l O P Publishing I Vietnam Academy of SdaiCB and Tedmofogy Attonces m Nahjral Soences NanosciBnce and NamHadinology A * . Nat Set: NanoscL NanolechnoL 7 (20IS1 025010 (9pp) doi:10 1 0 8 8 / 2 0 4 3 ^ 6 2 / 7 / 2 / 0 2 ^
In vitro evaluation of Aurora kinase inhibitor
—VX680—in formulation of PLA-TPGS nanoparticles
Thi Thuy Duona Le', Phuong Thu Ha^ Thi Hai Yen Tran'", DacTu Nguyen^ Hoai Nam Nguyen^ Van Khanh Bui^ and My Nhung Hoang^
•a^tamtl of Aihml Cell Technology, faflmte of Bioteclmology. Vietnan, Aerfmy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam
' taitllnte of Materials Science, Vietnam Academy of Science and Technology, 18 Hoann Onoc Viet.
Hanoi, Vietnam —e .<
"Faculty of Biology, VNU University of Science, 334 Nguyen Tiai Road. Hanoi, Vietnam Depaitmeot of Biological Science., Korea Advanced Instimte of Science and Technology 291 Daehak,ro, Yuseong-gu, Daejeon 34141, Korea
E-.jail: [email protected] Received 9 March 2016
Aceepttdforpublication4 April 2016 A f l \ Published 28 April 2016 \ H / Abstata °°"""'^
Mjmoric tmopaiScles prepared irom polyOacddeMocopheryl polyethylene glycol soccinate (PLA-TPGS) weiB used as potential dnig (amies with many advantages to oveirame the disadvantages of msoluble anticancer drags and enhance blood circulation time and tissues VX6S0 IS an Auroia kinase inhibitor and is also the foremost Aurora kinase inhibitor to be smdied m clinical trials. In this smdy, we aimed to investigate whether VX680-loaded PLA- TPGS nanoparticles (VX680-NPs) are able to effectively incnase tire toxicity of chemotheiapv Accordmgly, we Erst syntiiesized VX680-loaded nanoparticles and NP characterizations of moijphology, mean size, zeta potential, and encapsulation efficiency were spherical shape 63 nm - 3 0 mV and 76%, respectively. Then, we investigated flie effccls on H e U cells The cell cylotoucity was evaluated by Uie xCELLigence ical-tirae cell analyzer allowing measurement of Changes ,n clectttcal impedance on flie surface of Oie E-plate. Analysis of nucleus morphology and level othistone H3 phosphorylation was observed by confocal fluorescence scanmng llucroscopy. CeU cycle distribution and apoptosis were analyzed by flow cytometry. Om- lesults s h o « ^ that VX680-NPS reduced ceU viability witt, IC^o value lower 3.4 times compared to ftee VX680. CeU proliferation was inhibited by VX680-NPs accompanied by oUier effects such as high abnonnal changes of nucleus, a decrease of phospho-histone H3 at SerlO level, an increase M polyploid cells and resulted in higher apoptotic cells. These icsults demonsttated fliat VX680- NPs had more cytotoxicity tiran as tieated wifli VX680 alone. Thus. VX680.NPS may be considered as promising drag deUvery system for cancer oeatment.
Keywords: nanoparticle, VX680, PLA-TPGS, xCELLigence, Aurora kinase inhibitors Uassmcatton numbers; 2.04, 2.05
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©2016 Vietnain Academy oJ Soence & Tedvmogy
Attv NaL So Nanosci NamKecnnoi 712016) 025010
1. Introduction 2. A/laterials and methods
Aurora kinases are senne/threonin kinases that are essential in multiple functions of mitosis. In mammals, the Aurora kinases consist of A, B, and C and they are involved in spindle apparatus organization during cell division [1. 2].
