docetaxel ( dcx ) is 
 a second generation taxane , derived from the 
 inactive 10-deacetyl baccatin iii , extracted from the european yew 
 tree ( taxus baccata ) .
dcx has better 
 water solubility , pharmacokinetic profile , and anticancer activity 
 than paclitaxel .
current fda approved dcx products , 
 including taxotere , are essentially tween 80/ethanol - based solutions , which unfortunately are associated with various 
 significant side effects .
hypersensitivity reactions , which 
 are attributed to the tween 80 in the formulations , can vary from 
 simple skin rash to systemic anaphylaxis and necessitate 
 premedication with corticosteroids .
other 
 problems associated with the tween 80/ethanol - based dcx formulations 
 include the nonspecific accumulation of dcx in healthy organs , which 
 may lead to systemic toxicity and subsequent discontinuation of therapy .
nanoparticle - based , tween 80-free dcx formulations 
 are expected 
 to not only avoid tween 80-related side effects but also increase 
 the concentration of dcx in tumors due to the enhanced permeation 
 and retention ( epr ) effect .
data from many previous studies demonstrate that nanoparticles of 
 100200 nm are most successful in tumor vasculature extravasation , although there are disagreements in the literature .
the heterogeneous nature of tumor type , size , location , and metastasis 
 may contribute to the disagreements . in 
 order to improve the epr - related nanoparticles extravasation
, nanoparticles 
 should be designed to circulate longer in the blood , while the drug 
 of interest is retained within the nanoparticles .
pegylation is a strategy to render the surface of nanoparticles 
 hydrophilic , thus enabling the nanoparticles to evade early opsonization 
 and circulate longer in the blood . on the other 
 hand , for a drug to be retained within the nanoparticles ,
a strong 
 affinity between the drug and the excipient(s ) used to prepare the 
 nanoparticles is required .
solid 
 lipid nanoparticles ( slns ) have been extensively investigated 
 as drug carriers .
advantages of such nanocarriers include high compatibility with 
 lipophilic drugs , ease of fabrication , and controlled release .
reported the development of a pegylated lipid nanocapsule 
 formulation ( lnc ) for paclitaxel using a novel phase inversion - based 
 method .
the resultant lncs were made 
 of an oily medium - chain triglyceride core and stabilized with soybean 
 lecithin as a lipophilic surfactant , and peg hydroxystearate ( solutol ) 
 as a hydrophilic surfactant .
applied a high pressure 
 homogenization technique to prepare a sln formulation of paclitaxel 
 using triglyceryl myristate ( trimyristin ) and phospholipids .
the formulation showed improved in vitro activity , but the in vivo circulation time and biodistribution profile were not improved , 
 as compared to the market product taxol .
applied a factorial design to optimize formulation 
 parameters to prepare paclitaxel sln formulations using compritol 
 888 ato ( a mixture of mono- , di- , and triglycerides of behenic acid ) 
 and precirol ato5 ( i.e. , glyceryl palmito - stearate ) , and the final 
 optimized formulation demonstrated an improved in vitro cytotoxic activity against the murine breast cancer cell line mxt - b2 . the present study aimed at the rational 
 selection of a triglyceride 
 from a list of medium- and long - chain triglycerides for the development 
 of a sln formulation to ultimately improve the antitumor activity 
 of dcx . previously it was reported that low melting point triglycerides 
 are excellent solubilizers for dcx , prompting 
 us to hypothesize that high melting point triglycerides will be suitable 
 excipients for preparing dcx - incorporated slns .
triglycerides that 
 are solid at body temperature were selected to ensure formulation 
 stability and to avoid droplet coalescence .
an oil - in - water ( o / w ) emulsion - based method was applied , where dcx 
 and all lipid components were dissolved in the oil phase , and the 
 aqueous phase consisted of a 0.1% ( w / v ) poloxamer 188 aqueous solution . 

finally , the in vitro and in vivo antitumor activities of the selected formulation were evaluated .
the 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-[methoxy ( polyethylene glycol)-2000 ] ( dope - peg-2000 ) and 
 phosphatidylcholine from chicken egg ( epc ) were from avanti polar 
 lipids , inc .
sepharose 4b , mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 
 bromide ) kit , tween 80 ( t80 ) , poloxamer 188 ( pluronic f68 ) , trimyristin 
 ( tm ) , trilaurin ( tl ) , tristearin ( ts ) , tripalmitin ( tp ) , mannitol , 
 sucrose , phosphate buffer saline ( pbs , ph 7.4 ) , triglyceride assay 
 kit , and caspase 3 assay kit were all from sigma - aldrich ( st . louis , 
 mo , usa ) .
