neutralize the charge of 
 sali .
this would mean that sali can be trapped more stably inside 
 of the hydrophobic core of the nps .
consequently , we could see an 
 improvement of both loading efficiency and release profiles of the 
 coencapsulated sali .
the hypothesis was proven true as the coencapsulation 
 led to a very high loading efficiency , 75.0  6.17% ( figure 2c ) .
in addition , the newly generated nps , including 
 sali and two additives ( termed itep  sali np3s ) , have a mean 
 diameter of 179.9  43.0 nm and share a size similar to that 
 of np2 ( figure 2a ) .
importantly , the release 
 rate of sali from the np3s is remarkably slower than that of np2s 
 or np2s with dmha only ( figure 2b ) .
the release 
 half - life of np3 was 4.1 h , which is significantly longer than 2.9 
 h of the np2s with dmha only or 1.0 h of the np2s ( table 1 ) . together
, these results showed that the paired 
 additives , dmha and -tocopherol , improved both loading efficiency 
 and release kinetics of encapsulated sali in itep  sali nps . 

sali np3 solubilized sali in aqueous solution , 
 rendering a maximum solubility of 7,320  230 g / ml , which 
 represents a 430-fold increase , given that the intrinsic solubility 
 of sali is only 17 g / ml .
lastly , 
 the results of the in vitro cytotoxicity study showed 
 that itep 
sali np3s possess the same level of selective toxicity 
 to cscs as free sali ( figure 2d ) . here
, we 
 used cells harvested from 4t1 tumor mammospheres as a model for cscs 
 because these cells carry characteristic phenotypes of cscs , such 
 as a cd24/cd44 phenotype and a mammosphere - forming 
 capacity ( figure s9 in the supporting information ) .
the ic50 of the np3 to 4t1 mammosphere cells was 0.48 
 m , which , statistically , was the same as the ic50 of free sali , 0.49 m ( figure 2d ) . 

this similarity is understandable as we expect that the encapsulated 
 sali is released from the np3s during the incubation with the cells 
 and exerts its toxicity the same as free sali .
sali 
 np3s was administered to mice 
 at a dose of 5 mg / kg intravenously , plasma concentrations of free 
 sali or encapsulated sali were sampled up to 12 h ( figure 3a ) .
these concentration data fit best into a two - compartment 
 pharmacokinetics model . according to the fitting results ( table 2 ) , the auc of the np3-delivered sali was 192.10 
 m h , which was approximately 6 times greater than that of the 
 free sali , 30.70 m h. similarly , free sali s clearance 
 was 6 times faster than that of the encapsulated sali ( clsali , 216.87 l/(h g ) versus clnp3 , 34.66 l/(h 
 g ) ) .
the elimination half - life of the np3-delivered sali was approximately 
 5.2 times longer than that of the free sali .
meanwhile , in summary , 
 the results support a conclusion that the np3s significantly retard 
 the clearance of the encapsulated sali from the systematic circulation , 
 which , together with sizes of the np3s ( 179.9 nm in diameter ) , might 
 lead to greater tumor accumulation of sali via the epr effect . indeed , 
 there was a 2.4-fold increase of tumor sali accumulation resulting 
 from using the np3s .
at 12 h postinjection , the mean sali concentration 
 in tumors of those mice receiving free sali was 1.17  0.17% 
 id / g .
in contrast , the mean concentration for the np3-delivered sali 
 was 2.85  0.12% id / g ( figure 3b ) . at 
 the same time , the np3s reduced sali accumulation in heart and lungs 
 ( figure s10 in the supporting information ) .
( a ) plasma sali concentrations after they were administered as 
 free form ( blue line ) or as encapsulated form in itep  sali 
 np3 ( red line ) through intravenous injection .
the concentrations were 
 determined by hplc in combination with a precolumn derivatization 
 with dnph .
the sali plasma concentration was plotted on the log scale 
 based on 10 as a function of time postinjection .
( b ) tumor accumulation 
 of sali after they were administered as free form ( blue bar ) or as 
 encapsulated form in itep  sali np3 ( red bar ) through intravenous 
 injection .
the presented data are for tumor samples collected at 12 
 h post intravenous injection at 5 mg / kg .
the quantities of sali were 
 expressed as percentage of initial dose normalized by weight of tumor , 
 % id / gram .
the * indicates that the difference between sali and itep  sali - np3 
 is statistically significant with a p = 0.0031 analyzed 
 by one - way anova . since itep 
sali np3s extended the systematic 
 circulation of sali and increased tumor accumulation of sali , it was 
 intriguing to see whether such improvements could strengthen the selective 
 inhibition of cscs by sali in vivo . to test this 
 hypothesis , we inoculated murine 4t1 syngeneic tumor cells in mammary 
 fat pads of balb / c mice to establish an orthotopic tumor model .
once 
 the tumors grew to desired sizes , we treated the mice with 5 doses 
 of pbs as a control , 5 mg / kg free sali , or itep 
after 5 treatments , we analyzed csc 
 frequencies in these tumors using cd24 and cd44 markers .
the cscs 
 in tumors were defined operationally as those cells that have the 
 same cd24 and cd44 expression profiles as those cells collected from 
 4t1 mammospheres ( figures s9 and s11 in the supporting 
 information ) . for the control group ,
free sali was able to lower the frequency 
 significantly to 64.32  5.72% ( p < 0.05 ) . 