Studies have indicated that Aurora A and B are overexpressed in lung cancer (3], cervical cancer [4], colorectal cancer [5], prostate cancer [6], renal carcinoma [7], hepatocellular car- cinoma [8], ovarian cancer [9] and bladder cancer [10]. As one cf the main reagents of the chromosomal passenger complex which is a functional mitotic structure, Aurora B plays an important role in chromosomal bionentation, reg- ulating the association of kinetochores and microtubules as well as cytokinesis [2]. Particularly, the role of Aurora B in phosphorylating histone H3 which is believed to aid m chromatin condensation and separation has also been explored. When Aurora B localizes at the centiomere, it can control the activity of other kmases positioned along the chromosome aim [II]. In recent years, Aurora kinases are promising targets for cancer treatment This led to develop- ment of new anticancer drugs that could target Aurora kina- ses. Currently, inhibitors of Aurora kinase have been discovered as a novel class of anti-cancer drugs under pre- chnical and clinical testing [1, 2, 12, 13] including VX680, MLN8237, AT9283. AZD1152 and ZM447439. Among these inhibitors, VX680 can associate with the ATP-binding site of Aurora kinases and inhibit all three isoforms, specially, Aurora kinases A and B. In its mechamsm of action, VX680 delays cytokinesis, disrupts bipolar spindle foimation but allows the cells to entry through the other stages of mitosis, which results in polyploidy or massive apoptosis in some cancer cell lines. VX680 was also die foremost Aurora kinase inhibitor lo he studied in clinical trials. However, as the otiier anti-cancer drugs, VX680 is limited by its water-insoluble property [2, 14, 15].
The use of non-toxic biodegradable polymers for syn- thesizing nanoparticles can improve tiie solubility, die bio- compatibility. and treatment efficiency and control flie drug release ofanu-cancer drugs [16. 17] PolyOactic acid) (PLA), an aliphatic polyester, is one of most extensively investigated biodegradable polymers which approved by the US Food and Drug Administration [18], was chosen to entrap VX680 in nanoparticles. However. PLA was Umited by some chemo- physicai characteristics such as hydrophobicity, low degra- dation rate [19. 20] as used for drug delivery. The PLA surface modifications wifli o-a-Eocopheryl polyetiiylene gly- col 1000 succinate (TPGS) can Umit disadvantages [20, 21].
TPGS was demonstrated not only to improve flie dmg solu- bility and treatment effects, overcome multidrug resistance enhance cellular uptake but also to exhibit anti-cancer effects by P-g!ycoproiein inhibiUon mediated multidrug resistance [22-24]. In fliis study we developed PLA-TPGS nanoparticles for loadmg VX680 and investigated some cytotoxic effects of tins dmg delivery system compared to free VX680 on H e U cells.
2.1. Cell and reagents
HeLa cell Une was purchased ftom ATCC. T h l ' l
JM
cultured in Dulbecco's modified eagle's mediuia^l supplemented with 10% fetal bovine serum a penicillin/streptomycin at 37 °C in a humidified a contaiiung 5% C02. After cell growth reached, § confluence, logarithmic phase cells were useS 1 experiment. VX680 (Tozasertib. MK-0457) was pid from BioVision (USA) and was dissolved in d oxide (DMSO) (Sigma-Aldrich, St. Louis, M O . U a i ^ ^ at —80 °C and diluted in fresh medium i m m e d i ^ ^ T ^ use. Staining agents such as Hoechst and propicUuBl^P (PI) were obtained from Invitrogen. All other orgamc sol were analytical grade from Fisher Scientific.
2.2. Preparation of VX680 loaded PLA-TPGS nanopar^
VX680 loaded PLA-TPGS nanoparticles (VX680-NPs)«
prepared using emulsion solvent evaporation/difiiision tf nique and PLA-TPGS copolymer used in diis stiidy t synthesized in our previous studies [25, 26]. Briefly, lOmgrf.
VX680 were dissolved in lOml DMSO and 30mg of P L ^ . TPGS copolymer in 30 ml of distilled water and stirred fbr,^lj' to ensure complete dissolution of PLA-TPGS. The r was closed and stirred for 24 h. After fliat, flie ohta mixture was dialyzed against distilled water for 4 8 b | remove DMSO. Dialyzed product was cenaifugcd4 5600 rpm to remove un-encapsulated VX680. Transf ' solution containing VX680/PLA-TPGS NPs was oblai^
and stored at 4 °C for further uses.
