Research Article Effect of an Albumin-Coated Mesoporous ...downloads.hindawi.com/journals/jnm/2016/4086456.pdf · Effect of an Albumin-Coated Mesoporous Silicon Nanoparticle Platform

Research ArticleEffect of an Albumin-Coated Mesoporous SiliconNanoparticle Platform for Paclitaxel Delivery in HumanLung Cancer Cell Line A549

Yu Gao123 Xiaofang Che12 Chunlei Zheng12 Kezuo Hou12 Xiujuan Qu12

Yunpeng Liu12 and Zhitu Zhu3

1Department of Medical Oncology The First Hospital of China Medical University Shenyang Liaoning China2Key Laboratory of Anticancer Drugs and Biotherapy of Liaoning Province The First Hospital of China Medical UniversityShenyang Liaoning China3Department of Oncology The First Affiliated Hospital of Jinzhou Medical University Jinzhou Liaoning China

Correspondence should be addressed to Yunpeng Liu cmuliuyunpenghotmailcomand Zhitu Zhu zhuzhitu163com

Received 14 July 2016 Revised 20 September 2016 Accepted 3 October 2016

Academic Editor Martin J Sweetman

Copyright copy 2016 Yu Gao et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Albumin-coated paclitaxel-mesoporous silicon nanoparticles (APMSN) were prepared to improve the anticancer effect in lungcancer by means of regulating the dissolution rate of paclitaxel (PTX) PTX was absorbed into the mesoporous structure ofmesoporous silicon nanoparticles (MSN) which was defined as PMSN PTX was proved to exist in an amorphous state in PMSNwhich increased the dissolution rate of PTX Albumin was coated on the surface of MSN to form AMSN AMSN and PTX weremixed to form APMSN in order to achieve sustained release of PTX Then it was found that APMSN had more significantantiproliferate effects and induced more apoptotic proportion in comparison with PTX in A549 cells Furthermore the absorptionmechanism of APMSN into A549 cells was investigated Transmission electron microscopy (TEM) and laser scanning confocalmicroscopy (LSCM) showed that APMSN could cross the cell membrane and was taken into the cytoplasm quickly Taken togetherour results demonstrate that AMSN carriers have potential as nanodrug delivery systems in the treatment of lung cancer

1 Introduction

In recent years advances in nanotechnology have demon-strated that nanoparticles generally ranging from 10 to500 nm in size can be used as effective drug transport deliv-ery systems [1] Nanoparticles can be made from differenttypes of materials and include natural or synthetic polymernanoparticles liposomes micelles and silica nanoparticles[2] Mesoporous silica nanoparticles (MSN) are very suitablecarriers for nanodrug delivery systems due to their nanoscalesize relatively low density good fluxility and biocompatibil-ity [3] For nanoparticles made frommesoporous silica mate-rials it is well known that drug particle size can be decreaseddue to the spatial confinement of the mesoporous structure

and as a result the specific surface area of drug particlesincreases significantly Therefore the drugrsquos solubility anddissolution rate can be enhanced according to the OstwaldndashFreundlich and NoyesndashWhitney equations Moreover themesoporous structure could retain drug molecules in anamorphous form or in a microcrystalline state thus inhibit-ing the aggregation of drug particles and further improvingdrug stability [4 5]

It is well known that paclitaxel (PTX) is an effective drugfor the treatment of solid tumors such as lung cancer ovariancancer breast cancer and pancreatic cancer [6 7] HoweverPTX is insoluble in water which seriously restricts theabsorption of the drug [8 9] A nanodrug delivery system cantransport anticancer drugs directly to the cancerous tissue

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2016 Article ID 4086456 9 pageshttpdxdoiorg10115520164086456

2 Journal of Nanomaterials

and effectively improve the absorption of water-insolubledrugs A recent study has shown that porous silicon canimprove the oral bioavailability of celecoxib in vitro and invivo [10] Wang et al reported that modification of surfacechemicals affected the release of an antibacterial drug fromnanostructured porous silicon [11] Furthermore Wu et alhave demonstrated that novel synthesized dual-mesoporoussilica nanospheres with core-shell structures can enhance thedissolution rate of poorly water-soluble drugs [12]

Although MSN can significantly improve the dissolutionrate of a drug it could also cause recrystallization of thedissolved drug to reduce the efficacy of the drug Several waysare known to solve this problem Chitosan-functionalizedspherical nanosilica matrix is reported to be able to regulatethe release rate of the poorly water-soluble drug carvedilol[13] Additionally osmotic pump technology could also beused to control the release rate of fenofibrate absorbed inmesoporous silica materials [14]

In this study we used albumin-coated MSN (AMSN)as carriers in a nanodrug delivery system Albumin isan endogenous human substance and therefore has goodbiocompatibility [15] We predicted that when coated withalbumin an albumin-MSN-PTX delivery system (APMSN)would exhibit increased drug diffusion resistance and adecreased dissolution rate and obtained sustained release Sothe APMSN increased drug absorption and improved theeffect of PTX Finally through the solid state characterizationof PTX inAMSNMTT test flow cytometry and fluorescencemicroscopy we sought to explore whether AMSN is suitableas a carrier for a nanodrug delivery system in the treatmentof lung cancer

