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Advances in Natural Sciences- NanosciBnce and Nanotech i:10.1088/2043-6262/7/2/(
Development of chitosan graft
pluronic®F127 copolymer nanoparticles containing DNA aptamer for paclitaxel delivery to treat breast cancer cells
Kim Thach Nguyen', Due Vinh U ^ Dinh Ho Do' and Quang Huan Le=
I 2 ^ v 2 f r ^ aad Molecular Biology, Pham Ngoc Thach University of Medici.e, „ . Chi
; ^ » ^ o f P^asimlogy and Mycology, Pham Ngoc Thach Umveniity of Medieme, Ho Chi Mmh i l ^ ; l ^ V i ? J L " ' * ° ' ° - " " • = ° ' « ° « ^ 9 , o g y , Viemam Academy of Science and E-m.il: .gu,eald„tlach@p„tedu,vn and huanle<,u.„g@gm,,i,„m
Received 12 March 2016
Aetepled lot pnbBcadon 12 April 2016 / S \ PtihM«d6lBne2016 ( H ) ttSlratrt CrossMark S u ^ ' T ' ^ S t ^ ^ e - ^ S l S r ^ f ^ ™ ' T - factorrecepmrfa^ly, is
"«««• cancer, b t i s stiidy we a ^ T i l o r n N r " " " " ' ' ™ ' '^""""'^ • " « « & ' HER-2 overexpressuis SK-iiR 3^,™ b * " " " " ' * P ' ' ^ " 'P^'licaUy bmd to a
*«loped a noWmfloiCnch i " " " " " = ' " " "••«• "^i"* ^ELEX snategy. We . -JdeUvctyofpacUaxeTT^ K- . ' = " ° " ^ ' ' * ' " ° ° ' ' " ' " » i ' > ° ° f A . - P h o n i c • F 1 2 7 i , d c E Z ^ i . " " ^ ' " " " ' '^''='° "-"'^•'"S "f AP niodified K-ymeric m L S h ^ ^ n b ! t ' t " " ' " ^ ' ° " ^ " ^ "P»="> " " ' ™ « • ° ' - " ' ^'^»»"y- 'Wn«ilMely 8 « 2 ± ? 5 !?! "^ "'' " " " ' ' " - " " n i b h e s of block copolymer of A i e n e y (S3 28 ± 0 1 3 % f T i T""' " ^ " " " ' " » '°'«'='' " " • "'sl- =»«P™Iation 35% in the fits, I2h aid 8 5 ^ ^ ™ I " T f J " ' " " * ° " ' * ' - •^= '='=''»= " " • « " ™'= ^ 9 % - MTT assay showed fliT™,*" ' ^ ' ' " P " ' ' ^ of receiving media. Hie ICso doses by Med eeUs up t o l i % ! £ r , f : ™ " > ' <"""oparticles loaded pacUmxcl over ftee p.clitiutel J ' ^ « t i a s a n e , f i c i e n : t r t i o : ^ S * - X , r d : i " S o r a l t ^ ^ ^ ^ ^ ^ ^ ^
55~""«'sr5'r"''*^'"- ™"'™"=- -="=
1. NFoduction
« « * « e l (PTX), die fi„t of a new class of micrombule su- M M g agents, has demonstiated signUicam antimmor ravny m cluneal dials agaiist a broad mnge of soUd mmo„, nelndmg refiactoty ovarian cancer, memsatic breast cancer
, _ . ^ . ' ° ' * • ' ^ • " " " & ™ „ „ „ s Aontiu,™, 3.0 l,co.„ C n«Il«td«lnbunonofa]i.»x.riin,us,maintainanribiiri™,,„rt, . ^
2a«3«asz ie,'0isoi8-oes33co
non-small-cell lung cancer. AIDS-relau^ Kaposi's sareoma (1. 2|. PTX IS a highly hydrophobic diti;tpenoid pseudo alkaloid widl poor aqueous solubility of approxinntiely I PS ml '. To .solve dns problem. PTX has to be fonnulaled (Taxol) al timgml ' in a vehicle composed of 1:1 blend of Cremophoi EL and edianol. which can be admimsttred by innavenous ,njeciion |3[. However, this administiation mediod can lause great disness lo patienOi and is incon- ven,eni for diem Because of die relatively large amount of Cremophor EL used and die nonspecific biodistiibution of die dmg ,n lumon and nonnal tissues, Taxol b u also been m Acaciemy ol Sconce & Tedvywpy
associated widl serious side effects, uicluding severe hyper- sensitivity reactions, myelosuppression, and nemotoxicity Developing a convenient and safe deUvety system for F I X to maxunize die dlerapeutic eflicacy at tiunor sitis while nuni- mizmg die side effecc: is dierefore a chaUengmg endeavor