float - a - lyzer dialysis tubes ( mwco 50,000 ) were from spectrum 
 chemicals & laboratory products ( new brunswick , nj , usa ) .
tc-1 cells ( murine lung cancer 
 cell line ) were from the american type culture collection ( atcc , rockville , 
 md , usa ) and grown in rpmi 1640 , supplemented with 10% fetal bovine 
 serum ( fbs ) and 1% of 100 g / ml streptomycin and 100 iu / ml penicillin 
 ( 1% p / s ) .
m - wnt cells ( murine mammary gland cell lines ) were from 
 dr .
m - wnt cells were grown in a similar medium as tc-1 , with an 
 additional supplement of 1% glutamax . human breast adenocarcinoma 
 cells ( mda - mb-231 ) were from atcc and grown in dmem supplemented with 
 5% fbs and 1% p / s .
all cell culture reagents were from invitrogen 
 ( life technologies , carlsbad , ca , usa ) .
female c57bl/6 mice ( 68 
 weeks old ) were from charles river laboratories ( wilmington , ma , usa ) .
briefly , 1 ml of dichloromethane 
 ( dcm ) containing dcx , a triglyceride ( tm , tp , tl , or ts ) , egg pc , 
 and dope - peg-2000 in a weight ratio of 1:20:10:2 was added to 10 ml 
 of 0.1% poloxamer 188 aqueous solution in a glass vial , and the mixture 
 was sonicated using a probe sonicator , with a microprobe attached , 
 for 40 s , at a sonication intensity of 50% ( q - sonica llc , newtown , 
 ct , usa ) .
the glass vial was placed in an ice bath during sonication 
 to prevent heat accumulation .
the emulsion was stirred for 15 min 
 at 400 rpm in a water bath ( 65 c ) to evaporate dcm , and was 
 then stirred for an additional hour at room temperature .
the resultant 
 nanoparticle suspension was concentrated to 1 ml by ultrafiltration 
 using an amicon device ( millipore inc .
, 30,000 mwco ) ( 490 g , 25 min , 4 c ) as previously reported . finally , slns were briefly sonicated to eliminate aggregates due 
 to the concentrating process .
dcx - free slns were prepared similarly 
 without the addition of dcx . for slns that were used in animal studies , 
 the ultrafiltration period was extended to 60 min to further concentrate 
 the suspension .
the prolonged ultrafiltration did not result in any 
 significant particle size change ( data not shown ) .
kansas city , mo , usa ) 
 with 9.25% ( w / v ) sucrose as a cryoprotectant .
the tween 80/ethanol - based 
 dcx formulation ( dcx in t80/e ) was prepared by dissolving dcx in tween 
 80 ( 20 mg / ml ) .
this concentrate was then diluted with water / ethanol 
 solution to make a final dcx solution of 4 mg / ml .
the final concentrations 
 of tween 80 and ethanol in the solution were 20% ( v / v ) and 13% ( v / v ) , 
 respectively .
particle 
 size and zeta potential of the slns were measured using a malvern 
 zetasizer nano zs ( malvern instruments , worcestershire , u.k . ) .
briefly , 
 20 l of the concentrated slns in suspension were diluted to 
 1 ml with water , and the particle size and zeta potential were determined 
 at room temperature .
the slns were 
 examined using an fei tecnai transmission electron microscope ( fei 
 corporate , hillsboro , or , usa ) at the institute for cellular and molecular 
 biology , microscopy and imaging facility at the university of texas 
 at austin as previously reported .
the content 
 of dcx in the slns was determined using hplc after 
 extraction as previously reported with modifications .
briefly , slns in suspension were diluted 510 times 
 with methanol in a glass vial , which was placed in a water bath ( 65 
 c ) for 20 min to dissolve the lipids , and placed at 20 
 c for 45 min .
the supernatant was collected by centrifugation 
 at 18000 g for 10 min at 4 c ( beckman coulter 
 inc . ,
brea , ca , usa ) , and 5 l of the supernatant was used for 
 hplc assay as previously described .