2 Materials and Methods

21 Materials PTX was provided by the Tianfeng Bio-technology Company (Xirsquoan China purity gt 99) Anhy-drous ethanol chromatographic acetonitrile tetraethylor-thosilicate (TEOS) styrene monomer hexadecyltrimethyl-ammonium bromide (CTAB) (3-aminopropyl)triethoxysi-lane (APTES) 221015840-azobis[2-methylpropionamidine] dihy-drochloride (AIBA) lysine albumin hypromellose and oc-tane were obtained from the Jin Zhou Xing Bei ReagentCompany (Liaoning China) 3-[45-Dimethylthiazol-2-yl]-25-diphenyltetrazolium bromide (MTT) annexin V-FITC and propidium iodide (PI) were purchased fromNanjing KeyGEN Biotechnology Company (Jiangsu China)46-Diamidino-2-phenylindole (DAPI) was purchasedfrom Shanghai Beyotime Company (Shanghai China)Hoechst33342 and rhodamine-phalloidin were purchasedfrom Shanghai Univ-Bio Company (Shanghai China) Flu-orescein isothiocyanate (FITC) and formaldehyde werepurchased from Beijing Solarbio Company (Beijing China)Deionized water was used in all experiments

22 Cells and Cell Culture Human lung adenocarcinomaA549 cells were obtained from the Type Culture Collectionof the Chinese Academy of Sciences (Shanghai China) Cellswere cultured in RPMI 1640 medium (Sigma USA) contain-ing 10 fetal bovine serum (FBS) penicillin (100UmL) and

streptomycin (100mgmL) at 37∘C under an air atmospherecontaining 5 CO

2

23 Synthesis of MSN and MSN Coated by Albumin MSNwere prepared according to the process reported by Zhaoet al [16] Briefly the water phase (CTAB and water) andthe oil phase (octane) were mixed to form an oil-in-wateremulsion and then monomeric styrene lysine TEOS andAIBA were sequentially added to the emulsion at 60∘C underan atmosphere of N

2 Styrene monomer (039ndash55mgmL)

can regulate the pore size (5ndash16 nm)The reaction occurred at60∘C for 3 h and then remained at room temperature for 12 hParticles were obtained by filtration and drying The driedparticles were calcined at 550∘C to remove the templatesTheMSN were obtained and stored in a dryer

MSN surfaces were grafted with aminopropyl groupsusing APTES Briefly MSN (1 g) were placed into a three-necked flask and dehydrated at 100∘C under a nitrogenatmosphere for 1 h After that APTES (40mL) and ethanol(100mL) were added to the system while stirring Afterwardthe samples were refluxed at 77∘C under a nitrogen atmo-sphere for 12 hThe obtained product was washed three timeswith ethanol and dried at 90∘C The obtained MSN withamino groups (MSN-ap) were stored in a dryer

The principle of albumin coating of mesoporous siliconnanoparticles is based on electrostatic adsorption The pro-cess is shown in Figure 1 At pH 74 an albumin moleculehas more than 200 negative charges whereas the MSN withamino groups are positively charged Due to electrostaticinteractions the pores of MSN can be sealed by albuminThe coating process was performed as follows albumin wasdissolved in phosphate buffer (PBS pH 74) and MSN withamino groups (300mg) were suspended in the albuminsolution (01mgmL) for 5 h while stirring Afterward thesuspension was centrifuged at a speed of 10000 rpm andthe products were washed three times with deionized waterfollowed by threewashes with anhydrous alcoholThe residuecontaining albumin-encapsulated MSN was dried at 60∘Cunder vacuum The obtained samples (AMSN) were storedin a dryer

24 Drug Loading Procedure The anticancer drug PTX usedhere as a model drug was loaded into AMSN using theimpregnation-adsorption method (Figure 1) AMSN andPTX (mass ratio of 1 1) were mixed and dispersed indichloromethane The system was stirred at room temper-ature for 24 h until the adsorption process reached equilib-rium Finally the powder samples (APMSN) were obtainedby centrifugation and dried in a vacuum for 24 h PTXwas extracted from an accurately weighed amount ofAPMSN with methanol The drug content was determinedby high-performance liquid chromatography (HPLC L-2400HITACHI Japan) All measurements were performed intriplicate The drug loading procedure for MSN and MSN-ap was the same as that of APMSN Drug-loaded samplesof MSN and MSN-ap were defined as PMSN and PMSN-ap PMSN and PMSN-ap were used for comparison withAPMSN for in vitro dissolution experiments in order to show

Journal of Nanomaterials 3

APTES

MSN

AMSN

Albumin

PTX

APMSN

Drug release

+ +

+++

+

+

++ +

+

+ +

+

+ +

+

+

++

minus

minus

minus

minusminus

minus

minus

minus

minus

minus

minusminus

minus

minus

Figure 1 Preparation scheme of AMSN and APMSN

the effect of MSN on improving the dissolution of PTXThe equation about the calculation of drug content is asfollows Drug Loading Capacity (DLC) = PTX in APMSNweightAPMSN weight times 100

25 Characterization of MSN AMSN and APMSN Thestructure and morphology of carriers were characterizedby scanning electron microscopy (SEM) (JEOL JSM-7001Foperated at 20 kV) and transmission electron microscopy(TEM) (Tecnai G2F30 operated at 200 kV) The thermalanalysis of PTX was carried out by differential scanningcalorimetry (DSC) (DSC-60 Shimadzu Japan) from 30∘Cto 300∘C at a heating rate of 10∘Cmin under a nitrogenatmosphere X-ray diffraction analysis (XRD) (RigakuUltimaIV Japan) was used to evaluate the crystal changes of PTX inAMSN The scanning rate was 4∘min over a range from 3∘(2120579) to 60∘ with a step length of 002∘

26 In Vitro Drug Dissolution A dissolution apparatus withmagnetic stirring (DF-1 JintanYoulian Instrument ResearchInstitute) was used for dissolution testing and the dissolutionmedium was composed of phosphate buffer (pH 74) Acertain amount of PMSN-ap and PMSN powder containingan equivalent of 07mg raw PTX was respectively placedinto 200mL of dissolution medium at 37∘C and stirred witha paddle speed of 100 rpmmin Dissolution medium (2mL)was withdrawn at 5 10 15 20 30 45 and 60min and passedthrough a 022120583m microporous membrane filter for HPLCanalysis APMSN powder containing an equivalent of 07mgPTX was placed into 200mL of dissolution medium at 37∘Cand stirred with a paddle speed of 100 rpmmin Dissolutionmedium (2mL) was withdrawn at 2 4 8 12 24 36 4872 96 120 144 168 192 and 216 h and passed through a022120583mmicroporous membrane filter The drug content wasanalyzed by HPLC at a wavelength of 227 nm The mobilephase consisted of acetonitrile and water (50 50 VV)