Ul recem years, diere have been considerable mterestii ut developing biodegradable nanopanicles (NPs) as effective dmg deUvery systtms [4], AmphiphUic block copolymers have been widely investigated as hydrophobic drag solubi- Uzmg agents m drag deliveiy systtms. TUey can sponm- neously setf-assemble mto polymeric miceUes and NPs m aqueous envUonmentii [5J. Most polymeric miceUes are composed of a hydrophobic block as flie inner core and a hydrophiUc block as flie outer sheU. A hydrophobic drag can he encapsulated m die hydrophobic core of die miceUes to mcrease die wao^r solubUity. The hydrophiUc sheU is able to prolong flie cuculation time due to a decrease hi phagocytosis and renal cleanmce (6). The polymeric miceUes nonnaUy have average size of approximaKiy 10-lOOnm diameteis allowing flte panicles to accumulatt in tiutior tissue duough a mechamsm called enhanced penneation and retention effect radier dian m nonnal tissues (7). This is due to die fact fliat tiimor vessels are stiucmrally nngolar and leaky compared to nornid vessels. One of die most commonly used micelles m drag dehvety applications is pluronic, an amphiphUic ni- block copolymer, composed of poly(efliylene oxide) (PEO) and poIy(propyle„e oxide) (PPO). TTie hydrophiUc PEO and hydrophobic PPO blocks fomi die corona and die core of die micelles, respectively. Pluromc«*I27 (PF) has attracted a lot of attention because of dieir low toxicity hi die body and die abihty to encapsulate any hydrophobic agentii. However die major problem of ushig polymeric micelles is dieir instiibUity
oLlcZ7T *'' "°"'"'°°- ^^-^ "'•""•'i'^ ""h *i'-
osan Chl) to fomi a copolymer was suggested. Chi is die eationic polysaccharide derived ftom chitin which s t i m l t t s n biom"? T r r " "''°'^'"'' ™ " - " " - ^"-'y " 2 in biomedicd and phannaceudcal appUcations because of iK htocompatihiluy and biodegmdabiUty [9|. Aldiough Chi g r S P uromc has been used m many forais such I h y d r o ^ ^ nano-aggregation, and NPs, u has never been usS^ J a dehveo' vector fo, anti-cancer drags " » « «s a abouf I ' Z t V ^ h ' T f ' " * - « ™ ' ' « l oUgonucleotides of specifici^Tlot Th '"" '"'"' "'•^ " ' 8 " •^"'y »<!
nuctac I d •„ ^^ r '^°"'y ^yl"^-'"' b a s i on nuceic acid hbranes by a procedure tenned svsttmanc evolution of ,iga.d, by exponential emichmtt ELEX
m n k f ™ '"""" •" ' P " ™ ' " '"'"«li"E a relatively smaU physical size, lack of immunogenici^. ia vL 2TZ
Z'^ZtoZ'^^'r' '•'''"•"'"' '^"•'y »""'*™""
--^.rs2ri^rSu^-et^
"^^r^p^d^^^^fi^i^-r
gets of die aptameis were also bnefiy investig.ttd Using an
,; [ ••jgayen e l al ionic-gelation metiiod we synfliesized and characicn/cd novel NPs contiumng DNA aptiunere for PTX dclncri systems ctimprismg Chi and PF. The properties of diese polymeric miceUes such as their appearance and stabihty. encapsulation efficiency, loadmg capacity (LC), and in vitro drug release arc mvestigatixl, EspeciaUy, for die comparison of die cytotoxi- city level on SK-aR-3 bieast cancer cell-Une, NS-VN-67 colon cancer ceUs and HT-VN-26 stomach cancer cells of Viemamese patient were also evaluated.