, 
 santa clara , ca , usa ) , with rp - c18 column ( zorbax eclipse , 5 m , 
 4.6 mm  150 mm ; santa clara , ca , usa ) .
the mobile phase was 
 acetonitrile and water ( 1:1 , v / v ) .
the flow rate was 1 ml / min , and 
 the detection wavelength was 230 nm .
the dcx loading percentage was 
 measured using a similar procedure , with the exception that the slns 
 were lyophilized , and 5 ml of methanol was added to 5 mg of the lyophilized 
 slns .
the weight percentage of dcx in the slns ( % w / w ) was calculated 
 based on the following formula : to investigate 
 whether free dcx coexisted with dcx - slns in the nanoparticle preparation , 
 the slns ( 100 l ) were applied to a sepharose 4b column ( 6 mm 
  30 cm ) equilibrated with water , and the dcx - slns were eluted 
 with water .
fractions of 0.5 ml were collected , and 0.3 ml of each 
 fraction was lyophilized to determine the content of dcx as mentioned 
 above . in addition , the absorbance of each fraction ( 100 l ) 
 at 500 nm was measured using a biotek synergy ht multi - mode microplate 
 reader ( winooski , vt , usa ) to determine their turbidity , which was 
 used as an indication of the presence of nanoparticles in the fractions 
 collected .
finally , the concentration of triglycerides in each fraction 
 was also measured using a sigma triglyceride assay kit following the 
 manufacturer s instruction .
dcx - slns prepared using 
 different triglycerides were stored in parafilm - sealed vials at 4 
 c for eight days .
particle size , zeta potential , and dcx content 
 were measured as mentioned above shortly after the preparation and 
 on day 8 to monitor any change of these parameters .
the release of 
 dcx from the slns made with different triglycerides 
 was monitored using float - a - lyzer tubes ( mwco 50,000 ) .
briefly , dcx - slns 
 suspension was diluted to 1 ml with pbs and transferred to the dialysis 
 tube , which was then placed in a 50 ml plastic tube containing 20 
 ml of release medium ( pbs , 0.1 mm , ph7.4 , with 1% tween 80 ) .
the tubes 
 were then placed at 37 c in an orbital shaker at 100 rpm ( max - q 
 5000 , thermo scientific , waltham , ma , usa ) . at predetermined time 
 points , the whole release medium was replaced with fresh medium to 
 maintain sink condition , and dcx concentration was analyzed using 
 hplc as previously mentioned .
the release of dcx from the dcx in t80/e 
 formulation was evaluated similarly for comparison .
for mdsc , a ta instruments 
 model 2920 dsc ( new castle , de , usa ) was 
 used , and the data were analyzed using ta universal analysis 2000 
 software .
the ramp rate was 5 c / min , and the temperature range was 
 from 10 to 200 c .
samples included dcx , trimyristin , 
 dcx - slns ( prepared with trimyristin ) , blank slns , and the physical 
 mixture of dcx and blank slns . a philips model 1710 x - ray 
 diffractometer ( philips electronic instruments inc . ,
mahwah , nj , usa ) 
 available in the texas materials institute x - ray facility at the university 
 of texas at austin was used to analyze the crystallinity of dcx in 
 the slns .
samples included dcx alone , dcx - slns ( prepared with trimyristin ) , 
 dcx mixed with blank slns , and blank slns .
cells were seeded in 96-well 
 plates at a density of 3,000 cells / well and incubated at 37 c 
 with 5% co2 overnight .
they were treated with various concentrations 
 of dcx - slns ( prepared with trimyristin ) , dcx in t80/e , blank slns , 
 or t80/e alone for 72 h. cell viability was determined using an mtt 
 assay as previously described .
ic50 values were calculated using graphpad prism ( graphpad software , 
 inc . , la jolla , ca , usa ) .
caspase 
 3 activity was determined 
 using a sigma - aldrich caspase 3 fluorimetric assay kit . in brief , 
 tc-1 cells were seeded in 24-well plates at 25,000 cells / well and 
 incubated overnight .
the cells were treated with dcx - slns ( prepared 
 with trimyristin ) , dcx in t80/e , blank slns , or t80/e for 72 h. the 
 concentration of the dcx was 0.01 m .
the supernatant was transferred 
 to a clear - bottomed black plate and mixed with the assay substrate , 
 acetyl - asp - glu - val - asp-7-amido-4-methylcourmarin ( ac - devd - amc ) .
the 
 mixture was incubated for 6 h for the hydrolysis of the ac - devd - amc 
 by caspase 3 to release the fluorescent amc , which was quantified 
 by measuring the fluorescence intensity at 360 nm ( excitation)/460 
 nm ( emission ) according to the manufacturer s instruction . 