27 In Vitro Cytotoxicity Assay The MTT assay was used toassessmitochondrial activity of the cell by thiazolyl blue tetra-zolium bromide A549 cells (at a density of 3000 cellswell)were seeded in 96-well plates Various concentrations ofAMSN PTX and APMSN were dispersed in serum-free1640 culturemedium containing 2 hypromellose and addedto 96-well plates The suspension was withdrawn after anincubation period of 72 h Then 20120583L of MTT solution(5mgmL) was added to each well and the cells wereincubated for an additional 4 h at 37∘C Lastly 200120583L ofDMSO was added to each well and the optical density (OD)was measured using a microplate reader (Tecan Swiss) at490 nm

28 FlowCytometry Analysis Apoptosis was detected by flowcytometry (Becton Dickinson CA) A549 cells (1 times 105cellswell) were seeded in 6-well plates and incubatedovernight PTX and APMSN with concentrations of 5 ngmLand 10 ngmL respectively were added to the 6-well platesAfter 48 h of incubation A549 cells were harvested bytrypsinization Cells were collected by centrifugation andresuspended in 200 120583L of binding buffer Annexin V-FITC(5 120583L) and PI (5 120583L) were added and the cells were incubatedfor 15min in the dark The apoptosis rate of A549 cells wasevaluated by flow cytometry (Becton Dickinson CA)

29 Morphological Observation of Apoptosis A549 cells (1 times105 cellswell) were seeded in 6-well plates The next day theRPMI 1640 medium was replaced and various concentra-tions of PTX or APMSN were added to each well After 48 hof incubation A549 cells were fixed in a 4 formaldehydePBS solution After 30min the fixed cells were stained withDAPI The presence of apoptotic cells was determined basedon cell morphology as observed by fluorescence microscopy(Olympus Japan)


100nm

(a)

100nm

(b)

50nm

(c)

50nm

(d)

Figure 2 The SEMmicrographs of the MSN (a) and AMSN (b) TEMmicrographs of the MSN (c) and AMSN (d)

210 Laser Scanning Confocal Microscopy (LSCM) Obser-vation of AMSN Uptake in Cells FITC can combine withamino groups on the surface of AMSN Fluorescently labeledAMSN was defined as FAMSN FAMSN (50 120583gmL) wereadded to A549 cells (1 times 105 cellswell) for 05 1 or 2 hThen A549 cells were washed three times with PBS andfixed in a 4 formaldehyde PBS solution After removingthe formaldehyde solution the A549 cells were incubatedin a PBS solution containing 01 Triton X-100 and 10bovine serum albumin The cell nuclei were stained withHoechst33342 in a PBS solution for 10min and then the actincytoskeletons were stained by rhodamine-phalloidin in a PBSsolution for 20min Finally evaluation of FAMSNuptake intocells was performed by laser scanning confocal microscopy(Olympus Japan)

211 Transmission Electron Microscopy (TEM) Study of Nano-particle Uptake A549 cells (1 times 105 cellswell) were seededin 6-well plates The 1640 culture medium was removedafter 24 h to allow for cell attachment Drug-free AMSN(50120583gmL) were dispersed in serum-free 1640 culturemedium and added to 6-well plates After 4 h of incubationthe suspension was removed and the cells were washed threetimes with PBS A549 cells were then collected fixed with

a 2 glutaraldehyde PBS solution for 24 h and embeddedin 2 agarose gel Lastly cells were fixed in a 4 osmiumtetroxide solution and were embedded in epoxy resin afterdehydration After polymerization at 60∘C for 24 h the resinblock was sliced using an ultramicrotome (Leica Germany)and the obtained ultrathin sections were examined by TEM

3 Results and Discussion

31 Structure Characteristics and Drug Loading SEM imagesrevealed the porous appearance of MSN and demonstratedthat the MSN average particle size was 100 nm (Figure 2(a))The TEM image in Figure 2(c) showed themesoporous struc-ture with a radial pattern The pore size was approximately7 nm which is a suitable size for decreasing the particlesize of poorly water-soluble drugs The space limiting effectsof nanometer-scale pores could restrict drug particles intonanometer-scale spaces and prevent agglomeration of drugparticles [1] Furthermore high drug dispersity in a meso-porous structure could significantly improve the physicalstability of the drug [17] Images of AMSN were shown inFigures 2(b) and 2(d) It was obvious that a layer of albuminwas present at the surface of the particles due to electro-static adsorption Albumin has a hydrodynamic diameter of


10 20 30 40

(1) PTX

(2) Albumin

(3) MSN

(4) AMSN

(5) Physical mixture of PTX and AMSN

(6) APMSN

Inte

nsity

(au

)

2120579 (deg)

Figure 3 XRD profiles of PTX (1) albumin (2) MSN (3) AMSN(4) physical mixtures (5) of PTX and AMSN and APMSN (6)

14 nmtimes 4 nmtimes 4 nm [18] so albumin could only be adsorbedat the entrance of pores The albumin-coated layer couldincrease the diffusion resistance of a drug and produce asustained release effect

The drug content of APMSN was 3765 plusmn 0149 Theloading capacity of AMSN depended on the PTX concen-tration and increased at higher PTX concentrations butwhen the PTX concentration exceeded 300mgmL the drugadsorption capacity of AMSN remained stable Therefore300mgmL PTX was chosen as the optimal concentrationto obtain maximum adsorption These data suggested thatAMSN had a good adsorption capacity as a carrier of poorlywater-soluble drugs