2. Materials and methods 2.1. Matenals
Chitosan F-MMW 400 kDa was pmchased ftom Sigma Aldnch (USA). Plmt)nio*F127 (PF), paclitiixel (PTX) 3-(4 5
<Umethyldiia20l-2-yl)-2, 5 diphenyltettazoUmti bromide (MTT), McCoy's 5A medium, Dulbecco's modified eagle medium (DMEM), fetal bovUie seram (PBS) and Bypsin- efliylenedianunetetta-acettc acid (EDTA) were purehased ftom Ltfe Technologies (Smgapore). Slide-A-Lyzer™ dia- lysis cassettes wifli molecular weight cut-off (MWCO) of 3.5 kD was purchased ftom Thenno Fisher ScientiUc (USA) OUgonucleonde library 5'-TCA CCG GGA GGA GAC CCT GA-A/40-GTG GCT TGG TGG TGG TTC AA -3'. Forwani pnraer 5'-TCA CCG GGA GGA GAC CCT GA-3' and reverse primer 3'-TTG AAC CAC CAC CAA GCC AC-5'
" ' ! M - ' ' ' " ! S 2 ' ' ' ^y '^•^ <Si"e>Po") SK-BR-3 breast cancer celHine (HTB-30TM) was supplied ftom A T T C ' (USA) Cetaaxon was purehased ftom Bidiphar JSC (Viemam)' Sodium mtnte (NaNO,), hydrochloric acid (HCl), sodium hydroxide (NaOH), acemne, acetic acid, sodium tiiphotphatt S ^ i V r n ? ' ^ ' : ' " ' ' ' ' <^'^>' t-Umefliylammopyridme S ^ ? H ^ i T " " ' ™ * * ' W-UmefliyH-omamide m n S N ''*'"*<^-^™=*>'l»°^"opropyl)-earbodUmide S ' ,'^-''>'*>>">'™™1e (NHS), 2-mere.ptoeflianol, rea?/ ';; T * ^ ' ' " " ' ™ ' ' ' ' < ° " S 0 ) were all analytical reagent purchased ftom Merck Milipore Chemical Company (Gennany,. AU odier cUemicals were of analytical g,»le and
used wifliom fiirther purifiation. S«ueanu The colon cancer tissues and stiimach cancer tissues of
Hospital m Ho Chi Minh City. NS-VN-67 and HT-VN-S
* r m r ° T"" '°"'^"'°^ '"* W'> ^™^
resne^nvH ? " " " ^ ' ' * '^°' " I « * >>umidity, respecnvely, were culmred and coUected.
*tai
2.2 SELEX procedures
l ^ t ^ I o ' L i r " " " " ' " " " "'""'^ ''y ">'P«mization and 1 X 10 cells were recovered ut complete media at 37 -r far 30 mm. 200pmol ssDNA pool dissolved in S o o ^ . f J I - J buffer ( 4 . 5 g r ' glucose, 5mM M g C l / 0 ™ ' 1 ° ' ^ ' " ' ^ « IRNA, I mgml-' bovine se™m albumin " „ ' T ^ [ > T ' fered salme (PBS)) was denamred b y T e t l t l f ' ^ ' t ' ? ' ; 5 mm and cooled on ice for 10 min befnn. h i „ . Z. °^
SSDNA pool was incubated witb a c t ^ m o X ' ^ T T S
•111
Wesij
JSSc
rAteNBtSetrtfanoactWanttedwi?
