there 
 is growing evidence that drug combinations can be more effective 
 than the sum of individual drug effects .
one course of therapy that demonstrated this theory is the administration 
 of paclitaxel ( ptx ) combined with 5-fluoroucacil ( 5-fu ) to patients 
 with gastric , breast , pancreatic , and other types of cancer , each drug using a different mechanism to kill tumor cells .
although 
 the use of combined drugs appears to be promising in cancer therapy , 
 the problem of bioavailability is still encountered because the drugs 
 are often removed from blood circulation by macrophages or other molecular 
 components associated with drug clearance from circulation . in a conventional 
 anticancer drug combination regimen ,
the combined drugs that have 
 synergistic actions in vitro are injected into the blood together 
 but will distribute and be eliminated independently of each other . 

for example , ptx and 5-fu have half - lives of 1 h and 7 min , respectively , 
 in mice .
the different pharmacokinetic properties 
 make it impossible to control the molar ratio of the two drugs taken 
 up by the same diseased cells .
drug carrier systems have been proposed 
 to address this problem based on the knowledge 
 that the pharmacokinetic characteristics of individual drugs 
 will depend on the carriers .
oil in water ( o / w ) nanoemulsions 
 ( nes ) , a nanocarrier system , 
 can offer various advantages to therapeutics and targeting , for example , 
 improvements of the chemical and/or enzymatic stability of carried 
 therapeutic agents , long - term colloidal stability , and ease of manufacture 
 and scale - up .
however , o / w nes are designed 
 to deliver hydrophobic drugs , which limit their application for the 
 delivery of combined drugs , particularly for the combination of hydrophobic 
 and hydrophilic anticancer drugs . in order to improve the compatibility 
 between the cargo and the 
 core of nes , in the present study , ptx and 5-fu
were first conjugated 
 with the functional vitamin e ( ve ) and tocopherol poly(ethylene glycol)succinate 
 ( tpgs ) , with ve and tpgs representing the oil core and pegylated emulsifying 
 agent in the nes , respectively .
the conjugated ptx and 5-fu were then 
 loaded into the nes based on the core - matched technology ( cmt ) proposed 
 by our lab ( figure 1 ) .
the solubility and the 
 stability of ptx and 5-fu in the core - matched nes is increased due 
 to more favorable interactions with the core by the functional molecules 
 anchored to the nes .
ptx and 5-fu modified by ve and tpgs , respectively , 
 can easily come into contact with the matched ve core contributing 
 to the prevention of rapid drug release and the observation of satisfactory 
 pharmacokinetic properties .
in addition , ptx 
 is known to be a substrate of p - glycoprotein ( p - gp ) , which has been involved in the multidrug resistance 
 ( mdr ) of several cancers due to its overexpression .
tpgs has 
 been extensively investigated as an inhibitor of p - gp , and some tpgs - based 
 nanoparticles or copolymers with similar structures have been proven 
 to overcome mdr .
our previous study has also reported that ve causes 
 significant reversal of mdr due to the inhibition of atpase activity .
ve and 
 5-fu  tpgs can not only be encapsulated into a single nanocarrier 
 to produce synergism by cmt but also reverse mdr in cancer treatment 
 ( figure 1 ) .
schematic illustration of the core - matched 
 nanoemulsions 
 codelivering ptx and 5-fu to achieve the synergistic effects 
 and reversal of mdr .
our aim was to design core - matched nes with the following 
 functions : 
 ( 1 ) ability to codeliver hydrophilic and hydrophobic anticancer drugs 
 effectively ; ( 2 ) improvement of the poor pharmacokinetics 
 of ptx and 5-fu by promoting long circulation times and synergistic 
 effects in vivo ; ( 3 ) reversal of resistance to ptx by enhancing its 
 antitumor activity .
ve and 5-fu  tpgs were 
 synthesized in our lab ( figure 2 ) .
the synthesis 
 and characterization of ptx  ve and 5-fu  tpgs is provided 
 in the supporting information ( figures 
 s1s4 ) .