32 Solid State Characterization X-ray diffraction analysis(XRD) results demonstrated that the PTX adsorbed intothe mesoporous structure was in an amorphous form Asseen in Figure 3 the characteristic peak of raw PTX wasat 126∘ In contrast no crystalline PTX was detected inAPMSN whereas a peak at 126∘ was observed in a physicalmixture with the same proportion of PTX and AMSN Theabsence of distinctive peaks in APMSN was attributed to theamorphous state of PTX loaded into AMSNThe DSC resultsalso supported the conclusions of XRD The endothermicpeak of raw PTX was at 221∘C (Figure 4) However no PTXmelting peak was displayed in the DSC curves of APMSNIn contrast the endothermic peak of the physical mixturewith the same proportion of PTX and AMSN was observedat 221∘C This confirmed that PTX absorbed in AMSN wasin an amorphous state which has direct implications for thesolubility of PTX The combined results of XRD and DSCfurther proved that the space restriction imposed by themesoporous structure significantly inhibited the crystallinityof PTX causing amorphous PTX to be highly dispersed inthe mesoporous channels

100 200

(1) PTX

(2) Albumin

(3) MSN

(4) AMSN


(6) APMSN

Hea

t flow

(mW

)

Temperature (∘C)

Figure 4 DSC profiles of PTX (1) slbumin (2) MSN (3) AMSN(4) physical mixtures (5) of PTX and AMSN and APMSN (6)

33 In Vitro Drug Dissolution The effect of structural fea-tures on the dissolution of PTX was shown in Figure 5The cumulative dissolution of PMSN at 10min reached80 while APMSN reached 80 dissolution after 120 hObviously the dissolution rate of PTX in MSN was signif-icantly improved due to the nanometer-sized pores largespecific surface area and the spatial confinement effect ofthe mesoporous structure Furthermore according to thedissolution behavior of PMSN-ap amino chemical processof MSN had no effect on the dissolution of PTX Afteralbumin-coatedMSNwere prepared the dissolution rate waseffectively decreased As a result showed sustained releaseeffect which could prevent the recrystallization of drugsAlbumin is an endogenous substance meaning MSN coatedby albumin canmake it prolonged time in circulation system

34 In Vitro Cytotoxicity Assay AMSN concentrations rang-ing from 125 120583gmL to 500120583gmL were selected for toxicityevaluation A549 cells were incubated with various concen-trations of AMSN for 72 h As seen in Figure 6(a) incubationwith 500 120583gmL AMSN resulted in a slight reduction in cellviability and nomore than 4 apoptosis in the A549 cell lineThese data indicated that AMSN has good biocompatibilityand no toxicity According to the increased absorption andlower toxicity of AMSN an AMSN system may be a suitablecandidate for lung cancer treatment

TheMTTassay results for PTX- andAPMSN-treated cellswere shown in Figure 6(b) Treatment with APMSN (from10 ngmL to 320 ngmL) for 72 h resulted in a significantreduction in cell viability compared to treatment with PTX(from 10 ngmL to 320 ngmL) The IC50 dose was 4676 plusmn046 ngmL for APMSN at 72 h and 1092 plusmn 484 ngmL forPTX These results demonstrated that APMSN had signifi-cant in vitro antiproliferative effects compared to treatment


0 50 100 150 2000

20

40

60

80

100

Time (h)

Cum

ulat

ive d

issol

utio

n (

)

(a)

0 10 20 30 40 50 60Time (min)

PMSNPMSN-ap

0

20

40

60

80

100

Cum

ulat

ive d

issol

utio

n (

)

(b)

Figure 5 In vitro drug percentage cumulative dissolution profiles of APMSN (a) PMSN (b) and MSN-ap (b) (119899 = 3)

0 100 200 300 400 5000

20

40

60

80

100

Cel

l act

ivity

(100

)

Concentration (120583gmL)

(a)

0 40 80 120 160 200 240 280 3200

10

20

30

40

50

60

70

80

Concentration (ngmL)

PTXAPMSN

Inhi

bitio

n ra

tio (1

00

)

(b)

Figure 6 MTT results of AMSN (a) and PTX and APMSN (b) for evaluating carrier toxicity and cell inhibition rate of drug loading system(119899 = 3)

with PTX Due to the nanoscale properties of AMSNadsorption of PTX was increased significantly which led tothe enhancement of the antiproliferative effects of PTXWithan enhanced antitumor effect and lower toxicity APMSNappears to be a suitable choice for lung cancer treatment

35 Analysis of Apoptosis As shown in Figure 7 the apoptosisrate of APMSN (5 ngmL) was 3439 plusmn 1182 compared to911 plusmn 233 for PTX (5 ngmL) When the APMSN concen-tration was increased to 10 ngmL the apoptotic populationwas 4595 plusmn 1618 and correspondingly the apoptosis rate

at 10 ngmL PTX was 2290 plusmn 1163 Based on these datait was obvious that APMSN can induce apoptosis in A549cells and that the apoptosis rate was closely related to drugconcentration Furthermore the apoptosis rate of APMSN-treated cells was higher than that of cells treated with PTXThe analysis of apoptosis based on cell morphology furtherconfirmed the above results Fluorescencemicroscopy imagesin Figure 7 show that the nuclei of cells treated with APMSNorPTXpresent shrinkage and fragmentationwhile the nucleiof untreated cells display uniform complete blue fluores-cence With an increase in the PTX concentration from


Annexin V Annexin V Annexin V Annexin V Annexin V

PI

Control

104

103

102

101

100

104103102101100

FL2

-H

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104

103

102

101

100

FL2

-H104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H

PTX (5ngmL) PTX (10ngmL) APMSN (5ngmL) APMSN (10ngmL)