: (iBtget cells} at4 "Cja^ffl^'Ctens w ^ *asU«I twice tath washing buffer, and^^^^dodhtl RNA aptamer^ were dMed by healmg at 9 5 1 : ^rSam sai separated by phenoL (ffitnofomnisoamyl aIcohoI'(IitpiMe, Roche) and chloroform
^liactiDn. TUe obtamed DNA was polymerase chain reaction
^CR>anipUfied wifli pnmets (25 cycles o f 3 0 s a t 9 4 ° C , 3 0 s al 58 °C; and 30 s at 72 "C, foUowed by 5 nun at 72 "C; die Taq polymerase and deoxynucleotide tiiphospUate (dNTP) weiE obtained fiom Proniega. In flte fitst round selection, die ant of initial ssDNA pool was 12 nmoL dissolved m 1 ml (rfbindiag buffer; and die countiir selection step was eUmi- aalcd, fit onler to acquire aptamet^ wifli high affinity and
^rafidly. die wash stiengfli was enhanced graduaUy by eaending wash dme (fiom I to lOmin), mcreasmg flie wtane of wash buffer (from 1 tii 5 ml) and die number of : washes (ftom 3 to 5). AdditionaUy, 20% FBS and 50-300
| i B molar excess 45-mer random DNA Ubrary (a completely
« t o t hhtaiy) were added to flie mcubation solution to
"Since die nonspecUic bmdhig of die selecttd pool. After 20 imn^ of selection, flie selecttd ssDNA pool was PCR- afflphfied usmg unmodified primers.
anhydride as described in die reference [16) Briefly .--bi TPP-PF copolymer (5g, 0.75 mmol), succinic anhydride (0.56 g, 3 mmol), DIHAP (0.5 g), and TEA (0.5 nfl) were dissolved m 30 ml anhydrous 1,4-dioxane and stined over- mght at 30 C. Tien die 1,4-dioxane was removed mider a centtifiige vacuum. The residue was dissolved m chlorofonn precipimttd mto an excess of diefliyl eflicr, and Oien filtered to remove un-reacttd succiiic anhydride, DMAP and TEA Witti repeating die process twice, flie precipioite, cariioxyl- ttnmnated pluronic (about 85% yields), was obtitined after hltermg and drying in a vacuum for 24 h.
2.6. Preparation of core-shell NPs
IS, Preparalon of low-mdecular-welght {MW) Cht a otain a tow MW Chi, medium MW Chi was depoly- ' ^ m l ?> ?• ^ ^ <'""™''> •"' 1>»™lved m acetic
M o f NaNOi (7.0mg ml ') K flie dissolved Chi at room
^ n ^ under magnetic stining. After Ih, flie de^o™
S , l l e w h i , ^ i ' ^ " " " ^ S ' - i o " " a s lOOOOipm for
^ y o p h U i z e d product was th'^'il^red ^4% ^ . t -
M Prepatation of Chi-TPP-PF copolymer
^ P i e p a r a t i o n of Chi NPs was achieved via flie lomc :#totonmefliodbvCalv<i.,i«/ri,;i A r-c- .
