Microporous and Mesoporous Materials 190 (2014) 197–207

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Microporous and Mesoporous Materials

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Synthesis, characterization and application of gold nanoshells usingmesoporous silica core

http://dx.doi.org/10.1016/j.micromeso.2014.02.0031387-1811/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Tel.: +20 235676830 (Office)/222873230 (Home),mobile: +20 1001200674.

E-mail address: n_elbialy@hotmail.com (N. Elbialy).

Nihal Elbialy ⇑, Noha Mohamed, Ahmed Soltan MonemBiophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt

a r t i c l e i n f o

Article history:Received 14 October 2013Received in revised form 18 December 2013Accepted 2 February 2014Available online 12 February 2014

Keywords:Gold nanoshellsMesoporous silicaDoxorubicinPhoto heat conversionPassive targeting

a b s t r a c t

The present study developed a novel multifunctional nanoparticle capable of being targeted passively tothe tumor site, mediating sustained drug release as well as providing photothermal therapy. Thisfabricated nanoparticle is mesoporous silica-loaded doxorubicin covered with a thin layer of pegylatedgold (PEG-DOX-MPS-GNSs). The prepared nanoparticles were characterized using transmission electronmicroscopy, energy dispersive X-ray analysis, UV–VIS absorption spectroscopy, dynamic light scattering,zeta potential measurements and small angle X-ray diffraction. The prepared mesoporous silica nanopar-ticles (MPS) were approximately 150 nm in diameter and were characterized by its well-orderedmesoporosity of d-spacing �4.5 nm, which enabled a high doxorubicin-loading capacity. Laser scanningconfocal microscopy was used to study the dynamics and cellular uptake of PEG-DOX-MPS-GNSs, in addi-tion to its therapeutic efficiency upon NIR irradiation. Superior cytotoxicity in MCF-7 cells was obtainedfor irradiated PEG-DOX-MPS-GNSs compared with other experimental groups. Intravenous application ofPEG-DOX-MPS-GNSs (1 mg/kg), followed by NIR irradiation of the tumor area, inhibited the growth ofsubcutaneous Ehrlich carcinoma in vivo (p < 0.0001) and induced a stronger anticancer effect comparedto other applied oncological modalities. Moreover, histopathological examination demonstrated a highpercentage of necrosis in PEG-DOX-MPS-GNSs-treated group (97%) compared with NIR (34%) or control(18%) groups, which was consistent with the in vitro and in vivo findings. Thus, in this context, wepresent a novel strategy for preparing a photothermal responsive formulation (PEG-DOX-MPS-GNSs),demonstrating the controlled DOX-release behavior and its therapeutic effect. These prepared multifunc-tional nanoparticles can efficiently convert laser energy into heat, which in turn induces thermal damageand delivery of doxorubicin to the tumor site with a subsequent high therapeutic efficacy.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

In recent years, numerous nanosized drug-carriers have beendeveloped to improve delivery of anticancer drugs to target sites.These drugs, either attached to the surface or encapsulated withincarriers, are usually released at target sites by activation of trigger-ing motifs, including pH [1], temperature [2] or enzyme availability[3]. Doxorubicin hydrochloride is a widely used drug for the treat-ment of different types of cancers, such as leukemia, breast cancer,ovarian cancer, various lymphomas, etc. However, its clinicalapplication is limited by its harmful side effects, the mostsignificant of which is cardiac toxicity, which can result in cardio-myopathy and congestive heart failure [4]. Thus, efforts have beenmade to develop new drug delivery techniques to reduce these

undesirable side effects, which could alter the biodistribution ofthe drug, enhance its deposition at the tumor sites, and improveits therapeutic efficacy [5].