2.3. Characterization of VX680 loaded PLA-TPGS nanoparticles
Morphological characteristics of VX680-NPs were obser^^
by field emission scanning electron microscopy (FESEN^' (Hitachi S-4800 FE-SEM).'nie average size of VX680-I^;
was analyzed by dynamic light scattering (DLS). The ^ ' potential of these NPs was evaluated m deionized water US^^
flie electrophoretic mode of Zetasizer 3000 HS ( M a l ^ p - instruments Ltd, UK). Each sample was measured M
tripUcate. ^ The encapsulation efficiency (EE) of VX680 into P L ^
TPGS nanoparticles was calculated by following formulalii:^-, from Ehe HPLC results provided by sample solutions: •*1^
EE(%j ^ total drug content - free dmg content ^ - ; total dmg content ^•;
2.4. Evaluation of cellular cytotoxicity
To evaluate the effects of VX680 and VX680-NPS, ' developed a system using flie xCELLigence real-time c analyzer (RTCA). H e U cells were seeded on 96-4 E-plates. After 24 h seeding, cells were treated wifli VXf^
Adv. NaL Sd.. Nanosd- Nanolachnol. 7
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S c J i ^ j S " ^ " ^ " " " ' ^ ' ' ^ ' ° - ' ° ^ ^ ' * PLA-TPGS nanoparticles. (a) FESEM unage of VX680-NPs and (b) size distribution of
and VX680-NPS and incubated at 37 "C, 5% COj wifltin 120 h. The normaUze cell index (NCI) was evaluated and IC50 was determined by xCELLigence system (Roche).
2.5 Morphological examination
For ttie morphological examination of nuclear modification, cells were incubated witii eitiier VX680 or VX680-NPs at tiie concentration of 0.2 fiM (calculated by VX680) for 24 h and ttien cells were stained witti Hoechst 33342. Hoechst 33342 solution was added to flie culture medium at a concentration of 1.6 iM. After incubating for 30 nun, ceUs were coUected and washed witti phosphate buffered saline (PBS) and observed by confocal fluorescence scanning microscopy (Zeiss LMS 510 confocal microscopy).
2.6. Cell cycle analysis
CeUs were seeded at 1.5 x 10* in 2 ml of DMEM per petri dish witii diameter of 35 mm, allowed to grow for 24 h before being exposed to dmg. CeUs were treated witii VX680 or VX680-NPS at tiie concentration of 0.2 fM (calculated by VX680) for 24 h and DNA content was determined by flow cytometry Briefly, after incubation cells were coUected to cenirifiige ttibcs and fixed widi ettianol 70%. Then, cells wei«
stained wifli PI and treated wltii ribonuclease. CeU samples were diluted wifli PBS to appropriate concentration of 10^
ceUs ml ' to determine tiie DNA content by BD FACSCamo II Flow Cytometer.
annexin-binding buffer to get ttie density at 10* cells ml"'.
Incubate ceUs solution witii 5 /il of alexa fluor®488—annexin V and imp] of PI workmg solution for ISmin at room temperature. Gentiy mix witti 400/il of annexm-binding buffer and analyze ceUs solution on FACS Canto II sys- tem (BD).
2.8. Immunofluorescence
To investigate tiie effect of VX680 and VX680-NPs on phosphorylation level of histone H3 at SerlO, cells were grown on coverslips and treated witii VX680 or VX680-NPs at ttie concentration of 0 2 /iM (calculated by VX680) for 24 h. Then, cells were fixed in 2% formaldehyde (37 "C, 10 min), permeabiUzed in 90% ethanol ( - 2 0 "C, 15 min) and incubated witti primary anU-phospho-SerlO-histone H3 mouse monoclonal antibody (Abeam, MA) for 1 h at room temperature (RT) and anti-mouse secondary antibodies con- jugated witti alexa fluor®488 (Santa Cmz, CA) for 30 min at RT. Huorescently labeled cells were observed by confocal fluorescence scanning microscopy.