*) was ohf,i„-< I J. <" " ' [151. A Chl solution (0.1 % w / ) « l B 0 b t a e d by dissolvtiig low MW Chi Ul 1% v/v acetic
^ ^ " T - " " " " " ^ » P » - » » u s l y upon a d L r o f S c s t i n " ' " " ""^ ' « > " " ' " " ° 1 - send w S o T T r J i . ' " ™ ' ^ P ^ " " ™ for 1 h. Ul aU cases, tile
« " » = ratio of Chi-TPP solution was 2:1. PF was mcoroo Z u T s o T t i • " " ^ ^ "^"" -"- " - » i r o n to S
2.9. In vitro drug release
I e-S. Syr,thesis of caiboxyl-terminated Chi-TPP,PF copolymer ^^ ^ ' ^ / * ^ detennined by incubating the NPs in PBS at A caiboxyUc acid group was introduced to the h • ^^ ^ ^'^ °^' ^^^'"^"^"S free PTX in the supemMant was ffiO-PPO-PEO (PF. MW = 12500) by reaT" *'°'* °*^ measured for its absorbance al A = 280 nm by using UV-vis l^lenninaa hydroxy] group of plMonic*Fi27 W h " °^ * ^ spectrophoiometer. For each sample, the release medium was
^ w m succmic withdrawn at predetemiined time intervals at 12 d and -ITie procedure of the reaction between carboxyl-tenmnated Chi-TPP-PF copolymer and PTX was as follows- 4 g ( a ? mmol of -COOH grotips) of carboxyNterminated Chi- TPP-PF copolymer was dissolved in 2 ml of N N-dmiethylformamide (DMF) at room temperature 0 1 g of EDC and 0.2 g of NHS was added in carboxyl-tennmated Chi-TPP-PF copolymer solution. After 15 min reaction, 1.5 m] of 2-mercaptoethanol was added to quench the EDC followed by an addition various concentrations of PTX (0.2, 0.4, 0.6, 0.8 and LOmgrnl"') to the activated carboxyl-ter- imnated Chi-TPP-PF copolymer. The activated carboxyl-ter- minated Chi-TPP-PF copolymer in DMF was reacted with PTX in the presence of triethyJamine (1 mg) and stirred at room temperature for 12 h under nitrogen. The resultant was dialysed against DMF by using a dialysis cassette with MWCO of 3.5kD for 24 h and finally lyophilized Eo gain the product.
2.7. Characterization of NPs
The morphological examinations of NPs were made by scanning electron microscopy (SEM) (440, Leica Cambridge Ltd, Cambridge, UK). NPs were dried on an aluminum disfc at room temperature. The fixed NP& were coated with gold using a sputter coater (Desk II, Denton Vacuum, Moorestown, NJ).
Transmission electron microscopy (TEM) (JEM 1230, Joel Ltd, Tokyo, Japan) was used to examine and compare the topography of the NPs. Freeze dried NPs were suspended in milli Q water before observation.
2.8. Size measurements and determination of zeta potential Size and zeta potential (surface charge) of NPs were measured at least in tnplicate using nano paitica SZ-100 (Honba, Japan). For both of die above measurements, 0.2 mg NPs was suspended in 10 ml milli Q water.
I
Tabte 1. Ail of the oligonucleotides used in this woifc.
T Nauyen el al
Sequence
Ubt^ 5'-TCA CCG GGA GGA GAC OCT GA-/^40-GTG GCT TCG TCG TOG TTC AA 5' Aptan^r 5'-AAC TTG GTG GTC GTT CGG TOG CTG TTC AGG G T O ^ ^ ^ ^ Q I . V /^ is base number which is equal to the coiresponding aptamer sequences.
replaced by the same medium al the same condition [3]
PTXreleased(%) = _ amount of PTX released initial amount PTX
Tabte 2. Effect o f TPP ccmcentratioD on c
potential of Chi NPs. n diameter and zeta TPP concemradon (%
w/v) Mean dia-
meter (nm) Zeta potential (mV) 0.010
0.015 0.020 0.025 0.030 2.10. Evaluation of dmg encapsulation and LC
0.5 mg NPs were dissolved in 5 ml milli Q water and cen- -
^ g i n g at 14000rpm for 30min at 15 ''C. TTie amount of flK released m the supematant was measured by spectro- phoiometiy at 247-249 nm. Each sample was measured in ^- "^®"'*S tnplicate. TTie following equations were used to evaluate the LC and encapsulation efficiency (EE) of the NPs [6]:
26.24 ± 4.37 29.43 ± 1.23 37.11 ±5.42 53.26 ± 3.21 82.51 ± 7.46
49.18 ± 1.14 43.22 ± 0J8 40.31 ± L52 38.30 ± 0.92 35.82 ± 0.34
LCf%l - total PTX - free PTX
"-f^' r-; • X 100%