Many drug delivery systems have been studied, such as biode-gradable polymers [6], liposomes [7], gold nanoparticles [8],hydrogels [2] mesoporous silica nanoparticles [9,10] and others[11,12]. A successful drug carrier system would be a system thathas the smallest drug dosage, could be administrated once andcould be released in a controlled manner. Mesoporous silica nano-particle has been widely investigated, due to its extraordinarychemical and physical properties, e.g. tunable particle and poresize, large specific surface area, high chemical and thermal stabil-ity, excellent biocompatibility, and versatile chemistry for furtherfunctionalization [13,14]. Further, its pore walls have a highsurface density of silanol groups, which could be reactive towardspecific guest molecules [15].

Physiological media (water, blood, and tissue) are relativelytransparent in the near infrared (NIR) region of the spectrum,

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198 N. Elbialy et al. / Microporous and Mesoporous Materials 190 (2014) 197–207

enabling tissue penetration depths of up to 10 cm [16]. Within thepast decade, a wide variety of nanoparticles with high absorptioncross sections in the near-infrared have been developed [16–20].The most readily used in biomedical applications are gold nanocag-es [11,21], gold nanoshells [18,19], hollow gold nanospheres [22]and gold nanorods [20,21]. These gold nanoparticles stronglyabsorb in the NIR and efficiently convert photon energy to heatenergy, which produces a local increase in temperature at its accu-mulation sites [18–22]. These particles are characterized by longcirculation times in vivo and are chemically inert and non-toxic[21]. Silica–gold (SiO2–Au) consists of a silica dielectric core, whichis surrounded by a gold shell tailored to absorb in the near infrared(NIR) region of the spectrum [18,19]. The intracellular generatedheat by photo-heat conversion is not only used in cancer photo-thermal therapy, but is also triggered in the release of chemo ther-apeutic drugs when gold nanoshells serve as an anticancer-drugcarrier. Thus, it is possible to introduce a promising and novelnanostructure that achieves both chemo- and photothermal thera-pies. The combination of two oncological modalities will firmlyestablish an efficient therapeutic mode for cancer treatment[23,24,22,25,26]. The development of such a dual functionalsystem, with each individual function acting in a coordinatedway with the other, is critical to optimize the therapeutic efficacyand safety of therapeutic regimes.

In this study, we developed a promising category of gold nano-shells with mesoporous silica core-loaded doxorubicin (PEG-DOX-MPS-GNSs), which are approximately 170 nm in diameter. Thesenanoshells enable passive targeting to the tumor site and providecombined cancer chemo-photothermal therapy (CCPT). Upon NIRirradiation, these gold nanoshells convert light energy into heat,which markedly affects the release rate of doxorubicin and effi-ciently kills cancer cells photothermally. Further, the weak acidicextracellular matrix of tumor tissues offers sustained drug release.

2. Experimental

2.1. Materials

Gold (III) chloride (HAuCl4�3H2O, 99.99%), tetraethyl orthosili-cate (TEOS, 99%), cetyltrimethylammonium bromide (CTAB)(99%), potassium carbonate (K2CO3, 99%), sodium citrate dehydrate(HOC(COONa)(CH2COONa)2�2H2O, 99%), thiolated polyethyleneglycol (PEG-SH, MW5000), PRMI 1640, fetal bovine serum, trypsin,sodium chloride, Triton X-100, sodium dodecyl sulfate (SDS), Trisbuffer, concentrated HCl and doxorubicin hydrochloride were pur-chased from Sigma–Aldrich. 3-Aminopropyltriethoxysilane(APTES), solution of 0.1 M sodium hydroxide (NaOH), 2-ethoxyeth-anol (C2H5OCH2CH2OH, 99%), and sodium borohydride (NaBH4,98%) were purchased from Merck. Ammonia hydroxide (NH4OH,28%) was purchased from Fluka. The WST-1 proliferation assaykit was purchased from Cayman Chemical.