the unit of the caspase 3 activity was mol amc / min / ml . a caspase 3 
 inhibitor ( provided in the kit )
total protein concentration in the 
 cell lysates was determined using bio - rad dc protein assay kit following 
 the manufacturer s instruction .
all animal 
 protocols were approved by the 
 institutional animal care and use committee at the university of texas 
 at austin , and the national institutes of health ( nih ) guidelines 
 for laboratory animal use and care were followed .
animals were left 
 to acclimatize for at least 7 days upon arrival from the vendor .
each 
 mouse was injected with the murine tc-1 lung cancer cells ( 5  
 10 cells per mouse ) suspended in 100 l of fbs - free 
 rpmi 1640 medium subcutaneously in the shaved left flank .
six days 
 after the implantation ( day 6 ) , mice were randomized into 4 groups , 
 7 mice per group , and injected intravenously via the tail vein with 
 dcx - slns ( prepared with trimyristin ) , dcx in t80/e , blank slns , or 
 5% mannitol as a vehicle control .
tumor sizes were measured using a digital caliper , and tumor volumes 
 were calculated using the following formula : on day 21 , mice were euthanized to 
 harvest tumor tissues , which were weighed , fixed in zn formalin buffer 
 for immunohistochemistry . tissue sample 
 preparation for 
 immunohistochemical evaluation
was carried out in the histology and 
 tissue analysis core at dell pediatric research institute ( dpri ) at 
 the university of texas at austin .
the formalin - fixed tumor tissues 
 were embedded in paraffin wax , sectioned , and stained with an antibody 
 against cd-31 ( abcam , cambridge , ma , usa ) as a marker for angiogenesis 
 ( n = 3 ) .
slides were 
 then scanned , and images were taken using the scanscope xt ( aperio 
 technologies , vista , ca , usa ) .
three weeks after tumor 
 implantation , mice were divided into 2 groups ( n = 
 910 ) ; one group was injected with dcx in t80/e ( equivalent 
 to dcx dose of 16 mg / kg ) via the tail vein , and the other group with 
 dcx - slns prepared with trimyristin ( equivalent to dcx dose of 16 mg / kg ) . 

two or twelve hours later , 45 mice from each group were euthanized 
 to collect tumor , liver , kidney , spleen , heart , lung , and blood samples . 
 the organs and tumor tissues were weighed and then stored at 80 
 c
the blood samples were mixed with an edta solution and allowed 
 to stand for about 15 min and centrifuged ( 3300 g , 
 10 min , 4 c ) to separate the plasma , which was stored at 80 
 c .
dcx was extracted from the samples using ethyl acetate , and 
 dcx concentrations in the samples were determined using hplc .
statistical analyses were completed 
 by performing anova followed by fisher s protected least significant 
 difference procedure .
a p - value of 0.05 ( two - tail ) 
 was considered significant .
the nanoparticles were composed of dcx , a triglyceride , 
 epc , dope - peg-2000 , and pluronic f68 .
the average particle size of 
 slns produced using tristearin , tripalmitin , trimyristin , and trilaurin 
 as the triglyceride was 178.4  2.3 , 176.3  3.9 , 182.8 
  2.0 , and 150.7  14.5 nm , respectively ( table 1 ) .
the polydispersity indices of all nanoparticle 
 preparations were equal to , or below , 0.2 .
the content of dcx in the final 
 slns was 2.42.8% ( w / w ) ( table 1 ) .
the release of dcx from the slns was slower , relative to 
 the diffusion of dcx out of the dcx in t80/e formulation ( figure 1a ) .
only about 4.59% of the dcx was released 
 from the sln formulation within the first 6 h , whereas about 31% of 
 dcx diffused out of the t80/e formulation within the same time period 
 ( figure 1a ) .
the rate at which the dcx was 
 released from the dcx - slns prepared with trimyristin was the slowest 
 ( figure 1b ) .
( a ) the release of dcx from dcx - slns ( closed 
 circles ) prepared 
 using tristearin ( ts ) , tripalmitin ( tp ) , trimyristin ( tm ) , or trilaurin 
 ( tl ) . as a control , the release of dcx from dcx in t80/e ( open circles ) 
 was also included .
( b ) a comparison of the percent of dcx released 
 per hour from dcx - slns prepared using different triglycerides .
the 
 release rates were calculated with data in the initial 24 h period . 

each point represents mean ( n = 3 ) . during a short - term ,
8-day stability study at 4 
 c , no significant 
 change in particle size and dcx content in any of the four dcx - slns 
 preparations was found ( figures 2a , b ) . however , 
 the zeta potential of dcx - slns prepared with tripalmitin and trilaurin 
 changed significantly ( figure 2c ) . based on 
 data shown in figures 1 and 2 ,
the dcx - slns prepared with trimyristin were chosen for further 
 studies , because the release of dcx from the slns prepared with trimyrstin 
 as the triglyceride was the slowest , and the resultant dcx - slns were 
 also relatively more stable .
the particle sizes ( a ) , dcx contents ( b ) , and 
 zeta potentials ( c ) 
 of dcx - slns prepared with different triglycerides shortly after preparation 
 or after 8 days of storage at 4 c .
shown in figure 3a are 
 the gpc results for the dcx - slns prepared with trimyristin as the 
 triglyceride .
about 90% of the dcx that was eluted from the column 
 was associated with the triglyceride and with the fractions that contained 
 the nanoparticles ( calculated based on the area under curves of the 
 gpc profiles ) ( figure 3a ) .
the 
 slns were successfully lyophilized with 9.25% ( w / v ) sucrose as a cryoprotectant 
 ( data not shown ) .