007 813 102 2079 2945

Figure 7 Apoptosis results and fluorescence microscope images of PTX and APMSN for evaluating effect of drug loading system on A549cells (119899 = 3)

5 ngmL to 10 ngmL the level of apoptosis increased notablyThe number of apoptotic cells among APMSN-treated cellsincreased significantly relative to PTX-treated cells The EPReffect (enhanced permeability and retention effect) is oneof the characteristics of tumors The rapid growth of thetumor tissue leads to tumor vascular abnormality and highvascular permeability therefore nanoparticles of 10ndash200 nmcan penetrate the tumor tissue vessel wall and accumulate intumor tissue [19] Because of the EPR effect the circulationof a nanoscale drug system may act to enhance the drugconcentration in tumor tissue [20 21]

36 The Uptake of FAMSN into A549 Cells According tothe LSCM results the transport of FAMSN in A549 cellswas time-dependent As shown in Figures 8(a) 8(b) and8(c) A549 cells were incubated with FAMSN (50 120583gmL)in serum-free RPMI 1640 medium for 05 h 1 h and 2 h at37∘C LSCM observations showed that the uptake of FAMSNgradually increased with the extension of time At 05 hthere were only a few FAMSN in A549 cells when 1 h and2 h had passed more particles were observed in A549 cellsHowever FAMSN did not transport into the nucleus butdid aggregate in the cytoplasmic region The intracellulardistribution of FAMSN was further investigated by TEMAs shown in Figures 8(d) and 8(e) FAMSN was taken upinto A549 cells by endocytosis Untreated cells and FAMSN-treated cells were shown in Figures 8(d) and 8(e) respectivelyWith the extension of time FAMSN accumulated in thecytosolic compartment or in vesicles These results furthersuggested that the cellular uptake mechanism of FAMSNmaybe include phagocytosis nonspecific diffusion andorendocytosis Moreover with the added of advantages ofnanoscale delivery the drug loading and incubation times

were proportional to the amount of drug in the cells whichis one of the reasons for APMSN-accelerated cell apoptosis

4 Conclusions

We successfully prepared albumin-coated mesoporous sil-icon nanoparticles (AMSN) to improve the solubility ofPTX AMSN were suitable drug carriers due to their goodbiocompatibility hydrophilicity and lack of toxicity Becauseof the space confinement inherent to the AMSN structurePTX was present in an amorphous state in APMSN TheAPMSN delivery system resulted in sustained drug releasecompared to a PMSN delivery system Additionally cellapoptosis in cells treated with APMSN was greater thanthat in cells treated with PTX because APMSN transferredmore PTX into A549 cells due to the nanoscale properties ofAPMSN Based on these studies AMSN is a promising carrierto enhance the absorption of poorly water-soluble drugs

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Science and Tech-nology Major project of the Ministry of Science and Tech-nology of China (Grant no 2013ZX09303002) Science andTechnology Plan project of Liaoning Province (Grant no2014225013) National Natural Science Foundation of China(Grant no 81372546) and Liaoning BaiQianWan TalentsProgram (Grant no 2014921032)


5120583m 5120583m 5120583m 5120583m

Hoechst33342 FAMSN Rhodamine-phalloidin Merged

05

h

(a)


1h

(b)


2h

(c)

(d) (e)

Figure 8 Cell uptake results of FAMSN in A549 cells (a b and c was the LSCM images at 05 1 and 2 h after administrated 50 120583gmL ofFAMSN and FITC was used as a fluorescent probe d and e TEM images)

References

[1] F Tang L Li and D Chen ldquoMesoporous silica nanoparticlessynthesis biocompatibility and drug deliveryrdquo Advanced Mate-rials vol 24 no 12 pp 1504ndash1534 2012

[2] S Baek R K Singh D Khanal et al ldquoSmart multifunctionaldrug delivery towards anticancer therapy harmonized in meso-porous nanoparticlesrdquoNanoscale vol 7 no 34 pp 14191ndash142162015

[3] Z Zhao Y Gao C Wu Y Hao Y Zhao and J Xu ldquoDevel-opment of novel core-shell dual-mesoporous silica nanoparti-cles for the production of high bioavailable controlled-releasefenofibrate tabletsrdquo Drug Development and Industrial Phar-macy vol 42 no 2 pp 199ndash208 2016

[4] M J K Thomas I Slipper A Walunj et al ldquoInclusion ofpoorly soluble drugs in highly ordered mesoporous silicananoparticlesrdquo International Journal of Pharmaceutics vol 387no 1-2 pp 272ndash277 2010

[5] M Van Speybroeck R Mellaerts R Mols et al ldquoEnhancedabsorption of the poorly soluble drug fenofibrate by tuning its

release rate from ordered mesoporous silicardquo European Journalof Pharmaceutical Sciences vol 41 no 5 pp 623ndash630 2010

[6] S S RamalingamM Shtivelband RA Soo et al ldquoRandomizedphase II study of carboplatin and paclitaxel with either linifanibor placebo for advanced nonsquamous non-small-cell lungcancerrdquo Journal of Clinical Oncology vol 33 no 5 pp 433ndash4422015

[7] J A Yared and K H R Tkaczuk ldquoUpdate on taxane devel-opment new analogs and new formulationsrdquo Drug DesignDevelopment andTherapy vol 6 pp 371ndash384 2012

[8] A Cirstoiu-Hapca F Buchegger N Lange L Bossy R Gurnyand F Delie ldquoBenefit of anti-HER2-coated paclitaxel-loadedimmuno-nanoparticles in the treatment of disseminated ovar-ian cancer therapeutic efficacy and biodistribution in micerdquoJournal of Controlled Release vol 144 no 3 pp 324ndash331 2010