5-fluorouracil injection 
 was purchased from app pharmaceuticals , llc ( schaumburg , il ) .
antibodies 
 against p-53 , -tubulin , p - gp , gapdh , and horseradish peroxidase 
 ( hrp)-conjugated anti - mouse or anti - rabbit whole igg were obtained 
 from santa cruz biotechnology ( san diego , ca ) .
deadend fluorometric 
 tunel assay kits were obtained from promega ( madison , wi ) . other chemicals 
 were purchased from sigma - aldrich ( st .
chemical structures and 
 synthetic design of ptx  ve ( a ) and 
 5-fu  tpgs ( b ) .
kb-3 - 1 and kb-8 - 5 cells originally obtained from american 
 type 
 culture collection ( atcc ) ( manassas , va ) were maintained in rpmi 1640 
 and dmem medium ( invitrogen , carlsbad , ca ) containing 10% fetal bovine 
 serum ( invitrogen , carlsbad , ca ) , 100 unit / ml penicillin , and 100 
 g / ml streptomycin ( invitrogen , carlsbad , ca ) .
all experiments performed on animals were in accordance with 
 and approved by the institutional animal care and use committee at 
 the university of north carolina at chapel hill .
ve , 
 5-fu  tpgs , tpgs , and ve were first dissolved in chloroform . 

ve ( 10 mg / ml ) , tpgs ( 50 mg / ml ) , and 
 ve ( 150 mg / ml ) and 125 l of 5-fu  tpgs ( 15 mg / ml ) were 
 then separately withdrawn on ice and added to an eppendorf tube with 
 a screw cap .
the chloroform was evaporated under nitrogen gas , and 
 trace amounts of chloroform were further removed by keeping the mixture 
 under vacuum in a desiccator for 1 h. following the addition of 1 
 ml of distilled water , the mixture was sonicated using a sonic dismembrator 
 model 100 ( fisher scientific , pittsburgh , pa ) to produce the ne . in 
 order to obtain a more homogeneous and fine particle size ne , the 
 mixture was homogenized in a bullet blender ( next advance , averill 
 park , ny ) using zirconium oxide beads ( 1:2 ; 1.0 mm diameter/0.5 mm 
 diameter ) .
the instrument was set at the eighth speed for 15 min . 
 particle size and zeta - potential of the nes were determined by dynamic 
 light scattering measurements using a malvern zetasizer nano series 
 instrument ( westborough , ma ) .
the loading efficiency of ptx  ve 
 was assessed , following high - speed centrifugation to separate the 
 free drug from nes , using a liquid scintillation analyzer ( tri - carb , 
 packard bioscience company , waltham , ma ) .
the shape and surface morphology 
 of the nes were observed using a jeol 100cx transmission electron 
 microscope ( tokyo , japan ) . prepared samples ( 5 l ) were dropped 
 onto a 200 mesh carbon - coated copper grid ( ted pella , inc . , redding , 
 ca ) and then wicked off from the grid after 3 min .
grids were then 
 stained with 1% uranyl acetate ( 5 l ) for 1015 s and 
 wicked for tem .
kb-3 - 1 and kb-8 - 5 
 cells were seeded into 96-well plates at a density of 1  10 cells per well and allowed to adhere overnight .
various concentrations 
 of drug or formulation were added to the plates for 48 h. following 
 incubation , 20 l of mtt reagent ( 5 mg / ml in pbs ) was added 
 to the culture medium and the cells were incubated for an additional 
 4 h at 37 c .
the culture medium was then carefully removed , 
 and 200 l of dmso was added to the wells to dissolve the formazan . 

uv absorbance was measured at 570 nm using a bio - rad microplate imaging 
 system ( hercules , ca ) , and results were expressed as % cell viability 
 ( od of treated group / od of control group  100 ) .
kb-8 - 5 cells growing 
 exponentially were seeded at 1  10 cells / ml in 12-well 
 plates .
tpgs 
 ne , or combined ne for 48 h. the pbs solution served as the control . 

ice - cold 70% ethanol was used to fix the cells at 4 c overnight . 