2.2. Methods

2.2.1. Preparation of gold nanoshells loaded with doxorubicin (PEG-DOX-MPS-GNSs)2.2.1.1. Preparation of mesoporous silica nanoparticles. Mesoporoussilica nanoparticles (MPS) were synthesized using CTAB as theporogen and 2-ethoxyethanol as the co-solvent [14]. Typically,0.5 g of CTAB was dissolved in 70 ml distilled water, and aftercomplete dissolution, 0.5 ml of ammonia hydroxide (28%) and30 ml of the co-solvent were added. The mixture was vigorouslystirred in a closed vessel at room temperature for 30 min. Then,2.5 ml of TEOS was dropped into the mixture, which was thenvigorously stirred for 24 h. A white precipitate was collected using

centrifugation at 4472g for 15 min, washed 10 times with distilledwater. Then, the precipitate were further dispersed in ethanol solu-tion (60 ml) containing concentrated HCl (120 ll) and stirred at30 �C for 3 h to remove the template (CTAB). This surfactant extrac-tion process was repeated twice to ensure complete removal ofCTAB. The template removed mesoporous silica nanoparticles werewashed with water for three times then dried at 250 �C for 60 min.

2.2.1.2. Amino functionalization of mesoporous silica nanoparti-cles. Mesoporous silica nanoparticles were then surface functional-ized by grafting with 12 mM 3-aminopropyltriethoxysilane(APTES) at a volume ratio 3:7 under constant heat (80 �C) andvigorous stirring for 1 h. The amine-grafted mesoporous silicananoparticles were then cooled to room temperature, washed andsubjected to at least 7 cycles of centrifugation at 2862g in absoluteethanol and deionized water for 30 min. This process was necessaryto ensure that all residual reactants were removed. The aminegrafted mesoporous silica nanoparticles (AF-MPS) were then resus-pended in deionized water at a concentration of 0.3 g/ml.

2.2.1.3. Preparation of seed solution and Doxorubicin loading. Theamine grafted mesoporous silica nanoparticles were seeded withAu(OH)3 nanoparticles on their surfaces by the deposition precipi-tation method DP process [18,27]. The mixture was vigorously stir-red and heated at 70 �C for 30 min until the mild white solutionturned a light orange color, indicating successful loading ofAu(OH)3 nanoparticles on the amine grafted silica and the forma-tion of gold seeds. The seed solution was then centrifuged at 45gfor 60 min and washed with distilled water at least 5 times. Thefinal orange pellets were dispersed in phosphate buffer saline(PBS) at pH 7.4 to obtain a final precursor seed solution volumeof 40 ml. Four milligrams of doxorubicin hydrochloride (DOX)was dissolved into 1 ml mesoporous silica seed solution at pH 8,and the suspension was shaken in a dark bottle at 37 �C for 24 h.The drug-loaded MPS seeds were then separated using centrifuga-tion at 4472g for 15 min and washed with PBS several times. Thefree DOX contents in solution were calculated from the calibrationcurve at an excitation k of 480 nm and emission k of 585 nm usinga spectrofluorometer (Shimadzu, RF 5301pc, Japan). The amount ofloaded drug was (2.8 mg of DOX/1 ml MPS seeds) approximately70% of the loading percentage:

Loading efficiency ð%Þ

¼ Initial amount of DOX� Supernatant free amount of DOXInitial amount of drug

2.2.1.4. Preparation of pegylated gold nanoshells loaded with doxoru-bicin (PEG-DOX-MPS-GNSs). In growing the shell, the pH of HAuCl4was adjusted to around 10.1 by addition of 60 mg of K2CO3 to1.5 ml of 25 mM HAuCl4 diluted in 100 ml of water and allowingthe solution to stir in the dark overnight at room temperature forthe HAuCl4 to hydrolyze and age to give a colorless gold hydroxidesolution which called K-gold. The growth of the GNSs was progres-sively approached by mixing the DOX-MPS seeds with K-gold at aratio of 1:200 in the presence of sodium borohydride and sodiumcitrate. Sodium borohydride was used to reduce the complex goldhydroxide anions in the K-gold onto the Au(OH)3 seeds, whilesodium citrate was used to slow the reaction and stabilize the goldnanoshells by acting as a capping agent. This reduction resulted ina nearly immediate color change: red, purple, and blue dependingon the shell thickness and its degree of completeness.