[9] J-H Kim Y Kim K H Bae T G Park J H Lee and KPark ldquoTumor-targeted delivery of paclitaxel using low densitylipoprotein-mimetic solid lipid nanoparticlesrdquoMolecular Phar-maceutics vol 12 no 4 pp 1230ndash1241 2015

[10] J Riikonen A Correia M Kovalainen et al ldquoSystematic invitro and in vivo study on porous silicon to improve the oral


bioavailability of celecoxibrdquo Biomaterials vol 52 no 1 pp 44ndash55 2015

[11] M Wang P S Hartman A Loni L T Canham N Bodifordand J L Coffer ldquoInfluence of surface chemistry on the releaseof an antibacterial drug from nanostructured porous siliconrdquoLangmuir vol 31 no 22 pp 6179ndash6185 2015

[12] C Wu X Sun Z Zhao et al ldquoSynthesis of novel core-shell structured dual-mesoporous silica nanospheres and theirapplication for enhancing the dissolution rate of poorly water-soluble drugsrdquoMaterials Science and Engineering C vol 44 pp262ndash267 2014

[13] L Sun Y Wang T Jiang et al ldquoNovel chitosan-functionalizedspherical nanosilica matrix as an oral sustained drug deliverysystem for poorly water-soluble drug carvedilolrdquo ACS AppliedMaterials amp Interfaces vol 5 no 1 pp 103ndash113 2013

[14] CWu Z Zhao Y Zhao Y Hao Y Liu and C Liu ldquoPreparationof a push-pull osmotic pump of felodipine solubilized bymesoporous silica nanoparticles with a core-shell structurerdquoInternational Journal of Pharmaceutics vol 475 no 1 pp e298ndashe305 2014

[15] M Jupin P J Michiels F C Girard M Spraul and S SWijmenga ldquoNMR identification of endogenous metabolitesinteracting with fatted and non-fatted human serum albuminin blood plasma fatty acids influence the HSA-metaboliteinteractionrdquo Journal of Magnetic Resonance vol 228 pp 81ndash942013

[16] Z Zhao C Wu Y Zhao Y Hao Y Liu and W Zhao ldquoDevel-opment of an oral push-pull osmotic pump of fenofibrate-loaded mesoporous silica nanoparticlesrdquo International Journalof Nanomedicine vol 10 pp 1691ndash1701 2015

[17] Y ChenW Yang B ChangHHu X Fang andX Sha ldquoIn vivodistribution and antitumor activity of doxorubicin-loaded N-isopropylacrylamide-co-methacrylic acid coated mesoporoussilica nanoparticles and safety evaluationrdquo European Journal ofPharmaceutics andBiopharmaceutics vol 85 no 3 pp 406ndash4122013

[18] T Wang H Jiang Q Zhao S Wang M Zou and G ChengldquoEnhanced mucosal and systemic immune responses obtainedby porous silica nanoparticles used as an oral vaccine adjuvanteffect of silica architecture on immunological propertiesrdquo Inter-national Journal of Pharmaceutics vol 436 no 1-2 pp 351ndash3582012

[19] N Bertrand J Wu X Xu N Kamaly and O C FarokhzadldquoCancer nanotechnology the impact of passive and activetargeting in the era of modern cancer biologyrdquo Advanced DrugDelivery Reviews vol 66 pp 2ndash25 2014

[20] H Maeda ldquoToward a full understanding of the EPR effect inprimary and metastatic tumors as well as issues related to itsheterogeneityrdquo Advanced Drug Delivery Reviews vol 91 pp 3ndash6 2015

[21] T Stylianopoulos and R K Jain ldquoDesign considerations fornanotherapeutics in oncologyrdquo Nanomedicine NanotechnologyBiology and Medicine vol 11 no 8 pp 1893ndash1907 2015

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and effectively improve the absorption of water-insolubledrugs A recent study has shown that porous silicon canimprove the oral bioavailability of celecoxib in vitro and invivo [10] Wang et al reported that modification of surfacechemicals affected the release of an antibacterial drug fromnanostructured porous silicon [11] Furthermore Wu et alhave demonstrated that novel synthesized dual-mesoporoussilica nanospheres with core-shell structures can enhance thedissolution rate of poorly water-soluble drugs [12]

Although MSN can significantly improve the dissolutionrate of a drug it could also cause recrystallization of thedissolved drug to reduce the efficacy of the drug Several waysare known to solve this problem Chitosan-functionalizedspherical nanosilica matrix is reported to be able to regulatethe release rate of the poorly water-soluble drug carvedilol[13] Additionally osmotic pump technology could also beused to control the release rate of fenofibrate absorbed inmesoporous silica materials [14]

In this study we used albumin-coated MSN (AMSN)as carriers in a nanodrug delivery system Albumin isan endogenous human substance and therefore has goodbiocompatibility [15] We predicted that when coated withalbumin an albumin-MSN-PTX delivery system (APMSN)would exhibit increased drug diffusion resistance and adecreased dissolution rate and obtained sustained release Sothe APMSN increased drug absorption and improved theeffect of PTX Finally through the solid state characterizationof PTX inAMSNMTT test flow cytometry and fluorescencemicroscopy we sought to explore whether AMSN is suitableas a carrier for a nanodrug delivery system in the treatmentof lung cancer