2.9. Statistical analysis
Statistical analyses were perfonned witti GraphPad Prism 5 and RTCA software (Roche). The results were expressed as mean ± SD.
2.7. Apoptosis analysis 3. Results
Annexin V conjugated alexa fluor®488/PI apoptosis assay
(Invitrogen) was employed to detect early apoptotic cells Characteristics of VXeaO-NPs
m s r r i t ' ^ c e r " " ' " ' ! , " ' ' ^ ' ' ^ ^ ' ' ' ' " ' ' ^ ' ' * ' ^ ^ ^ moiphology and size of VX680-NPs were examined a ™ f r T "'"* ""^ P'^f'^*^ ^"^ "^^"g ^SEM (figure 1(a)) and DLS (figure 1(b)) -^n^
S " s t ^ r e w a : : e d ' ' : i r p T s " r ' ""^ TH"^^- ^ ^ ' - ^ P P ^ - ^ - ^ ^ P ^ ^ - a l s h a p e a n d L e s i f e L ^ e f r m
nneny, cells were wasncd with PBS, then suspended in 50 to 100 nm with Ihe mean size of 63nra
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Rgure 2. xCELLigence RTCA biosensor technology revealed die profile of cells treated wifli VX680 and VX680-NPs.
VX680-loaded PLA-TPGS nanoparticles prepared wifli emulsion solvent evaporation/diffusion technique resulted in a high dmg EE of 76%. EE was calculated by measuring flie concentration of free VX680 in supernatant from tiie ultra- centiiftigation of nanopaiticles. The concentration of VX680 in ttie supematant was determined by comparing flie con- centration to a constmcted standard calibration curve. A standard caUbration curve of VX680 wifli ttie absorbance was studied at 253 nm.
3.2. Evaluation of cellular cytotoxicity using xCELLigence biosensor technology
Cell indexes (CIs) were normalized at ttie time point of adding compound. The NCI values were used to create ttie cellular profiting upon dmg treattnenL
The comparison of HeLa ceUular profiles between VX680 and VX680-NPs showed similar results at ttie higher doses: NCI values were higher tiian tiie control at ttie beginning and quickly dropped at tiie early stage of treatment (figure 2). RTCA profiles generated witii two dmgs treated on HeLa ceUs with indicated concentrations for 5 d The CI profiles reflected flie initial cell attachment, logaritiimic growtti phase, and responded to tiie compound treatment CIs were normalized at flie time point of addition compound TTie normalized time point is indicated by tiie venical line Cell profiles at concentration of 0.05 and 0.2/iM showed sig- nificant difference between VX680 and VX680-NPs ( P < 0.01). Each data pomt was calculated from triplicate values. Data represent tiie average ± standaiti devia- tion (n = 4).
At die dose of 0.05 ,,M flie NCI values of VX680-NPs
«eated-cells were higher ttian flie control and graduaUy decreased after 60h of mcubation, meanwhile ttie VX680- treated NCI values kept equa] to tiie control during whole
time of experiment. At tiie dose of 0.2 fM after 30h of incubation, cells treated witii VX680-NPs showed a dramalio decrease in cell grovrth. In ttie meantime, VX680-treated cdi growTh curve kept at a constant value fi«m time point tff 24-80 h of treatment then sUghtiy decreased (figure 2). Using RTCA software, we got flie time-dependent half raaxililfl inhibitory concentration QCso) values of HeLa ceUs treated witii eitiier VX680 or VX680-NPs for 24, 48, 72 and 961i (figure 3(a)), and flie dose-response curve of fliese dmgs on day 4 of treatinent (figure 3(b)). Notably, ttie ICso values of VX680-NPS were always lower ttian tiie VX680's one follow time of incubation. At time points of 72 h and 96 h htatineDt, tiie IC50 values of VX680-NPs were botti smaUer 3.2 and 4.3 times (respectively) ttian VX680 (figure 3(a)). TTie rcsDlB,-^.
indicated ttiat nanoparticle form not only conserved the activity but also increased ttie effect of dmg on HeLa cells.