Gold nanoshells surfaces were coated with polyethylene glycolfor intravenous injection, by adding 1 ll of 25 mM PEG-SH (MWT5000) to each 2 ml of gold nanoshells solution and incubatingthe mixture for 12 h at 4 �C. The suspension was then centrifuged

N. Elbialy et al. / Microporous and Mesoporous Materials 190 (2014) 197–207 199

at 952g for 30 min to remove residual PEG-SH from the formula-tion. Prior to the injection, the solution was centrifuged and thebillet was suspended in a sterile 0.9% saline solution. The concen-tration was then adjusted until its optical density was 2 at 805 nm.Finally, DOX concentration was 120 lg/1 ml GNSs. Preparationsteps was summarized in Scheme 1.

Samples of the pegylated mesoporous gold nanoshells (PEG-MPS-GNSs) were also prepared without the loading of DOX.

2.2.2. Sample characterizationThe size and morphology of the MPS, MPS seeds and MPS-GNSs

were determined using transmission electron microscopy (TEM)(FEI Tecnai G20, Super twin, Double tilt, LaB6 Gun) operating at200 kV. Energy-dispersive X-ray spectroscopy analysis (EDX) ofMPS seeds was performed to confirm the binding of gold ions tothe mesoporous silica surface. The absorption spectra of MPS,MPS seeds, DOX-MPS seeds, free DOX, MPS-GNSs and DOX-MPS-GNSs were measured using a UV–VIS spectrophotometer (JenwayUV-6420; Barloworld scientific, Essex, UK) in the wavelength rangefrom 400 to 900 nm.

The dynamic light scattering apparatus (Zeta Potential/ParticleSizer NICOMP TM 380 ZLS, USA) was used to measure the size dis-tribution of the prepared MPS and PEG-DOX-MPS GNSs. Further-more, the zeta potential of the MPS, AF-MPS, DOX-MPS seeds,DOX-MPS-GNSs, PEG-DOX-MPS-GNSs were measured in deionizedwater.

Small-angle powder X-ray diffraction (XRD) was used to mea-sure the diffraction pattern of MPS using the XPERT-PRO-PAN ana-lytical, Nether land (operating at 45 kV and 30 mA) with a CuKaradiation (k = 1.54056 Å). The sample was scanned from 0.52� to9.96� (2h), at a scan step time of 3.00 s and a step size (2h) of 0.04�.

A Basic Vector, FT/IR-4100 type A (Fourier Transform Infrared(FTIR)) (Germany) was used for obtaining the infrared spectra ofMPS, AF-MPS, MPS seeds, DOX, DOX-MPS seeds, DOX-MPS-GNSs,PEG- DOX-MPS-GNSs and PEG-SH in the range of 4000–400 cm�1.

2.2.3. Drug release from DOX-MPS seeds and PEG-DOX-MPS-GNSsSterilized dialysis bags with a dialyzer molecular-weight cut-off

of 12,000 Da were used to perform the drug release experiments.These dialysis bags were soaked overnight in the release medium.

Scheme 1. Summary of PEG-DOX-

Two phosphate buffered saline (PBS) solutions of pH 7.4 and pH 5.8were used as the drug release media to simulate normal blood/tis-sues and tumor environments, respectively. Half milliliter of DOX-MPS seeds (2.8 mg/ml) and PEG-DOX-MPS-GNSs (120 lg/ml) werecentrifuged and redispersed into release media, and the solutionwas placed into the dialysis bags. The sealed dialysis bags wereplaced into brown bottles, and 40 ml of release media was addedto each bottle. These bottles were shaken at a speed of 105 rpmat 37 �C under a light-sealed condition. At specific time intervals,three milliliters of the release media were removed to quantifythe concentration of the released drug using a spectrofluorometer.It was then returned to the original release media. The concentra-tions of the released drug were calculated from the calibrationcurve at an excitation k of 480 nm and emission k of 585 nm usinga spectrofluorometer.