2 Materials and Methods

21 Materials PTX was provided by the Tianfeng Bio-technology Company (Xirsquoan China purity gt 99) Anhy-drous ethanol chromatographic acetonitrile tetraethylor-thosilicate (TEOS) styrene monomer hexadecyltrimethyl-ammonium bromide (CTAB) (3-aminopropyl)triethoxysi-lane (APTES) 221015840-azobis[2-methylpropionamidine] dihy-drochloride (AIBA) lysine albumin hypromellose and oc-tane were obtained from the Jin Zhou Xing Bei ReagentCompany (Liaoning China) 3-[45-Dimethylthiazol-2-yl]-25-diphenyltetrazolium bromide (MTT) annexin V-FITC and propidium iodide (PI) were purchased fromNanjing KeyGEN Biotechnology Company (Jiangsu China)46-Diamidino-2-phenylindole (DAPI) was purchasedfrom Shanghai Beyotime Company (Shanghai China)Hoechst33342 and rhodamine-phalloidin were purchasedfrom Shanghai Univ-Bio Company (Shanghai China) Flu-orescein isothiocyanate (FITC) and formaldehyde werepurchased from Beijing Solarbio Company (Beijing China)Deionized water was used in all experiments

22 Cells and Cell Culture Human lung adenocarcinomaA549 cells were obtained from the Type Culture Collectionof the Chinese Academy of Sciences (Shanghai China) Cellswere cultured in RPMI 1640 medium (Sigma USA) contain-ing 10 fetal bovine serum (FBS) penicillin (100UmL) and

streptomycin (100mgmL) at 37∘C under an air atmospherecontaining 5 CO

2

23 Synthesis of MSN and MSN Coated by Albumin MSNwere prepared according to the process reported by Zhaoet al [16] Briefly the water phase (CTAB and water) andthe oil phase (octane) were mixed to form an oil-in-wateremulsion and then monomeric styrene lysine TEOS andAIBA were sequentially added to the emulsion at 60∘C underan atmosphere of N

2 Styrene monomer (039ndash55mgmL)

can regulate the pore size (5ndash16 nm)The reaction occurred at60∘C for 3 h and then remained at room temperature for 12 hParticles were obtained by filtration and drying The driedparticles were calcined at 550∘C to remove the templatesTheMSN were obtained and stored in a dryer

MSN surfaces were grafted with aminopropyl groupsusing APTES Briefly MSN (1 g) were placed into a three-necked flask and dehydrated at 100∘C under a nitrogenatmosphere for 1 h After that APTES (40mL) and ethanol(100mL) were added to the system while stirring Afterwardthe samples were refluxed at 77∘C under a nitrogen atmo-sphere for 12 hThe obtained product was washed three timeswith ethanol and dried at 90∘C The obtained MSN withamino groups (MSN-ap) were stored in a dryer

The principle of albumin coating of mesoporous siliconnanoparticles is based on electrostatic adsorption The pro-cess is shown in Figure 1 At pH 74 an albumin moleculehas more than 200 negative charges whereas the MSN withamino groups are positively charged Due to electrostaticinteractions the pores of MSN can be sealed by albuminThe coating process was performed as follows albumin wasdissolved in phosphate buffer (PBS pH 74) and MSN withamino groups (300mg) were suspended in the albuminsolution (01mgmL) for 5 h while stirring Afterward thesuspension was centrifuged at a speed of 10000 rpm andthe products were washed three times with deionized waterfollowed by threewashes with anhydrous alcoholThe residuecontaining albumin-encapsulated MSN was dried at 60∘Cunder vacuum The obtained samples (AMSN) were storedin a dryer

24 Drug Loading Procedure The anticancer drug PTX usedhere as a model drug was loaded into AMSN using theimpregnation-adsorption method (Figure 1) AMSN andPTX (mass ratio of 1 1) were mixed and dispersed indichloromethane The system was stirred at room temper-ature for 24 h until the adsorption process reached equilib-rium Finally the powder samples (APMSN) were obtainedby centrifugation and dried in a vacuum for 24 h PTXwas extracted from an accurately weighed amount ofAPMSN with methanol The drug content was determinedby high-performance liquid chromatography (HPLC L-2400HITACHI Japan) All measurements were performed intriplicate The drug loading procedure for MSN and MSN-ap was the same as that of APMSN Drug-loaded samplesof MSN and MSN-ap were defined as PMSN and PMSN-ap PMSN and PMSN-ap were used for comparison withAPMSN for in vitro dissolution experiments in order to show


APTES

MSN

AMSN

Albumin

PTX

APMSN

Drug release

+ +

+++

+

+

++ +

+

+ +

+

+ +

+

+

++

minus

minus

minus

minusminus

minus

minus

minus

minus

minus

minusminus

minus

minus









100nm

(a)

100nm

(b)

50nm

(c)

50nm

(d)








10 20 30 40

(1) PTX

(2) Albumin

(3) MSN

(4) AMSN


(6) APMSN

Inte

nsity

(au

)

2120579 (deg)





100 200

(1) PTX

(2) Albumin

(3) MSN

(4) AMSN


(6) APMSN

Hea

t flow

(mW

)

Temperature (∘C)






0 50 100 150 2000

20

40

60

80

100

Time (h)

Cum

ulat

ive d

issol

utio

n (

)

(a)

0 10 20 30 40 50 60Time (min)

PMSNPMSN-ap

0

20

40

60

80

100

Cum

ulat

ive d

issol

utio

n (

)

(b)


0 100 200 300 400 5000

20

40

60

80

100

Cel

l act

ivity

(100

)


(a)

0 40 80 120 160 200 240 280 3200

10

20

30

40

50

60

70

80


PTXAPMSN

Inhi

bitio

n ra

tio (1

00

)

(b)







PI

Control

104

103

102

101

100

104103102101100

FL2

-H

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104

103

102

101

100

FL2

-H104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H


007 813 102 2079 2945





4 Conclusions


Competing Interests


Acknowledgments



5120583m 5120583m 5120583m 5120583m


05

h

(a)


1h

(b)


2h

(c)

(d) (e)