after centrifugation and removal of the supernatant , the cells were 
 resuspended with 1 ml of staining buffer and washed once .
after repeat 
 resuspension , the cells were incubated with 10 l of rnase a 
 ( 10 mg / ml ) at 37 c for 30 min and then stained with 5 l 
 of propidium iodide ( 1 mg / ml ) at room temperature for 30 min .
the 
 cell cycle analysis was performed using a facscanto flow cytometry 
 system ( bd biosciences , san jose , ca ) .
adherent cells 
 in culture dishes were washed with ice - cold pbs , scraped using a cold 
 plastic cell scraper , and then gently transferred , in suspension , 
 to a precooled microcentrifuge tube .
whole cell lysates were 
 extracted with ripa buffer , and the protein concentration was measured 
 using a pierce bca protein assay kit ( thermo scientific , rockford , 
 il ) . for each sample ,
approximately 50 g of protein was separated 
 on a nupage 12% sds  polyacrylamine gel and then transferred 
 to a polyvinylidene difluoride ( pvdf ) membrane ( bio - rad , 
 hercules , ca ) .
the membrane was blocked with 5% skimmed milk in pbs 
 for 1 h. after incubation with primary antibody at 4 c overnight , 
 it was washed with pbst ( 0.2% tween 80 in pbs ) and then incubated 
 with secondary antibody for 1 h. antibodies against p - gp , p-53 , -tubulin , 
 and gapdh were used at 1:2000 , 1:200 , 1:500 , and 1:2000 dilutions , 
 respectively . an anti - mouse antibody conjugated with hrp at a dilution 
 of 1:10000 or an anti - rabbit igg at a dilution of 1:2000 served as 
 the secondary antibodies in the experiment .
the specific protein bands 
 were visualized using a chemiluminescence kit ( thermo scientific , 
 rockford , il ) .
chemiluminiscent signals were detected using 
 high - performance chemiluminescence film ( ge healthcare bio - sciences , 
 pittsburgh , pa ) . in vivo 

antitumor activity was evaluated in kb-3 - 1 and kb-8 - 5 bearing nude 
 mice .
nude mice were inoculated with 5  10 kb-3 - 1 cells 
 or kb-8 - 5 cells injected subcutaneously into their right or left flanks 
 to establish the xenograft model .
once the tumor mass in the xenograft 
 was established , mice were randomly divided into 5 groups ( 5 mice 
 per group ) and were injected , in the tail vein , with normal saline 
 ( the control group ) , taxol , ptx  ve ne , 5-fu  tpgs ne , 
 or ptx  ve combined 5-fu  tpgs ne .
drug doses of 5 mg / kg 
 ptx , 8 mg / kg ptx  ve , and 30 mg / kg 5-fu  tpgs were used 
 for all treatments .
therapy was continued at days 3 , 5 , 7 , and 9 ( five 
 doses in total ) .
tumor volumes were calculated as ( length  width)/2 from measurements taken every second day .
toxicity of the formulations 
 was determined by monitoring mice behavior and weight loss and by 
 he staining .
kb-3 - 1 
 and kb-8 - 5 tumor - bearing 
 nude mice were given iv injections of the five formulations on days 
 1 , 3 , 5 , 7 , and 9 .
tumors were fixed in 10% formalin for at least 24 h before being embedded 
 in paraffin and sectioned at a thickness of 5 m . in vivo tumor 
 cell apoptosis
the tunel staining 
 was performed as recommended by the manufacturer ( promega , madison , 
 wi ) , and dapi mounting medium was dropped on the sections for nucleus 
 staining .
images of tunel - stained tumor sections were taken with a 
 fluorescence microscope ( nikon corp . ,
the tunel - positive 
 cells were counted using imagej software ( national institutes of health , 
 bethesda , md ) .
female cd-1 mice 
 ( 1822 g ) were intravenously injected with ptx  ve and 
 5-fu  tpgs combined ne , taxol , or 5-fu injection at a volume 
 of 0.2 ml ( 8 mg / kg ptx - ve , 5 mg / kg ptx , and 2.22 mg / kg 5-fu ) .
blood samples were digested in tissue solubilizer 
 ( ge healthcare bio - sciences , pittsburgh , pa ) at a ratio of 1 mg/10 
 l . then
, 200 l of hydrogen peroxide ( 30% ) was added 
 to 100 l of sample for decolorization . following this ,
a scintillation 
 cocktail ( ultima gold xr , perkinelmer , waltham , ma ) was added and 
 the decolorized samples were stored in the dark for 40 min .
the radioactivity 
 was then quantified using a liquid scintillation analyzer ( tri - carb , 
 packard bioscience company , waltham , ma ) .
the concentrations of drugs 
 in the samples were determined , following subtraction of blank background 
 radioactivity , by extrapolation from standard calibration curves . 