nanoparticle weight EE(%) = 'oal PTX - treo PDC
2.11. In vitro cytotoxicity
on SK-BR-3 hmnan bmasl cancor cell-line, NS-VN-67 colon cancer cells and HT-VN-26 s.omach cancer cells of v S n "
me«e panents. The cell-lines weie c „ W m Mc'Coy M » d cn r n °° ™'""°° " " I ' " " ! « ' " at 37 -C at 5%
S t a t win r "i" """"^ °"^ °™ ™ * »•'« P*
:z^tZT^ £eTS"i^„str.r ™
1 , , r ^ ''^^ " ™ • " " " " « " f ° ' » additional 4 h and
2.12. Statistical analysis
f i T ^ L r * ° " ' ° = * " « " * " " I s r t delation and each lyzes were perfomicd by applying one-way ANOVA tesis and P-valne <0 05 was considcnd significanL
3,1. SELEX for entichment ol DNA aptamer candidates for target cells
To isolate aptamers with high affinity and specificity to the naove membrane presented form of HER-2, we performed SELEX nsmg a well-known HER-2 ovensxpressing breast cancer cell Ime, SK-BR-3. We started die SELEX procednre by nsing a DNA library of 45 nt iMdomized region, with 1 0 "
complexity We performed posieve selecdon by retrieving aptamer, that bind to SK-BR.3 cells. After 20 rounds of posidve selecdon, die binding affinity of DNA library reached samnioon (data not shown). After the successfiil ennchment of aptameis widi high affinity to SK-ER-3ceUs, mdividual aptamers were sequenced (table I).
3.2 Preparation of Chl-TPP NPs
The NPs were prepared by ionic-gelation upon die addition of r o o m t ' " " ' " S ; ' = " ' " - < ' " S"t>« tnagnetic sUning at room ttmperamre. T i e NPs widl smaller size have valufble charactensucs such as more miproved drug delivery lonn«
expe^nents were earned on, a. die concenuations of low
^l^nm.i'^'l "^ " " " ' ' " ' • ' " " " P » • » » . O-OIS'S.
U.02%, 0,025% and 0.03% (table 2),
Dne to dissociation, TPP dissociation in water and
™ s and ion P , 0 , „ ' had been competitive reaction with NH, group of Chi, The low MW Chi fiagmenls w ™ n ^ T cipated in die reaction widl TPP moleculTs °„ni7 , 1 combined w i * the a g i « o n to fo™ s Z ^ Z ^ ' ^ ^ ^ ^ ' '
Chl NPs did not geneiatt delammation after ->A . ^ ' 4-C, suggesting dia. NPs fomied h i te„ L . n * ' ? ' "
larger NP size would have been cansedleSi,^ '' '^
phase sepamnon during stomge, ' " " " " " t a u o n and SEM images showed dial die shape of r t material each large polymer particle size Chi NP, ""^^
ill's generated tin
%
'Ni
•IIS)
' • s i
"sure 2, Panicle azemddisnibndonofChiNPs.
tea'l^wl'SnT^' ' ' " •""""""^ *•'"'»' *»•
^ ^ f^n^parafibn of Chi-TPP-PF NPs
; ^ ^ n ^ u l t s of pluronic«F127 (PF) in decreased size
^ c o u l d be attnbuted to the formation of a rigid gel S S « i „ T * ^ ^ " P ^ - Slighter swelling c a L ^ a
; , ^ ^ " ^ l e 2). Smce there was not significant chalge in
; > < ^ ^ - "^ ' " " ^ ^ ^ * " concentrations of PF
> ^ < a M 8 ) , ,t could be concluded that PF was mosdy
" f q » r a t e d mside the NP matrix, the Chi 1%.TPP 0 025'i
" i ^ were mixed in different concentiations of PF (table 3).
l a * Charactenzation of the conjugation af aptamer to PF
^ . t a n c T modified piumnic • F I 2 7 (PF-Ap) was synthesized fby tbe reacnoo of caiboxylated phnonic *FI27 wiUi the
32.14 ± 0 36 28.47 ± I 42 30.14 ± 0.91
amino groups at the ends of aptamers (figure 3(a)). PF-Ap containing a scrambled aptamer was obtained by the same method. To confirm the conjugation and determine the maximum conjugation content of Ap on pluronic ®FI27, novex®I2% tris-glycine mini gel electrophoresis was earned out. The samples including free Ap, Chi-TPP-PF, and Chi- TPP-PF-Ap with or withoul EDC-NHS used m the conjuga- tion reaction were separately subjected to 2% tris borate edta sodium dodecyl sulfide poiyaciylamine gel electrophoresis (TBE SDS PAGE). Then the electrophoresis was perfoimed at 125 V for 30 min, and the base pair band on die gel was displayed by SYBR®green nucleic acid stain (figure 3(b)).