Cumulative release ð%Þ ¼ Amount of DOX releasedAmount of DOX in the nanoparticles

� 100%

To evaluate the drug release behavior as a function of NIR exposureand incubation time, eight glass tubes each containing 1.5 ml ofPEG-DOX-MPS-GNSs (120 lg/ml) at pH 5.8 were exposed to a NIRlaser at different time intervals (5, 10, 15, 20, 30, 40, 50, and60 min). The release experiments were repeated every 24 h for5 days using the dialysis bag method, as previously described.

2.2.4. Inoculation of the mice with tumor cellsThe Ehrlich ascites tumor was chosen as a rapidly growing

experimental tumor model where various experimental designsfor anticancer agents can be applied [18,28,29]. Ehrlich ascitescarcinomas cells were obtained from National Cancer Institute‘‘NCI’’- Cairo University and were intraperitoneally injected intofemale balb mice. The ascites fluid was collected on the 7th dayafter injection. The Ehrlich cells were washed twice and thenresuspended in 5 ml saline. Female balb mice (20–25 g in bodyweight and 6–8 weeks old) were obtained from the animal houseof NCI and were injected subcutaneously in their right flanks,where the tumors had developed into a single solid form. Tumorgrowth was monitored post-inoculation until the desired volume

MPS-GNSs preparation steps.

200 N. Elbialy et al. / Microporous and Mesoporous Materials 190 (2014) 197–207

was approximately 0.3–0.6 cm3. All animal procedures and carewere performed using the guidelines for the Care and Use of Labo-ratory Animals and was approved by the Animal Ethics Committeeat Cairo University [30].

2.2.5. Thermal transient measurementsThermal transient measurements of the Ehrlich tumor intersti-

tia were obtained using (Wahl TM-410, K-type, USA) a needle ther-mocouple. The tip of the thermocouple was positioned at thetumor center-of-mass. Twenty-four hours post-intravenous injec-tion (IV) of 200 ll PEG-MPS-GNSs (case I) and PEG-DOX-MPS-GNSs(case II), tumors (n = 3) were exposed extracorporeally to the nearinfrared (NIR) laser (continuous-wave (CW), compact infrared807 nm, single mode crystal laser, 300 mW/cm2, 5 mm spot size)for 1 h. Tumor temperatures were recorded during and followingthe NIR exposure until achieving the initial tissue temperature.Twenty-four hours post-intravenous injection with 200 ll PBS,tumors (n = 3) exposed to the NIR laser and tumor temperatureswas recorded (case III). The temperature change was plotted as afunction of the NIR irradiation time.

2.2.6. In vitro cytotoxicity of PEG-DOX-MPS-GNSsThe breast cancer cell line MCF-7 and human amnion wish cells

were cultured in RPMI 1640 containing 10% fetal bovine serum.Cells were maintained at 37 �C in a humidified and 5% CO2 incuba-tor. For all experiments, cells were harvested using 0.25% trypsin inEDTA and resuspended in fresh medium prior to plating. In vitrocytotoxicity was assessed using the WST-1 Cell Viability and Prolif-eration assay.

MCF-7 cells were seeded into 96-well plates at a density of 100cells per well. After incubation for 24 h at 37 �C in 100 ll of RPMI1640 medium containing 10% FBS, 50 ll culture medium was dis-carded then the cells were treated with 50 ll of PEG-DOX-MPS-GNSs (DOX concentration 120 lg/ml) group A and B. Group Cand D were treated with 50 ll PEG-MPS-GNSs, group F was treatedwith free DOX (120 lg/ml) and group E was exposed to the NIRlaser with no drug administration. Twenty-four hours post incuba-tion groups A, C and E were exposed to the NIR laser for 60 min.