the statistical significance of group differences 
 was analyzed using the student s t - test , and 
 a value of p < 0.05 was considered significant .
in the study , the average 
 particle size of the core - matched nes was 82 nm with a pdi of 0.165 , 
 which was considered effective for passive targeting of tumors .
the 
 morphology of the core - matched nes examined by tem ( figure 3 and figure s5 ) exhibited a uniform and spherical 
 shape .
the loading efficiency of ptx  ve in the core - matched 
 nes was > 95% , 6-fold more than that of ptx alone , due to the solubility 
 of ptx  ve in the ve oil phase .
the combined core - matched nes 
 had a negative zeta - potential of 11.6 mv .
the in vitro release of 
 ptx  ve in the core - matched nes was slow ( figure s6 ) , and no 
 burst effect occurred .
therefore , the core - matched nes effectively 
 avoid problems reported with other ptx formulations , including low 
 entrapment efficiency , drug instability , and rapid drug leakage .
many therapeutic agents hitherto 
 have not been successful due to 
 their limited ability to reach the target tissue .
thus , a drug delivery 
 strategy that selectively targets the malignant tumor is necessary .
the core - matched nes have properties suited 
 to synchronously delivering , in vitro and in vivo , both hydrophobic 
 and hydrophilic anticancer drugs because of their nanoscale size and 
 their stability within the matched core . data 
 in figure 4a , b show that ptx 
ve and 
 5-fu  tpgs had a toxic effect in both ptx - sensitive and -resistant 
 cells .
in particular , the combined core - matched nes had the most significant 
 cytotoxicity in both cell lines compared with the ne of the individual 
 prodrug .
ve ne in the resistant 
 kb-8 - 5 cells was 1.27 m , lower than that in the sensitive kb-3 - 1 
 cells ( 2.67 m ) .
however , the ic50 of ptx in resistant 
 cells was as much as 3 times that determined in sensitive cells ( see 
 table 1 ) .
it was shown that the codelivery 
 of ptx  ve and 5-fu  tpgs in one single drug carrier could 
 improve the sensitivity of ptx in resistant cells through the reversal 
 of mdr via ve and tpgs inhibiting the atpase activity .
the in vitro 
 cytotoxicity assay revealed that the combined core - matched nes improved 
 the ability of the individual prodrugs to cause decreased viability 
 of both sensitive and resistant kb cells .
ve core - matched 
 nes ( see figure 4c ) due to its inability to 
 transport through the cell membrane following its precipitation from 
 dmso after addition to the 96-well plate .
the result also showed that 
 the core - matched ne carrier plays a vital role in the delivery of 
 water - insoluble ptx - ve in vivo and in vitro .
ve 
 ne , 5-fu  tpgs ne , and the 
 combined ne against ( a ) kb-3 - 1 cells , ( b ) kb-8 - 5 cells , and ( c ) free 
 ptx  ve and ptx  ve ne in kb-8 - 5 cells ( mean  sd , n = 6 ) .
the concentrations on the x - axis 
 represent the concentrations of ptx  ve .
the synergism 
 of ptx  ve and 5-fu  tpgs in cytotoxicity 
 was assessed by the chou - talalay method .
the combination index was calculated by the following equation : 
 ci ( combination index ) = ( d)1/(dx)1 + ( d)2/(dx)2 , where dx is the 
 dose of one compound alone required to produce an effect ( ic50 ) and ( d)1 and ( d)2 are the doses of compounds 1 and 2 necessary 
 to produce the same effect in combination . the ci values calculated 
 for kb-8 - 5 and kb-3 - 1 cells , respectively , were 0.9 and 1.0 .
the ci 
 theorem of chou - talalay offers quantitative definition for additive 
 effect ( ci = 1 ) , synergism ( ci < 1 ) , and antagonism ( ci > 1 ) 
 in 
 drug combinations . the results indicated 
 that the combined nes have a synergistic effect on resistant kb-8 - 5 
 cells and an additive effect on sensitive kb-3 - 1 cells .