The detailed synthesis procedure, chemical compositions, and characterization of the matenals were given in supplementary information.
(
3.5. Nanoparticle EE and LC drug
The general addition of PTX increased ihe sizes of Chi NPs, but did not affect theu" zeta potential significandy (P < 0.02).
The effect of PTX concentration on particle size is more significant than when the concentration rises 0 . 2 - 0 4 m g m l " ' (P < 0.015). The PTX-loaded Chi-PF NPs size did grow significantly at concentrations up to 0.6-1 mgml, but djere was a change in size when die concentrMion of PTX 0 4 mg ml were increased maximum (EE = 83.28 ± 0 I3%- LC = 9 12 x 0.34%) and mean diameter 86.22 ± 1.45 nm.
PTX ua.. a lo^* MW anu-cancer drug. Therefore, it might not be possible to mcrease the PTX parUcle diameter severely
"H nt Scj.; HmM Minottdviol. 7 iaoi6i 025018 (•I
PF.COOH "
.X'ni?ctrr?sr;is:riie?rt=i?''™''='^'""^^
™ * 4, Characimzanon, encapsulation etiiciency and PTX (mgml'') Mean diameter
g capacity of PTX encapsulated core-shell NPs.
(nm) Zela potential (mV) EE (%) 65.11 ± 3.81 23.22 ± I 56 86.22 ±1.45 60 12 ±0.55 89.55 ±2.05 48.35 ± 0 24 99.32 ±7.14 40.21 ± 1 4 3 110.46 ±4.32 38,33 ±1.12
LC(%) 67.31 ± 0,51 3,94 ± 1 19 83.28 ±0.13 9.12 ±0.34 68 01 ± 3.77 8.15 ± 3.63 8062 ±1.28 6.41 ± 1 2 3 59.36 ± 1.53 5.85 ± 1.57
B , n . 4 . ,a, SEM and ,b, THM , „ . , „ of Ch, 1 %-TPP „.„25,-PP 15* „ , „^„, ^ „ . , „ ^ „ , -
until it reached its maximum capacity inside die NPs The ^'^^ "<»J"«"o3y °"VfS
prepa^d-N^™ ro4rcr:™^X"2'?^?pirN';:: T^. ™ . r - - ™ - -^ -*- «.h ^
were loaded in dilferen, concentiations of m 7Jtl7'' T^.ttlS!:^';;::^T^^" ' ° ' ' " « - "
" ^''iih mean
I Mv. Nat Sd- Nanoaa. NanotedwA 7(B t 3.7. In Vitro ilrug n teolPTX
Tlie amount of released PTX was presented as die percentage of cumulative release at 37 "C over a period of 12 d. The dmg
•fsoncentration was detennined by a fluorescence spectin- photometer at excitation wavelengdi of 280nm. Nanoparticle
» solution was used for die experiments widi concentiations of I ! 5*, 7%, and 10%. In die first 12h, followed by sustained
? Idease of 29%-35% and 85%-93% after 288 h at pH 7 5 result suggested dmt there were two phases of PTX se profile. Firsdy, die imtial burst release of die PTX ftom die NPs in die first 12h, Bmst release was die phenomenon of drag which a greater amomit of imtial bulky fclg were umnediately released prior to arriving at die steady level of die release profile. This direction affeetirf die effec i m exposme time of nano-eaniers. In die next phase a T ^ 4 " ' S ' , ° i " " ' ^ ^ ^ ' ' ^ f T ^ was shot™ aAer With PTX 0.4 mg ml ' (figure 5).