Human amnion wish cells were seeded into 96-well plates at adensity of 55,000 cell per well. After incubation for 24 h at 37 �C in200 ll of RPMI 1640 medium containing 10% FBS, 50 ll culturemedium was discarded and the cells were treated with 50 ll ofvarious concentrations of samples (free DOX, PEG-DOX-MPS-GNSs)then cells were incubated for 24 h. Four hours post-treatment,10 ll of the WST-1 solution was added into each well. The cellswere incubated for another 4 h, and the absorbance was monitoredat 450 nm on an Elisa micro-plate reader (TECAN). Culture mediumwithout nanoparticles was used as the blank control. The cytotox-icity was expressed as the percentage of the cell viability comparedwith the blank control.

2.2.7. Laser scanning confocal microscopyLaser scanning confocal microscopy (Zeiss, LSM 510, German)

was used to study the dynamics and cellular uptake of PEG-MPS-GNSs and PEG-DOX-MPS-GNSs in addition to their therapeutic effi-ciency upon NIR irradiation. MCF 7 cells were incubated with500 ll PEG-DOX-MPS-GNSs (120 lg DOX/ml) and PEG-MPS-GNSs,in a glass-bottom dish for 24 h, followed by exposure to the NIRlaser for 1 h. Four hours post-treatment, the cells were excitedusing 488-nm excitation light and the fluorescence emission fromDOX was imaged at a wavelength between 565 and 630 nm. Thefluorescence yields were obtained by normalizing the integratedfluorescence intensities to the cellular area (as indicated usingtransmission microscopy). Cell cycle progression was monitoredby the flow cytometric measurement of DNA content. Analysis ofDNA content in cells stained with propidium iodide was performed

using FACScan (Becton Dickinson, USA). The percentage of cells ineach phase of the cell cycle was evaluated using the ModFit soft-ware (Cell quest).

2.2.8. In vivo NIR laser photo-thermal therapyAs tumors reached the desired volume (0.3–0.6 cm3), the treat-

ment began. Sixty mice were initially used and divided into fivegroups: A, B, C, D and E. Prior to treatment, the mice were anesthe-tized via an intraperitoneal injection with thiopental (48 mg/kg).Mice of group A were Intravenously injected with 200 ll PEG-DOX-MPS-GNSs (1 mg/kg) via the tail vein, and after 24 h the tumorwas exposed extracorporeally to the NIR laser for 60 min. Mice ingroup B were intravenously injected with 200 ll PEG-MPS-GNSs,and the same previous conditions were performed as group A. Micein group C were intravenously injected with 40 ll of free DOX(4 mg/kg) (therapeutic dose of free doxorubicin) [31]. The skin atthe tumor site for groups A, B and D was shaved to maximize theradiation transmittance to the target area. Mice in group D (positivecontrol) were intravenously injected with 200 ll PBS, pH 7.4 andfollowed the same irradiation conditions as group A and B. Micein group E (negative control) received no injections (neither GNSsnor buffer) or subsequent laser irradiation.

2.2.9. Tumor size measurementsDue to the high growth rate in the Ehrlich tumor model,

changes in the tumor volume (DV) were monitored over a21-day period for the five groups (A, B, C, D and E). The ellipsoidaltumor volume (V) was assessed every 3 days and calculated usingthe formula V ¼ ðp=6ÞðdÞ2ðDÞ, where D and d represent the longand short axes, respectively, as measured with a digital caliper(accuracy 0.01 mm).

The statistical evaluation of the tumor size data was performedusing Fisher’s LSD (least significance difference) multiple-compar-ison test. A p-value less than 0.05 was considered statistically sig-nificant. Each data point was represented as the mean ± standarderror (SE). In addition, SPSS version 17 was used for the statisticalanalyses.

2.2.10. Histopathological examinationTreatment groups A, B and C were sacrificed immediately after

laser exposure to investigate the percentage of tumor cell necrosis.The tumors were excised, fixed in 10% neutral formalin, embeddedin paraffin blocks and then sectioned. Tissue sections wereobtained directly after treatment and stained with hematoxylinand eosin (H&E). Previous procedures were repeated for the controlgroup E. All tissue sections were examined using a light micro-scope (CX31 Olympus microscope) that was connected with a dig-ital camera (Canon).