more importantly , the nps further reduced the value to 55.62  
 3.73% , which was significantly lower than those of both the control 
 group and free sali - treated group ( p < 0.05 ) ( figure 4a ) .
these comparison results suggest that sali is 
 able to kill cscs in vivo just as they are able to in vitro ( figure 2b ) , and , more significantly , 
 itep 
( a ) the 
 csc frequencies of the 4t1 orthotopic tumors treated by 
 controls ( pbs ) , free sali , or itep  sali np3 .
the * symbols 
 indicate that the mean css frequency of the np - treated tumor is significantly 
 lower than that of control- or free sali - treated tumors ( p = 0.0004 and p = 0.0252 , respectively ) , and that 
 the mean frequency of the sali - treated tumors is significantly lower 
 than that of the control - treated tumors ( p = 0.0133 ) . 

( b ) mammospheres formed 
 from 4t1 orthotropic tumors after these tumors were treated by controls 
 ( pbs ) , free sali , or itep 
the * symbols indicate 
 that the mammosphere - formation ability of the np - treated tumor is 
 significantly lower than that of control- or free sali - treated tumors 
 ( p = 0.0035 and p = 0.0125 , respectively ) , 
 and that the mammosphere - formation ability of the sali - treated tumors 
 is significantly lower than that of the control - treated tumors ( p = 0.0358 ) .
( c ) the viability profile of regular 4t1 cells versus 4t1 mammosphere 
 cells after they were exposed to different concentrations of ptx for 
 72 h. the ic50 of ptx to regular 4t1 cells 5.79 nm with 
 a 95% ci = 4.906.86 nm .
the ic50 of ptx to 4t1 
 mammosphere cells 40.48 nm with a 95% ci = 37.6447.30 nm .
the capacity is relevant to the 
 drug resistance of tumors , thus they are very significant to cancer therapy . to investigate 
 functional significance of the np3s effect in reducing cscs 
 in 4t1 orthotopic tumors , we examined mammosphere - forming ability 
 of the cells collected from 4t1 tumors after these tumors were treated 
 by the np3 , free sali , and pbs .
we found that the np3-treated tumors 
 had a significantly lower ability to generate mammospheres than the 
 other two treatment groups ( figure 4b ) .
there 
 were on average 42.2 mammospheres formed per 500 np3-treated tumor 
 cells , which were significantly less than those formed from free sali- 
 or pbs - treated tumors ( 54.7 and 61.0 mammospheres per 500 tumor cells , 
 respectively ) .
putting together , the superior effects of the np3 over 
 free sali in eliminating cscs and impeding the mammosphere - forming 
 ability of 4t1 tumors are significant because they suggested that 
 the np3 was effective to alter cell composition of the tumors , and , 
 more importantly , to diminish tumorigenesis and drug resistance potential 
 of the tumors . in a separate study , we found that the cells 
 isolated from mammospheres 
 were more resistant to ptx than general 4t1 cells cultured as monolayers 
 ( figure 4c ) .
the ic50 of ptx to 
 the mammosphere cells was 7 times higher than the ic50 to 
 regular 4t1 cells , 40.50 nm versus 5.79 nm ( p < 
 0.05 ) .
the results are consistent with earlier reports that cscs play 
 a critical role in promoting the tumor mdr .
therefore , the aforementioned enhancing 
 effect of itep  sali nps to the csc - specific toxicity of sali 
 may have implications for overcoming the mdr .
since cscs play a role in 
 tumorigenesis and maintain the hierarchy 
 of tumors , depletion of cscs in tumors may lead to a tumor regression 
 or stabilization .
thus , we examined whether the superiority of the 
 np3-delivered sali over free sali in depleting cscs can translate 
 into an advantage of tumor growth inhibition or tumor regression . 

our results showed that neither the np3s nor free sali were able to 
 abolish tumors or to stop tumor growth ( figure 5a ) .
the growth - inhibition 
 effect was especially prominent for the nps as the mean tumor weight 
 of the np3-treated mice at day 16 was significantly smaller than that 
 of the control group : 436.8  137.9 mg versus 762.6  193.8 
 mg ( p < 0.05 ) , respectively ( figures 5b and 5c ) . while it is clear 
 the np3-delivered sali had an impact on overall tumor growth , the 
 impact was not as potent as its impact on cscs inside the 4t1 tumors . 