3.8. In vitro cytotoxicity
tit IPP 0,025%-PF 15% NPs loaded widl PTX at con cennaBonsofO.2,04 0 6 O R a n H I ™ i - i ""'
•^,o,<+,u.o, U.Sandlmgml ', usmg die MTT
mediod on SK-BR-3 cells. The NPs widiout P K were shown
..chrr.r24-ra::;rh.-°:c'^pSf=:
P^w up , „ „ „ ^ .^^^^^_ .^ _^ J^ ^ ^2^
r l , . l Z T " °^''^™» « ' " ' ' " ™ " *ecytotoxicity t ? t Z f ° T,"'^"f' ™= ••'=""'™ '"' viability after 6, 2 2 : 3 ? % , ' t e : r v e r "' ' " ™ - ' " ' ^ ' ' ' ' ' ' " "
I m n ^ ^ ' ^ ^ t " ."^ *''"' " ^ "S™"' SK-BR-3 cell was S ^ ,h t T * ° ° ' "™ " ^ ' ^ '•"»=•• •'>»° NPs loaded PTX On tiie odier hand, die NPs non-loaded FTX processhin
^yopMized drag stored when reconstimted in d l e ^ h i water, ttiey were not toxic to cells.
With die effect of NPs Ch, 1%-TPP 0.025%-PF 15%- PTX 0,4mgml ', die SK-BR-3, NS-VN-67 and HT-VN-26 were d«id91«.95%, 52%-56% and 26%-28%, res^rely S e fib ^ t P " " " - " ™» * » ' " y = ' ' " I lod to broken celis after 6 h. PTX was a hydrophilic anti-cancer drag dial needed membrane Msponers to enter tiie cells. Cells could uptake NPs by endocytosis mediated by a claOlrin-mediated process, f«LtlKd'd " " " " " " ' * ° 8 leliveiy sysmms diat M i t a t e d drag enounce mto die cells. Widi die effect of fiee P I X 1 mgml die cell dead proportion was 13%-14% ll did not affect die cells efficienUy. The above-mentioned results of die m vitro cytotoxicity test showed diat die NPs were biocompatible and used as anti-cancer drag cairieis
Time (h) ngur, 5, ITX release proBle, ,„ PBS . , pH „f 7.5
4. Conclusions
hi diis snidy, using SELEX, we i.solated DNA aptamer which cm bmd widi high affinity and specificity to HER-2 Chi-
fZ'^-!' " • " """ "• i^ynlfesi^ anew aplamer-plur- onic F127 conjugate. A nanopaniculate fonnnlation of PTX was fonnulaled successfully. The drag loaded Ap-m,celles showed higher cylotoxicily which compared to blank
\
' " ' " " c m r m o n l ^ ^ y F J j m 6. Cell vnbiliiy of (,) SK-BR-S v e , , „ , HUT
PIX at differem times. " " » " m m coneenranons of FTX and (b) SK BR-3, NS-VN-67, HT-VN-26 widi A micelle
Ap-mieeles and free drag. The Ap-mieelles loaded FTX were sphencal and unifonn witii mean diameter 86.22 nm and zeK potential 60,12 mV. EE and LC were 83.28% a T d T u T respectively. Tlie amount of released PTX was profile at' pH 7.5 over 12d. The PrX-A|^micelles were more cytotoxic against SK-BR-3 cells dian was free PTX about five ti™
We successMy developed a novel aptimier micelle assembly for efficient detection and deUve^' system for PTX tingeting specific breast cancer cells. This apnmier micelle e n h a n i die bmding abihty of the aptimie, moiety at physiological tern,, emmre, even diough die eomisponding ftee aptimier loses iB bmdmg abihty under tile same condition. Afi of tiiese advantages endow dns unique assembly widl die capacily lo fiinclion as an eflici.m deti^tion and dehveiy vehide m die biological hving system.
Acknowledgements
This work was supponed by Slate Progtam -Appficanon-
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