( a ) a representative gpc profile of dcx - slns prepared 
 with trimyristin . 

collected fractions were divided into three portions : one was analyzed 
 for dcx content ( red ) , one was used to measure turbidity ( od 500 nm ) 
 ( blue ) , and the other one was used to measure trimyristin concentrations , 
 and the visible absorbance of the formed colored component was measured 
 ( od 540 ) ( black ) .
( b ) a representative tem image of dcx - slns prepared with 
 trimyristin ( bar = 200 nm ) .
dsc analysis of the dcx - slns , free dcx , blank slns , and blank 
 slns 
 mixed with dcx showed that dcx exhibited a characteristic melting 
 peak at 167.4 c ( figure 4a ) .
the physical 
 mixture exhibited an endothermic melting peak at 143.4 c , which 
 can be attributed to the presence of free dcx , as the blank slns did 
 not show any distinct peak at that temperature .
a distinct dcx endothermic 
 melting peak was also absent in the dcx - slns ( figure 4a ) .
on the other hand , the presence of the endothermic melting 
 peak of trimyristin at approximately 5558 c confirmed 
 the solid state of the lipid within the slns ( figure 4a ) .
finally , xrd showed that a characteristic dcx peak was 
 present in the physical mixture , but absent from the dcx - sln composition 
 ( figure 4b ) .
dsc thermograms ( a ) and x - ray diffractograms 
 ( b ) of dcx - slns , dcx 
 alone , trimyristin ( tm ) alone , blank slns , or the physical mixture 
 of blank slns and dcx .
mtt assay revealed 
 that both dcx - slns and dcx in t80/e inhibited the proliferation of 
 tumor cells , including murine mammary gland cancer cells ( m - wnt ) , 
 murine lung cancer cells ( tc-1 ) , and human breast adenocarcinoma cells 
 ( mda - mb-231 ) .
however , the ic50 values of the dcx - slns 
 were significantly lower than that of the dcx in t80/e in each of 
 the three cell lines ( figure 5a ) . at the highest 
 equivalent dcx concentrations tested ( i.e. , 0.01 , 1 , and 0.05 m 
 in m - wnt , tc-1
, and mda - mb-231 , respectively ) , both the blank slns 
 and the t80/e vehicle control did not show any significant toxicity 
 in all three cell lines ( data not shown ) .
( a ) the ic50 values of dcx - slns and dcx in t80/e in 
 mda - mb-231 , m - wnt , and tc-1 cells .
cells were incubated with the dcx 
 formulations for 72 h. ( b ) caspase 3 activity ( in nmol amc / min / ml ) 
 in tc-1 cells following 72 h of incubation with dcx - slns or dcx in 
 t80/e ( dcx , 0.01 m ) .
caspase 3 activity was also measured 
 in tc-1 cells treated with 
 dcx - slns , dcx in t80/e , blank slns , and t80/e at a dcx concentration 
 of 0.01 m .
caspase 3 activity in cells treated with the dcx - slns 
 was significantly higher than in cells treated with dcx in t80/e ( p < 0.005 , figure 5b ) .
the total 
 protein contents in the cell lysates in all four groups were not significantly 
 different ( data not shown ) .
the antitumor activity 
 of dcx - slns was evaluated in tc-1 murine lung cancer model pre - established 
 in c57bl/6 mice .
as shown in figure 6a , both 
 dcx - slns and dcx in t80/e significantly inhibited the growth of the 
 tc-1 tumors in mice .
however , the dcx - slns were significantly more 
 effective than the dcx in t80/e formulation , starting on day 15 ( figure 6a ) .
the average body weights of mice that were injected 
 with blank slns or 5% mannitol ( as a vehicle control ) increased slightly 
 ( 10% ) during the 21 days after tumor cell implantation , while 
 the average body weight of mice that were treated with dcx - slns or 
 dcx in t80/e did not show any significant change ( figure 6b ) .
finally , the average weight of tumors in mice 
 that were treated with the dcx - slns was also significantly lower than 
 that in other groups at the end of the study ( figure 6c ) . anti - cd31 staining ( i.e. , angiogenesis marker ) showed 
 that the extent of cd31 staining tended to be lower in 
 tumors in mice that were treated with the dcx - slns , as compared to 
 in other groups ( figure 6d ) .