in contrast to the result that the np3-treated 4t1 tumors had a significantly 
 smaller csc population than free sali - treated 4t1 tumors after five 
 treatments ( figure 4a ) , the mean weights of 
 the np3-treated and the free sali - treated tumors were not statistically 
 different ( figure 5c ) .
one possible reason 
 for the discrepancy is that cells in 4t1 tumors might , on average , 
 be less sensitive to sali than the csc subpopulation in the tumors . 

indeed , our results showed that sali was 10 times less toxic to regular 
 4t1 cells than to cells isolated from 4t1 mammospheres ( figure 5d ) .
thus , it is possible that , as a result of lower 
 toxicity of sali to differentiated tumor cells and stromal cells , 
 the increased sali accumulation did not have the same degree of impact 
 on the overall tumor growth as on cscs .
in addition , the np3s did 
 not completely eradicate the csc subpopulation in the tumors .
the 
 surviving cscs could reignite tumor growth that counteracted any tumor 
 growth inhibition contributed by the np3s . as a result
( a ) the size changes 
 of 4t1 orthotopic tumors after they were treated 
 by five doses of controls ( pbs ) , free sali , or itep  sali np3 . 

both free sali and the np - encapsulated sali were administered at 5 
 mg / kg .
the tumor 
 dimensions were measured at the same day of dosing except for the 
 last measurement , which was performed at day 16 post tumor inoculations . 

( b ) photos of the tumors collected at day 16 after the inoculations . 

( c ) the mean tumor weights of each group of mice at the day 16 post 
 tumor inoculations .
the * indicates a significant difference between the connected two 
 means ( p = 0.0307 ) .
( d ) the viability profile of 
 regular 4t1 cells versus 4t1 mammosphere cells after they were exposed 
 to different concentrations of itep  sali np3 for 48 h. the 
 ic50 of the np3 to regular 4t1 cells 4.56 m with 
 a 95% ci = 3.495.95 m .
the ic50 of the np3 
 to 4t1 mammosphere cells 0.48 m with a 95% ci = 0.350.59 
 m .
in 
 this study , we designed and characterized an itep - based sali 
 delivery system that improved the pharmacokinetics and tumor accumulation 
 of sali , as well as strengthened its specific csc - inhibition effect 
 of sali . the results , in principle , validate the use of nanocarriers 
 to boost the efficacy of csc - specific drugs . because cscs were suggested 
 to play a role in causing tumor mdr and recurrence , this enhancement 
 effect of nanocarriers represented by itep 
iteps were chosen as building 
 blocks for the delivery system because 
 they bring three primary advantages to the system .
first , the nonimmunogenic 
 nature of iteps could save the carrier from the risk of any carrier - specific 
 immunogenicity and allow multiple dosing of the carrier .
in contrast , 
 if a pharmaceutical protein or polypeptide induces immune responses 
 during its application , the responses could severely compromise its 
 function .
second , recombinant polypeptides such as 
 iteps are biodegradable so the carriers built upon the polypeptides 
 are biodegradable and biocompatible as well .
indeed , itep carriers 
 have not revealed any adverse effect in our in vivo studies .
finally , the sequences , length , and hydrophobicity of iteps 
 can be definitely controlled and adjusted using genetic engineering 
 approaches .
this property of iteps becomes an advantage since it allows 
 for the generation of a wide spectrum of new iteps or itep nps to 
 precisely meet a specific delivery need .
the versatility of itep carriers 
 offers the possibility to further optimize delivery systems not only 
 for sali but also for other csc - targeting drugs .
examples of such 
 optimization include appending itep with protein - based targeting ligands 
 such as those antibodies recognizing csc - specific antigens .
the sali delivery system reported here represents a novel strategy 
 to deliver and use sali .
sali contributed the hydrophobicity to the 
 itep  sali conjugates and served as payloads at the same time . 