3.3. The effect of VX680-NPs on cell cycle The DNA content was assessed by flow cytometty analysisof ceUs stamed wifli PI. Compared vrifli ttie control group, <&»
yX680 group showed change in DNA content after incute- tion of 24 h (figure 4). However, ttiis change was even mrae.
significant in VX680-NPs treated samples witii ceUs COT- tained almost DNA content of 4N (3.1 folds higher) and >#I - (1.2 folds higher) flian tiie VX680. Most of cells treated wift VX680-NPS were arrested at diploid and polyploid s£a(B
^4N, accounted for 66.5%, meanwhile ttiis number ill VX680-treated cells was 33.3%. Thus, VX680 alone caas«d.
less polyploidy ttian VX680-NPs at tiie same time and con^
centration tieatment.
These results were consistent witti nucleus morphok^j analysis. The fluorescent images reflected tiie abnormal in t l ^ nucleus shape and size. The nucleus tumwl into muld-lobeS morphology associated wifli appearing several restiditioft
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Time oT treatnwnt (hour) log o f concsntnition of VX680 (M) S r , i 2 T ^ " ^ ' ^ "5 ^ ^ ^ ^ '"'^ VX68a-NPs in HeLa ceUs. {a)Time dependem IC« values of VX680 and VX680-NPs dunns 96 h
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Figure 5. Huorescent images of nucleus morphology stained with Hoechst. HeLa ceUs in VX680-NPs (a) tieated sample showed laigcst anfl • most muln-lobed nuclear in compare with ihe VX680-ireated (b) and control (e) samples.
pSerlOH3 Merge
Figure 6. Pho.phcrylauon of hisione H3 a. SerlO deiecied by immunofluorescence staining. Scale ban 10 ^m.
*«h. Nat Sd- Nanosa. Nanolachnol. 7 (2016) a
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T ^ p t o i i c population in HeL^ cells analyzed by flow cytometry after treatmenl of 48 h with (a) control sample, (b) VX680 and (c)
3.4. The eftect of VXmO-NPs on the level of phospho-histone HS at serine 10
Tb& rfiiUty of VX680 and VX680-NPs in inhibiting Aurora lonase B activities was examined via detection of phospho- histone H3 at serine 10 (pSerlO histone H3) immuno- fluoTKccnt staitung. The results showed fliat control sample had a clear and strong fluorescent signal of pSerlO histone H3 wheiEas samples treated witii VX680, VX680-NPs had a decrease in die expression level of pSerlO histone H3 (fipire 6). The fluorescent images showed a decrease in die ex^ESsiffli level of histone H3 phosphorylation at SerlO in samples treated witti VX680 and VX680-NPs at concentra- tion of 0.2 pm for 24 h. However, cells suffered from VX680- NPs tTEatment had tiie lowest expression of pSerlO histone H3. Therefore, we suggested tiiat VX680-NPs inhibited tiie phosphorylation of H3 at SerlO stronger ttian VX680 alone.
3.5. The effect of VX680-NPs on cell apoptosis Most chemotherapeutic anti-cancer drugs used in ttie cUnic today include agents fliat tai^et flie ceU cycle in order to
inhibit ttie hyper-proUferation state of tumor cells and sub- sequentiy to induce apoptosis, which is tiie desired outcome of chemotiierapy. Checking flie ability of VX680-NPs in increasing VX680—induced apoptosis is one of major aims of our work.
At 48 h post-treatment of dmgs, cells treated witti VX680-NPS and VX680 were collected and analyzed in FACS Canto U (BD). Dot plot map reflected tiie highest number of ceUs induced apoptosis in samples treated witti VX680-NPS. 2.3 folds higher than VX680 alone. Obviously.
ttie nanoparticulate formulation increased ttie dmg-induced apoptosis in H e U ceUs. Figure 7 shows flie apoptotic popu- lation in HeLa cells analyzed by flow cytometry after treat- ment of 48 h, in which flie quadrant Q1 represented for cells in early stage of apoptosis witii high intensity of Alexa Fluor® 488 and low mtensity of PI. In comparison witti sample treated wifli VX680 alone (23%), cells suffered from treatment of VX680-NPs were forced lo enter program cell deatii at higher level, accountmg for 51,9% of all events.