( a ) the growth curves 
 of tc-1 tumors in c57bl/6 mice ( p < 
 0.05 , dcx - slns or dcx in t80/e vs 5% mannitol , p < 0.05 , dcx - slns vs dcx in t80/e ) . ( b ) 

( d ) representative images 
 of tumor tissues after anti - cd31 staining ( bar = 100 m ) .
mice 
 were iv injected with dcx - slns or dcx in t80/e at a dcx dose of 15 
 mg / kg via the tail vein on days 6 , 9 , and 12 after tumor implantation . 

figure 7 shows the concentration of dcx 
 in tumors and other organs in tc-1 
 tumor - bearing mice 2 and 12 h after the mice were injected intravenously 
 with either dcx - slns ( prepared with trimyristin ) or dcx in t80/ethanol . 

the concentration of dcx in tumors in mice that were injected with 
 dcx - slns was about 50% higher than in mice that were injected with 
 the dcx in t80/ethanol formulation , 12 h after the injection ( p < 0.05 ) ( figure 7a ) .
however , 
 the concentration of dcx in the liver , spleen , kidneys , heart , and 
 lungs of mice that were injected with the dcx - slns were lower than 
 in mice that were injected with the dcx in t80/e formulation ( figures 7b  f ) . finally , 2 h after intravenous injection , 
 the concentration of dcx in the plasma in mice that were injected 
 with the dcx - slns was about 5-times higher than in mice that were 
 injected with the dcx in t80/e formulation ( figure 7 g ) .
the concentrations of dcx in tumor ( a ) , liver ( b ) , lungs ( c ) , heart 
 ( d ) , kidneys ( e ) , spleen ( f ) , and plasma ( g ) of tc-1 tumor - bearing 
 c57bl/6 mice 2 or 12 h after iv injection of either dcx - slns or dcx 
 in t80/e .
since the discovery of taxanes in the mid-1980s , 
 the fervent search 
 for more efficacious and less toxic taxane formulations has led to 
 the fda approval of three market products , namely , taxol ( bristol - myers 
 squibb , princeton , nj , usa ) , taxotere ( sanofi - aventis u.s .
llc , bridgewater , 
 nj , usa ) , and abraxane ( celgene corporation , summit , nj , usa ) . in 
 addition
, a polymeric pegylated micelle formulation of paclitaxel 
 ( genexol - pm ) has been marketed in south korea since 2007 , and others ( e.g. , bind-014 ) are in the pipeline .
taxotere used to be the only fda - approved dcx formulation 
 on the u.s .
include formulations launched by hospira , sagent , and accord , 
 which are all dcx in tween 80/ethanol solutions .
docefrez is a lyophilized 
 dcx powder to be reconstituted with 3.54% ethanol in tween 80 before 
 injection .
dcx has low water solubility ( 5 g / ml ) , and tween 80 and ethanol are used in the current 
 dcx formulations to solubilize dcx .
the main aim in the present 
 study was to rationally design a tween 
 80-free formulation of dcx that also improves the antitumor activity 
 of dcx .
, where the authors reported 
 that dcx solubility in low melting point triglycerides , such as tributyrin , 
 tricaproin , and tricaprylin , is 10,00020,000 times more than 
 in water , we postulated that triglyceride - based 
 sln formulations may exhibit attractive drug 
four different 
 medium- and long - chain saturated triglycerides that are solid at body 
 temperature were employed in this study ; namely , trilaurin ( mp 46 
 c ) , trimyristin ( mp 57 c ) , tripalmitin , ( mp 66 c ) , 
 and tristearin ( mp 69 c ) with fatty acid chain lengths of 12 
 ( c12:0 ) , 14 ( c14:0 ) , 16 ( c16:0 ) , and 18 ( c18:0 ) carbon atoms , respectively . 