this could 
 be ascribed to the reversal of mdr produced by tpgs and ve .
cell cycle checkpoints are used to monitor and regulate the progress 
 of the cell cycle .
it is known that ptx can arrest sensitive tumor cells at 
 the g2/m phase by an unusual stabilization of the microtubule . when resistant kb-8 - 5 cells were treated with 
 ptx  ve ne
, cells of the g2/m phase increased from 
 27.3% in the untreated group to 52.6% ( figure 5 ) .
ve was converted to the parent 
 drug ptx , which blocked the g2/m phase in the cell cycle , 
 even within the resistant cells , resulting in the reversal of mdr .
typical flow cytometric diagrams and g2/m phase 
 distribution of the kb-8 - 5 cells treated with ptx  ve 
 nes , 5-fu  tpgs nes , and the combined nes for 48 h ( mean  
 sd , n = 3 )
. it has been reported that 5-fu exhibits three modes of cell 
 growth 
 modulation , namely , loss or accumulation of s phase cells , g2/m block , and g1/s arrest . in our study , the g2/m phase arrest was evident in kb-8 - 5 
 cells treated with 5-fu  tpgs ; 53.2% of cells were arrested 
 probably because cells proficient in dna mismatch repair showed marked 
 modulation in the g2/m phase .
furthermore , the combined cme showed the most significant effect 
 on the cell cycle , with 61.4% of cells being arrested at the g2/m phase .
tubulin is the target for anticancer drugs 
 such as taxol , tesetaxel , 
 vinblastine , and vincristine .
previous studies have shown that ptx can 
 enhance the expression of -tubulin in sensitive kb-3 - 1 cell 
 lines but not in ptx - resistant cell lines . in the present study
ve cme and the combined 
 core - matched nes up - regulated the level of -tubulin in the 
 resistant kb-8 - 5 cell line ( figure 6 ) .
the 
 result is consistent with the cell cycle assay above , which showed 
 g2/m phase enhancement .
p - gp , a marker for mdr , showed 
 no change in both the untreated and treated groups .
previous studies 
 have elucidated that ve and tpgs reverse mdr by inhibiting the atpase 
 activity of p - gp rather than by regulating p - gp expression .
western blotting assays for p - gp , -tubulin , and 
 p53 protein 
 extracted from kb-8 - 5 cells treated with ptx  ve nes , 5-fu 
tpgs 
 nes , or the combined nes for 48 h. the p53 tumor suppressor is a principal mediator of growth 
 arrest , 
 senescence , and apoptosis in response to a broad range of cellular 
 damage
5-fu exerts its anticancer effects 
 through incorporation of its metabolites into rna and dna interfering 
 with their normal functions .
our previous studies have shown that 
 both 5-fu and 5-fu  pgs can increase the expression of p53 .
figure 6 shows that 5-fu  pgs 
 core - matched nes and the combined core - matched nes facilitate the 
 up - regulation of p53 .
this up - regulation is consistent with the reported 
 ability of p53 to induce cell cycle arrest at the g2/m 
 boundary of the cell cycle . as shown 
 in figure 7a , the reductions in tumor volume 
 observed in nude mice treated with ptx 
ve or 5-fu  tpgs 
 core - matched nes compared to control were not significant .
a synergistic 
 effect was observed in the combined core - matched nes , which exhibited 
 significant inhibition of tumor growth compared to the control group . 

however , taxol showed a significantly reduced kb-3 - 1 tumor volume 
 when compared to the other four groups .
one was that kb-3 - 1 is a ptx - sensitive cell line , and the 
 other is that the slow hydrolysis rate of ptx 
tumor growth inhibition in subcutaneous 
 tumor models of ( a ) kb-3 - 1 
 cells and ( b ) kb-8 - 5 cells .
tissue he staining ( c ) after injection 
 of the combined nes , via the tail vein , every second day for 10 days . 