Adv N3L SCI ' Nanosci. NanotecMnol 7 (20161 D^OIO 4. Discussion
In pubUshed researches, nanoparticles smaUer than 10 nm can be rapidly cleared by kidneys or ttirough extravasation in contrast to larger nanoparticles which can be rejected by the immune system [27]. Nanoparticles smaller than 100 nm were demonstrated to be ideal size to enhance circulation time in the blood and reduce hepatic clearance [28]. This suggests that nanoparticles we designed have suitable size for use in cancer treatment witii flie zeta potential of VX680-NPs o f - 3 0 m V .
In ttie form of nanoparticles, VX680 was first checked its abihty m inhibition of H3 phosphorylation at Ser 10, a natural substrate of Aurora B that is used as an indicator of Aurora B inhibition [11, 29-31]. The results revealed that the nano- particle form not only kept flie activity but also had more effect on ttiis substrate of Aurora kinase B,
Previous smdies on antimitotic drugs, such like VX680, have shown that treating cells at low concentrations can firstiy cause mitotic shppage. Tliis means ceUs escape ttie mitotic checkpoint and turn back to Gl phase without nuclear dividing. Some of cells die because of apoptosis, but otiier cells can continue to ttie next M phase with flie second tune of doubling flie DNA content. After two or more rounds of mitotic slippage, most of cells wfll eventually go to apoptosis [29. 30, 32]. Possibility, in our research, VX680-NPs wifli higher solubility and smaller size could come inside cells easier and reach ttie adequate amount to induce apoptosis earlier ttian VX680,
xCELLigence RTCA equipment was used to evaluate cell viability and cytotoxicity based on changes in electrical impedance on ttie surface of a plate wifli electnc nodes. CI values were relative to cellular changes including viabUity, morphology and adhesion degree. A normalized cell index (NCI) is introduced to reduce flie influence of inter-exper- imental variations [33-35]. The higher NCI values of treated cells in compare to control could be explained by tiie mech- anism of VX680 as an anti-mitotic drug. At tiie lower dose, flie compound cause mitotic slippage several rounds before cells died. Mitotic slippage leads to flie polyploidy in ceUs. At high doses, tiiis phenomenon was shortened and cell died sooner. VX680 is a potent inhibitor of Aurora kinases tiiat induces ttie accumulation of cells witti >4N DNA content followed by cell deatfi. In ceils lacking p53. endoreduphca- tion and apoptosis in response to VX680 are markedly enhanced [15, 30, 36] H e U cell is reported to have in-acti- vated p53 [37]. We suggested fliat botti treated-cell profiles showed an increase in NCI value due to tiie mcrease in ttie size but not ttie number of cells. The expansion of site attachment probably led to elevauon of die CI of treated cells whereas ttie increase in cell number, cell viabihty or attach degree is accountable for increasing Cl values of tiie untreated sample [34], Treatment witti VX680-NPs showed ttiat tiiese cells were more hkely to undergo endoreduplication and resulted m higher NCI values, sooner and stronger flian cells treated wifli VX680 alone lead to more polyploidy and multi- lobed nuclear. TTie results of real-time analysis expenment were coincidence wifli cell cycle and apoptosis analysis
profile. Clearly, VX680-NPs showed more effective onB cefls ttian VX680.
S. Conclusion
VX680-loaded PLA-TPGS nanoparticles showed t ness as a pan—Aurora kinase inhibitor by inhibiting pi
—H3, permrbed cell cycle, lead to polyploidy and ii apoptosis in cervical H e U cancer ceUs. Moreover, our si revealed ttiat VX680-loaded PLA-TPGS nanoparticlwl only conserved but also enhanced the activity of \ Thus, VX680-NPS may be considered as promising d ^ delivery system for cancer treatment.
Acknowledgments
TTiis research is funded by Vietnam National Foundation for Science and Technology Envelopment (NAFOSTID) code 106.02-2010.55 and TWAS Grant code: 09-077 RQ/
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