it was reported that triglycerides with melting points lower than 
 room temperature form nanodroplets that are prone to coalescence during 
 preparation or storage .
the use of high melting 
 point triglycerides may significantly decrease the mobility of the 
 drug molecules within the lipid core and thus reduce immature drug 
 leakage .
therefore , tricaprylin ( c8:0 , mp 9 
 c ) , tricaprin ( c10:0 , 
 mp 31 c ) , and trolein ( c18:1 , mp 5 c ) were excluded .
the 
 trimyristin - based sln formulation was chosen from the four tested 
 formulations because the rate at which the dcx was released from them 
 was the slowest ( figure 1 ) , and the slns were 
 also more stable ( figure 2 ) . in a mouse model 
 with pre - established tc-1 mouse tumors ,
the dcx - slns were significantly 
 more effective than dcx in t80/e in inhibiting the tumor growth ( figure 6 ) , likely because the dcx - slns significantly increased 
 the accumulation of dcx in tumor tissues ( figure 7a ) . circulating nanocarriers usually take advantage of the 
 leaky vasculature and/or poor lymphatic drainage in tumor tissues 
 to accumulate in tumors . since dcx accumulation 
 in tumor was only significantly higher at 12 h , but not at 2 h , following 
 the administration of slns , as compared to the dcx in t80/e , it is 
 likely that the enhanced retention of the dcx - slns in tumors due to 
 poor lymphatic drainage was responsible for the improved accumulation .
small molecules may also exhibit enhanced permeation to tumors as 
 well due to the leaky neovasculature in the tumor tissues , however , 
 they can easily diffuse out of tumors as well , while macromolecules 
 and nanocarriers are entrapped and consequently accumulate .
dcx not only induces apoptosis due to microtubule assembly 
 stabilization , 
 but also is antiangiogenic .
inhibition of the formation 
 of new blood vessels that provide rapidly growing tumors with increasing 
 nutritional demand is expected to stunt the tumor growth .
furthermore , the hastily formed 
 tumor - associated vasculature is characterized with various fenestration 
 and imperfections , and almost lacking any intact lymphatic drainage .
nanoparticles are known to take advantage of this leaky tumor vasculature 
 to extravasate into the tumor microenvironment , where they can accumulate .
the most widely 
 used lipid excipients are soy lecithin , glycerides , or a mixture of 
 the two .
formulated dcx into a nanostructured 
 lipid carrier based on soy lecithin , glyceryl monostearate , and fatty 
 acids , but the dcx in nanostructured 
 lipid carrier was only slightly more effective than a dcx in a t80/e 
 formulation ( duopafei ) against b16 tumors in mice .
xu et al . designed a trimyristin - based sln formulation 
 for dcx to treat hepatocellular carcinoma
instead , 
 their surface was galactosylated to target asialoglycoprotein receptors 
 overexpressed on the surface of hepatoma cell lines .
we previously reported the formulation of dcx - loaded 
 lecithin - based 
 pegylated slns .
the resultant slns showed 
 an improved in vitro cytotoxic activity , in addition 
 to improved tumor accumulation in a mouse model .
however , the capacity 
 of this formulation in incorporating dcx was limited , which may be 
 attributed to the limited affinity between dcx and the excipients . in the present study , in order to rationally 
 select the most suitable excipient , four dcx - sln formulations were 
 prepared using four different high melting point triglycerides . as discussed previously , a successful formulation for taxane is 
 the one that exhibits ( a ) long plasma circulation time , ( b ) long drug 
 retention within the delivery carrier , which requires high drug 
excipient 
 affinity and slow release , ( c ) high tumor accumulation , and ( d ) favorable 
 biodistribution profile , as less drug goes to healthy tissues .
drug release behavior from the dcx - slns and 
 short - term stability were the criteria for triglyceride selection 
 in this study . in this
regard , the absence of a burst release of dcx 
 from the dcx - slns in the first 2 h and a subsequent slower release 
 rate predict a limited drug leakage from the nanoparticles in the 
 blood circulation before reaching tumors .
the release of dcx from the tristearin - based and trilaurin - based 
 dcx - slns was relatively faster ( figure 1 ) , 
 and the tripalmitin- and trilaurin - based dcx - slns showed instability , 
 as the zeta potential changed significantly following a short - term 
 storage ( figure 2 ) . change in zeta potential 
 has been used as an indicator of nanoparticle instability .
several reports indicated that trilaurin does not exist in the solid 
 state within the slns , but rather as a 
supercooled - liquid 
 state  that resembles o / w emulsions , even at 4 c , which 
 was not the case with triglycerides having higher melting points .
the relatively faster release 
 of dcx from the trilaurin - based slns may also arise as a result of 
 the same phenomenon .
phenomenon was not reported , 
 therefore , the reasons to which this sign of instability may be attributed 
 need to be investigated . on the other hand ,
the absence of a burst 
 release of dcx from the dcx - slns prepared with trimyristin may infer 
 a strong dcx  trimyristin interaction .