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




APTES

MSN

AMSN

Albumin

PTX

APMSN

Drug release

+ +

+++

+

+

++ +

+

+ +

+

+ +

+

+

++

minus

minus

minus

minusminus

minus

minus

minus

minus

minus

minusminus

minus

minus









100nm

(a)

100nm

(b)

50nm

(c)

50nm

(d)








10 20 30 40

(1) PTX

(2) Albumin

(3) MSN

(4) AMSN


(6) APMSN

Inte

nsity

(au

)

2120579 (deg)





100 200

(1) PTX

(2) Albumin

(3) MSN

(4) AMSN


(6) APMSN

Hea

t flow

(mW

)

Temperature (∘C)






0 50 100 150 2000

20

40

60

80

100

Time (h)

Cum

ulat

ive d

issol

utio

n (

)

(a)

0 10 20 30 40 50 60Time (min)

PMSNPMSN-ap

0

20

40

60

80

100

Cum

ulat

ive d

issol

utio

n (

)

(b)


0 100 200 300 400 5000

20

40

60

80

100

Cel

l act

ivity

(100

)


(a)

0 40 80 120 160 200 240 280 3200

10

20

30

40

50

60

70

80


PTXAPMSN

Inhi

bitio

n ra

tio (1

00

)

(b)







PI

Control

104

103

102

101

100

104103102101100

FL2

-H

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104

103

102

101

100

FL2

-H104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H


007 813 102 2079 2945





4 Conclusions


Competing Interests


Acknowledgments



5120583m 5120583m 5120583m 5120583m


05

h

(a)


1h

(b)


2h

(c)

(d) (e)


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




100nm

(a)

100nm

(b)

50nm

(c)

50nm

(d)








10 20 30 40

(1) PTX

(2) Albumin

(3) MSN

(4) AMSN


(6) APMSN

Inte

nsity

(au

)

2120579 (deg)





100 200

(1) PTX

(2) Albumin

(3) MSN

(4) AMSN


(6) APMSN

Hea

t flow

(mW

)

Temperature (∘C)






0 50 100 150 2000

20

40

60

80

100

Time (h)

Cum

ulat

ive d

issol

utio

n (

)

(a)

0 10 20 30 40 50 60Time (min)

PMSNPMSN-ap

0

20

40

60

80

100

Cum

ulat

ive d

issol

utio

n (

)

(b)


0 100 200 300 400 5000

20

40

60

80

100

Cel

l act

ivity

(100

)


(a)

0 40 80 120 160 200 240 280 3200

10

20

30

40

50

60

70

80


PTXAPMSN

Inhi

bitio

n ra

tio (1

00

)

(b)







PI

Control

104

103

102

101

100

104103102101100

FL2

-H

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104

103

102

101

100

FL2

-H104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H


007 813 102 2079 2945





4 Conclusions


Competing Interests


Acknowledgments



5120583m 5120583m 5120583m 5120583m


05

h

(a)


1h

(b)


2h

(c)

(d) (e)


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




10 20 30 40

(1) PTX

(2) Albumin

(3) MSN

(4) AMSN


(6) APMSN

Inte

nsity

(au

)

2120579 (deg)





100 200

(1) PTX

(2) Albumin

(3) MSN

(4) AMSN


(6) APMSN

Hea

t flow

(mW

)

Temperature (∘C)






0 50 100 150 2000

20

40

60

80

100

Time (h)

Cum

ulat

ive d

issol

utio

n (

)

(a)

0 10 20 30 40 50 60Time (min)

PMSNPMSN-ap

0

20

40

60

80

100

Cum

ulat

ive d

issol

utio

n (

)

(b)


0 100 200 300 400 5000

20

40

60

80

100

Cel

l act

ivity

(100

)


(a)

0 40 80 120 160 200 240 280 3200

10

20

30

40

50

60

70

80


PTXAPMSN

Inhi

bitio

n ra

tio (1

00

)

(b)







PI

Control

104

103

102

101

100

104103102101100

FL2

-H

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104

103

102

101

100

FL2

-H104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H


007 813 102 2079 2945





4 Conclusions


Competing Interests


Acknowledgments



5120583m 5120583m 5120583m 5120583m


05

h

(a)


1h

(b)


2h

(c)

(d) (e)


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0 50 100 150 2000

20

40

60

80

100

Time (h)

Cum

ulat

ive d

issol

utio

n (

)

(a)

0 10 20 30 40 50 60Time (min)

PMSNPMSN-ap

0

20

40

60

80

100

Cum

ulat

ive d

issol

utio

n (

)

(b)


0 100 200 300 400 5000

20

40

60

80

100

Cel

l act

ivity

(100

)


(a)

0 40 80 120 160 200 240 280 3200

10

20

30

40

50

60

70

80


PTXAPMSN

Inhi

bitio

n ra

tio (1

00

)

(b)







PI

Control

104

103

102

101

100

104103102101100

FL2

-H

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104

103

102

101

100

FL2

-H104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H


007 813 102 2079 2945





4 Conclusions


Competing Interests


Acknowledgments



5120583m 5120583m 5120583m 5120583m


05

h

(a)


1h

(b)


2h

(c)

(d) (e)


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





PI

Control

104

103

102

101

100

104103102101100

FL2

-H

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104

103

102

101

100

FL2

-H104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H

104

103

102

101

100

FL2

-H


007 813 102 2079 2945





4 Conclusions


Competing Interests


Acknowledgments



5120583m 5120583m 5120583m 5120583m


05

h

(a)


1h

(b)


2h

(c)

(d) (e)


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




5120583m 5120583m 5120583m 5120583m


05

h

(a)


1h

(b)


2h

(c)

(d) (e)


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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Research Article Effect of an Albumin-Coated Mesoporous ...downloads.hindawi.com/journals/jnm/2016/4086456.pdf · Effect of an Albumin-Coated Mesoporous Silicon Nanoparticle Platform