all data are expressed as mean  sd ( n = 5 ) ; 
 * p < 0.05 for combined nes vs the individual nes 
 and p < 0.05 for combined nes vs the 
 group treated with taxol .
advantageous properties of the core - matched nes loaded with 
 both 
 prodrugs were observed in the nude mice bearing resistant kb-8 - 5 tumors 
 ( figure 7b ) .
the results showed that the core - matched 
 nes were able to significantly inhibit the kb-8 - 5 tumor growth compared 
 with the other four groups ( tumor growth was reduced by 57.7% [ t - test , p < 0.05 ] ) .
first , 
 the core - matched technology prolonged the circulation of drug in vivo 
 ( data provided in pharmacokinetics study ) , facilitating 
 greater drug accumulation in the tumor .
second , the codelivery of 
 ptx and 5-fu in the prodrug form based on core - matched technology 
 produced a synergistic effect .
third , ve and tpgs included in the 
 prodrug and core - matched ne formulations help to reverse mdr .
side effects are one of the major problems of cancer chemotherapy . 
 in this study , the toxicity of the combined core - matched nes on liver , 
 spleen , and kidney was investigated using he staining after long - term 
 treatment .
figure 7c showed no obvious damage 
 observed in these tissues after treatment with the core - matched nes . 

consistently , there was no decrease in body weight or noticeable change 
 in activity .
ve 
 and 5-fu  tpgs 
 core - matched nes caused the resistant tumor cell death .
as illustrated 
 in figure 8 , ptx  ve and 5-fu  tpgs 
 core - matched nes induced the most effective apoptosis of cells in 
 kb-8 - 5 xenograft tumors compared with the control and individual prodrug 
 nes .
the percentage of apoptotic cells ( 32.1  7.6% ) for the 
 combined nes was 7-fold higher than for taxol ( figure 8) .
the tunel assay for the sensitive kb-3 - 1 tumor cells indicated 
 that taxol was able to induce significant cell apoptosis compared 
 with other groups ( figure s7 ) .
the results were consistent with the 
 antitumor effect seen in the sensitive cell tumor ( figure 7a ) .
( a ) tunel assay of the 
 tumor sections from kb-8 - 5 tumor - bearing nude mice after a schedule 
 of 5 doses .
tunel - positive cells are shown as green dots , and the 
 nuclei stained by dapi are blue .
( b ) qualification of apoptosis ; p < 0.05 vs the control , p < 0.05 vs the group treated with taxol , and p < 0.05 vs the group treated with individual 
 nes . the p53 is involved in chemosensitivity , 
 and the loss of 
 p53 function has been reported to enhance cellular resistance to a 
 number of chemotherapeutic agents .
the extraordinary enhancement of cell apoptosis , and hence 
 reversal of mdr , observed in our study was ascribed to the up - regulation 
 of p53 and the arrest of the g2/m phase in the cell cycle .
anticancer drugs gain 
 access to solid tumors via the circulatory system and must penetrate 
 through the extravascular space to reach cancer cells in sufficient 
 concentration to cause lethal toxicity .
these considerations will most 
 likely result in limited distribution of chemotherapy drug in solid 
 tumors and potentially give rise to clinical resistance .
it is therefore 
 necessary to improve delivery of the drug to the tumor and to prolong 
 the blood circulation of the drug so that , aided by the epr effect , 
 lethal concentrations in the tumor cells can be achieved .
it is known 
 that peg is the most widely used moiety for surface modification of 
 nanoparticles , and pegylation can effectively retard the rapid uptake 
 of nanoparticles by the mononuclear phagocyte systems .
for this reason , 5-fu in our study was conjugated with tpgs and 
 tpgs was also included in the core - matched nes . as figure 9a , b and table 2 show , the 
 auc(0 ) of both ptx and 5-fu following the 
 administration of the core - matched nes was much higher than after 
 administration of taxol or 5-fu injection .
the cmax of ptx in nes was found to be 64.9 mg / l , whereas the cmax following administration of taxol was 1.9 

the mrt(0-t ) of ptx from 
 the core - matched nes was 29.6 h , significantly higher than that following 
 taxol administration ( 1.3 h ) .
this showed that ptx from core - matched 
 nes existed in vivo much longer than ptx from taxol .
data in table 2 show that the elimination of 5-fu after administration 
 of core - matched nes was also slower than after 5-fu injection .