2.2.11. Quantitative determination of the amount of doxorubicin intumor tissue

The accumulated DOX in tumor tissues were assessed forPEG-DOX-MPS-GNSs, and free doxorubicin (1 mg/kg). Tumorbearing mice (n = 3/time point) were intravenously injected withPEG-DOX-MPS-GNSs and free doxorubicin, then tumors were col-lected at 3, 6, 24, 48 and 72 h. Then, 0.1 g of tumor was homoge-nized in 1 ml lysis buffer (150 mM sodium chloride, 1.0% TritonX-100, 0.1% SDS and 50 mM Tris, pH 8.0). Tissue lysate were centri-fuged at 2000g for 10 min. The supernatant were then isolated andquantitative analysis of DOX was performed using spectrofluorom-eter at slit width 15. The concentrations of DOX were calculatedfrom the calibration curve at an excitation k of 480 nm andemission k of 585 nm. The final doxorubicin concentrations wereexpressed as the amount of DOX with nanogram per gram oftissue.

50 nm

(b)(a)

0 1 2 3 4 50

100

200

300

400

500In

tens

ity (c

ts)

2θ (degree)

Fig. 1. Small angle X-ray diffraction pattern of the MPS nanoparticles (a) and itsTEM image (b).

N. Elbialy et al. / Microporous and Mesoporous Materials 190 (2014) 197–207 201

3. Results and discussion

Mesoporous silica nanoparticles (MPS), amine functionalizedmesoporous silica nanoparticles (AF-MPS), mesoporous silicaseeds (MPS seeds), doxorubicin-loaded mesoporous silica seeds(DOX-MPS seeds), gold nanoshells (MPS-GNSs), pegylated goldnanoshells (PEG-MPS-GNSs) and pegylated doxorubicin-loadedgold nanoshells (PEG-DOX-MPS-GNSs) were characterized usingseveral techniques. Small-angle X-ray diffraction analysis was usedto confirm the formation of the ordered mesoporous silica struc-ture. The diffraction pattern of the prepared MPS showed a narrowand strong peak at 2h = 1.94� with d spacing of 45.3 Å indicating awell-ordered mesoporosity (Fig. 1a).

TEM images revealed that the MPS nanoparticles exhibit chan-nel-like pores on their surface (Fig. 1b). In addition, the mesopor-ous silica nanoparticles appeared to be fully covered with smallgold particles (Fig. 2a).

(a)

10 nm

(b)

Fig. 2. TEM image of the MPS seeds (a), and its

(a)

200 nm

(

Fig. 3. TEM image of the DOX-MPS-GNSs (a) an

To confirm the binding of gold ions to the mesoporous silicasurface, Energy-Dispersive X-ray spectroscopy analysis (EDX) ofthe MPS seeds was performed. The EDX spectrum of the MPS seedsshowed the main constituents of MPS, including silicon, oxygenand gold (Fig. 2b). The detected signals of copper and carbon wereattributed to the TEM grid while the appearance of the chloridesignal was due to the use of gold chloride trihydrate during thepreparation of the MPS seeds. The morphology of the final formedgold nanoshell on the mesoporous silica template clearly showedthe formation of a uniform gold shell (Fig. 3a and b).

FTIR spectroscopic measurements were carried out to investi-gate the removal of surfactant CTAB (Fig. 4). Owing to thelarge amount of CTAB exist in the channel, CTAB coated MPSshowed the characteristic C–H stretching vibrations at 2925 and2856 cm�1 and C–H deformation vibration around 1475 cm�1

(Fig. 4a). After the removal of CTAB, the characteristics CTAB peaksdisappeared, suggesting the successful removal of CTAB (Fig. 4b)[32,33].

Using dynamic light scattering, the measured mean diametersof the MPS nanoparticles and DOX-MPS-GNSs were 152 ± 30 nmand 168 ± 39 nm, respectively, which is appropriate for use as drugdelivery vehicles in cancer therapy (Fig. 5a). Particles with a diam-eter of approximately 200 nm are able to penetrate the cell mem-brane and enter the cytoplasm via endocytosis [34]. In addition,the size of the PEG-DOX-MPS-GNSs enables the selective accumu-lation of MPS in the extracellular medium of tumor tissue due to anenhanced permeability and retention effect (EPR) of canceroustissue [35].