it was reported that 
 the solubility of dcx in tributyrin ( 4 c chain ) is about 108 mg / ml , 
 and it gets lower with higher chain length , reaching about 56 mg / ml 
 with tricaprylin ( 8 c chain ) .
based on 
 this , and since dsc and xrd data ( figure 4 ) 
 implied that there is a strong interaction between dcx and the excipients , 
 it is speculated that dcx exists in the slns in either a noncrystalline 
 state or a dissolved state , or both , within the lipid matrix .
existence 
 of dcx in the amorphous state within lipid - based matrices was also 
 reported previously . the disappearance 
 of the characteristic dcx - related peaks in dsc and xrd was previously 
 shown to be related to the loss of dcx crystallinity .
in fact , we also found that the characteristic melting peak of dcx 
 at 167 c completely disappeared upon analyzing the thermal behavior 
 of dcx 
trimyristin mixtures at dcx to trimyristin ratios of 
 1:5 , 1:2 , and 1:1 using dsc ( data not shown ) , suggesting the prevalence 
 of a strong interaction between the dcx and trimyristin .
the relatively 
 slower release of dcx from trimyristin - based dcx - slns may also be 
 attributed to the strong dcx 
long - chain triglycerides in soybean oil and egg 
 yolk phospholipids are commonly used in intravenous fat emulsions 
 as components of parenteral nutrition for patients who are not able 
 to receive nutrition via oral diets ( e.g. , intralipid , b braun medical 
 inc . , bethlehem , pa ) .
triglycerides are metabolized in the blood by 
 lipases into corresponding fatty acids , which are cleared from the 
 blood within about 30 min .
phosphatidylcholine 
 and pegylated phosphoethanolamine are used in products that are approved 
 for intravenous infusion in humans ( e.g. , doxil ) .
therefore , we expect 
 that our new dcx - sln formulations will likely have a favorable safety 
 profile .
in fact , the body weights of the tumor - bearing mice that 
 were treated with the dcx - slns did not significantly change by the 
 end of the efficacy study ( figure 6b ) .
in addition , 
 the concentrations of dcx in vital organs such as liver , spleen , kidneys , 
 lungs , and heart of mice that were injected with the dcx - slns were 
 significantly lower than in mice that were injected with the dcx in 
 t80/e formulation ( figure 7 ) , indicating that 
 our dcx - slns may be less damaging to those vital organs than taxotere .
in the present study , by taking advantage of the high solubility 
 of dcx in triglycerides , we successfully prepared several dcx - incorporated 
 slns using various medium- and long - chain triglycerides . the dcx - sln 
 composition prepared with trimyristin
was selected for further evaluation 
 because the resultant dcx - slns were stable in a short - term stability 
 study , and the rate at which the dcx was released from them was the 
 slowest .
the dcx - slns showed a stronger antitumor activity than dcx 
 solubilized in a tween 80/ethanol mixture in cell culture and , more 
 importantly , in a mouse model with pre - established tumors , likely 
 because the dcx - slns significantly increased the accumulation of the 
 dcx in tumor tissues .
the decreased accumulation of dcx in vital organs 
 after iv injection of dcx - slns , relative to after injection of dcx 
 solubilized in a tween 80/ethanol mixture , suggests that the dcx - slns 
 may have a favorable safety profile .
 docetaxel 
 ( dcx ) is a second generation taxane . 
 it is approved by 
 the u.s . food and drug administration for the treatment of various 
 types of cancer , including breast , non - small cell lung , and head and 
 neck cancers . however , side effects , including those related to tween 
 80 , an excipient in current dcx formulations , can be severe . in the 
 present study , we developed a novel solid lipid nanoparticle ( sln ) 
 composition of dcx . 
 trimyristin was selected from a list of high melting 
 point triglycerides as the core lipid component of the slns , based 
 on the rate at which the dcx was released from the slns and the stability 
 of the slns . 
 the trimyristin - based , pegylated dcx - incorporated slns 
 ( dcx - slns ) showed significantly higher cytotoxicity against various 
 human and murine cancer cells in culture , as compared to dcx solubilized 
 in a tween 80/ethanol solution . 
 moreover , in a mouse model with pre - established 
 tumors , the new dcx - slns were significantly more effective than dcx 
 solubilized in a tween 80/ethanol solution in inhibiting tumor growth 
 without toxicity , likely because the dcx - slns increased the concentration 
 of dcx in tumor tissues , but decreased the levels of dcx in major 
 organs such as liver , spleen , heart , lung , and kidney . 
 dcx - incorporated 
 slns prepared with one or more high - melting point triglycerides may 
 represent an improved dcx formulation . 
Introduction
Materials
and Methods
Results
Discussion
Conclusion