the 
 half - life of 5-fu from the nes was nearly 3.5 times longer than that 
 of 5-fu following administration of the 5-fu injection .
the mice were injected intravenously 
 with taxol , 5-fu injection , or core - matched nes ( equal dose of 5 mg 
 ptx / kg and 2.2 mg 5-fu / kg ) containing 1 ci of tritium - labeled 
 ptx and 5-fu .
the results indicated 
 that the core - matched nes facilitate the 
 long circulation of ptx and 5-fu , which is closely associated with 
 the high accumulation of chemotherapeutic agents within tumors 
 and the improvement of antitumor efficacy .
although the elimination 
 of 5-fu in the core - matched nes was more rapid than the elimination 
 of ptx , it was still much improved when compared with the 5-fu injection 
 ( figure 9b ) .
the antitumor activity observed 
 has also shown the benefit of the combinational delivery with respect 
 to the pharmacokinetics . in order to prolong the circulation 
 of 5-fu even more , in future studies
, it is planned that the 5-fu 
 be conjugated with ve in order to avoid its loss through shedding 
 of peg derivative from the interface of nes .
based on the core - matched technology proposed 
 by our lab , ptx and 
 5-fu were coencapsulated into a single nanoemulsion in the form of 
 prodrugs with high entrapment efficiency ( > 95% ) , good stability , 
 and 
 fine particle size ( < 90 nm ) .
the combined core - matched nes facilitate 
 the long circulation of anticancer drugs in vivo , prevent drug loss 
 through rapid clearance and metabolism , and render more drug accumulation 
 in the tumor
. moreover , the codelivery of ptx and 5-fu in the single 
 core - matched nes contributes to reversal of mdr and the production 
 of profound synergism .
all of these advantages result in the significant 
 inhibition of resistant tumor growth with no obvious toxicity .
 in 
 this study , we developed the core - matched nanoemulsions 
 ( nes ) functionalized by vitamin e ( ve ) and tocopherol poly(ethylene 
 glycol)succinate ( tpgs ) to codeliver hydrophobic and hydrophilic drugs , 
 paclitaxel ( ptx ) and 5-fluoroucacil ( 5-fu ) , in order to achieve 
 synergistic effects and overcome ptx resistance in a multi - drug - resistant 
 ( mdr ) human epidermal carcinoma cell line kb-8 - 5 . 
 antitumor effect 
 of the combination therapy based on core - matched technology ( cmt ) 
 was evaluated in vitro and in vivo in mice . 
 the core - matched nes showed 
 entrapment efficiency of > 90% and were of nanoscale particle size 
 and negative zeta - potential . 
 the combined core - matched nes exhibited 
 concentration and time - dependent cytotoxicity against ptx - sensitive 
 kb-3 - 1 cells and ptx - resistant kb-8 - 5 cells as well as an obviously 
 increased g2/m phase block . 
 the improvements in therapeutic 
 response over either ptx  ve or 5-fu  tpgs therapy alone 
 were demonstrated by the ability to effectively induce the apoptosis 
 of tumor cells via up - regulation of tumor suppressor p53 and -tubulin 
 and by the significant inhibition of cell cycle progression . 
 the combination 
 therapy led to dramatic inhibition of tumor growth with little toxicity 
 in vivo , especially in the ptx - resistant kb-8 - 5 tumors , whereas taxol 
 had little therapeutic effect . 
 this was mainly ascribed to the synergism 
 of ptx and 5-fu and the reverse of mdr by the inhibition of atpase 
 activity by ve and tpgs . 
 coencapsulation of two chemotherapeutic 
 agents with different mechanisms allows simultaneous interruption 
 of diverse anticancer pathways , resulting in increased therapeutic 
 response and low toxicity . 
 the cmt markedly facilitated the long circulation 
 of ptx and 5-fu , which was closely associated with the high accumulation 
 of chemotherapeutic agents within the tumors and the improvement 
 of antitumor efficacy . 
 the current study demonstrated the feasibility 
 of incorporating ptx and 5-fu targeting to different pathways into 
 a single core - matched ne for the reversal of mdr and synergism in 
 cancer therapy . 