previously , hydrophobic drugs were used as a hydrophobic component 
 of micelle - forming amphiphilic conjugates and an encapsulated payload 
 in the micelle .
what we are reporting 
 is the first application of this delivery strategy to sali .
a stable 
 encapsulation represents the primary advantage of this strategy , which 
 happens when a hydrophobic drug is packed inside a hydrophobic core 
 contributed by the same drug .
when 
 charged hydrophobic drugs such as sali are trapped inside 
 a hydrophobic core of micelle - like nps , the charges may destabilize 
 the encapsulation , which was observed in both previous studies and 
 this current study . to cancel out the 
 charges , it is reasonable to coencapsulate an oppositely charged , 
 hydrophobic molecule with the charged drugs .
dmoa was believed to 
 be a good candidate to serve as coencapsulate additives for negatively 
 charged drugs such as sali , because it is hydrophobic and positively 
 charged .
however , our data revealed strong toxicity of 
 dmoa , especially in the comparison to sali , which excluded dmoa for 
 the coencapsulation with sali .
dmha , a positively charged analogue 
 of dmoh with no apparent toxicity , is a reasonable alternative .
however , 
 dmha is not as hydrophobic as dmoa due to its short hydrocarbon chain . 
 to address the insufficient hydrophobicity of the sali 
dmha 
 coencapsulation system , we tried using a second hydrophobic chemical 
 to supplement more hydrophobicity .
we observed improvements in both the loading efficiency and the 
 release profile of sali when dmha and -tocopherol were used 
 in combination .
we believe that this innovative , dual - additive approach 
 may find its application in many encapsulation - based delivery systems , 
 given that there could be many cases where the drugs to be encapsulated 
 do not have an ideal charge .
sali 
 nps , has a higher accumulation 
 in the tumor and a longer system circulation than those of free sali . 

consistent with these advantages , itep  sali nps deplete cscs 
 and decrease mammosphere - forming capacity of the 4t1 tumors more efficiently 
 than free sali .
however , the nps do not inhibit tumor growth more 
 effectively , a result consistent with a previous report that was obtained 
 by using a xenograph tumor model .
in 
 fact , neither the nps nor free sali stabilized or abolished tumors . 

there are at least two possible reasons for the discrepancy between 
 the csc eradication effect and the overall tumor inhibition effect 
 of the nps .
first , itep  sali nps did not eradicate cscs completely , 
 a deficiency that may contribute to tumor re - regrowth .
the regrowth , 
 in turn , may have canceled out some tumor inhibition effect of the 
 nps . given this possibility , it is important to develop new carriers 
 to expand the advantage of the sali carrier in accumulating sali in 
 tumors .
the carriers that could meet this need may be either a carrier 
 that offers more stable encapsulation and slower release or a carrier 
 that recognizes tumor cells through its targeting moieties .
the second 
 reason stems from the sali s toxicity characteristics , its 
 higher toxicity to cscs compared to differentiated cells in tumors . 

so , it is reasonable to find that cscs , rather than bulk tumors , respond 
 well to the increased sali accumulation caused by the itep 
the toxicity characteristics advocate for a combination therapy 
 that , concurrently or sequentially , uses the nps together with conventional 
 chemotherapeutics such as ptx .
the combinational therapy promises 
 to target all cell types in tumors , and hence is able to either stabilize 
 or abolish the tumors .
sali 
 nps is their application in preventing tumor recurrence or mdr , given 
 that the nps have been shown to strengthen the toxicity of sali to 
 cscs and that cscs have been shown as one factor driving the tumor 
 mdr .
 salinomycin 
 ( sali ) has selective toxicity to cancer stem cells 
 ( cscs ) , a subpopulation of cancer cells that have been recently linked 
 with tumor multidrug resistance ( mdr ) . to utilize its selective toxicity 
 for cancer therapy 
 , we sought to devise a nanoparticle ( np ) carrier 
 to deliver sali to solid tumors through the enhanced permeability 
 and retention effect and , hence , to increase its exposure to cscs . 
 
 first , hydrophobic sali was conjugated to a hydrophilic , immune - tolerant , 
 elastin - like polypeptide ( itep ) ; the amphiphilic itep  
 sali 
 conjugates self - assemble into nps . 
 next , free sali was encapsulated 
 into the nps alone or with two additives , n , n - dimethylhexylamine ( dmha ) and -tocopherol . 
 the 
 coencapsulation significantly improved the loading efficiency and 
 release profile of sali . 
 the resulting nps of the coencapsulation , 
 termed as itep  
 sali np3s , have an in vitro release half - life of 4.1 h , four times longer than itep  
 sali 
 np2s , the nps that have encapsulated sali only . 
 further , the np3 formulation 
 increases the plasma area under curve and the tumor accumulation of 
 sali by 10 and 2.4 times , respectively . 
 lastly , these improved pharmacokinetic 
 and tumor accumulation profiles are consistent with a boost of csc - elimination 
 effect of sali in vivo . in np3-treated 4t1 orthotopic 
 tumors , 
 the mean csc frequency is 55.62% , a significant reduction 
 from the mean frequencies of untreated tumors , 75.00% , or free sali - treated 
 tumors , 64.32% . 
 the csc - elimination effect of the np3 can further 
 translate to a delay of tumor growth . 
 given the role of cscs in driving 
 tumor mdr and recurrence , it could be a promising strategy to add 
 the np3 to conventional cancer chemotherapies to prevent or reverse 
 the mdr . 
Introduction
Material and Methods
Results
Discussion