The measured zeta potential of the MPS was +33 mV because ofthe presence of the CTAB covering the MPS surface. The aminofunctionalized MPS exhibits a surface charge of +21 mV, and thereduction of positivity was attributed to a replacement of thequaternary amine of CTAB with one amine from APTES duringthe surface functionalization process. In contrast, the MPS seeds

Energy Dispersive X-ray (EDX) spectrum.

b)

d a high magnification of the gold shell (b).

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

2925 cm-1

2856 cm-1

1475 cm-1a

b

Fig. 4. FTIR Spectrum of MPS nanoparticles (a) and AF-MPS nanoparticles (b).

202 N. Elbialy et al. / Microporous and Mesoporous Materials 190 (2014) 197–207

showed a significant reduction in surface charge (�3.5 mV) due tothe screening effect of gold layer [36]. These results confirmed theformation of a gold layer around the amino functionalized MPSforming MPS seeds. After growing, a gold shell DOX-MPS-GNSshas an average surface charge of �18 mV. PEG coating of theDOX-MPS-GNSs increased the negativity of the gold nanoshell

(a)

0 50 100 150 200 250 3000

20

40

60

80

100

Num

ber (

%)

Diameter (nm)

Fig. 5. Dynamic light scattering size distribution of the MPS nanoparticles (j), DOX-nanoparticles (b).

(a)

400 500 600 700 800 900

0.2

0.3

0.4

0.5

0.6

0.7 MPS nanoparticles MPS seeds Dox-MPS-seeds

Abs

orba

nce

(a.u

)

Wavelength (nm)

Fig. 6. UV–VIS absorption spectra of the MPS nanoparticles, MPS see

surface charge, which achieved a high stability and preventedaggregation [37] (Fig. 5b).

The absorption spectrum of the prepared MPS nanoparticlesshowed a decrease in the optical absorption with an increase inwavelength, indicating that the MPS did not exhibit any surfaceplasmon resonance. After the attachment of small gold ions onthe AF-MPS and the formation of MPS seeds, an optical responseappeared at 550 nm. Interestingly, the characteristic peak of doxo-rubicin appeared at 500 nm when DOX-loaded MPS were formed(Fig. 6a). The MPS-GNSs with K-gold to seed ratio 200:1 werefound to have a surface plasmon resonance in the wavelengthranging from 610 to 900 nm (Fig. 6b). When DOX-MPS-GNSs wereused in the formation of gold nanoshells, two absorption peaks at(500 nm and 610–900 nm) appeared. These two peaks confirmedthe formation of gold nanoshells loaded with doxorubicin.

In vitro release profile of doxorubicin from MPS seeds and PEG-DOX-MPS-GNSs at pH 7.4 and 5.8 PBS solutions was shown inFig. 7. The results indicated that DOX release from PEG-DOX-MPS-GNSs exhibited a relatively slow profile at pH 7.4 suggestingthat gold shell was acting as a structurally stable phase at physio-logical pH in addition to the strong electrostatic attraction betweenthe DOX molecules and MPS pores which prevent DOX diffusion(Fig. 7b). The ability of PEG-DOX-MPS-GNSs to release DOX duringthe NIR laser exposure was demonstrated when a suspension ofPEG-DOX-MPS-GNSs was irradiated with the NIR laser in vitro atroom temperature and at pH 5.8 (simulation of tumor environ-ment) (Fig. 7a). Under this condition, 16.5% of doxorubicin wasreleased after 1 h (exposure time). When the laser beam wasswitched off, a continuous release of DOX was observed up to120 h at a pH value of 5.8 and temperature of 37 �C (Fig. 7b). A

(b)

MPS AF-MPS

Dox-MPS seeds Dox-

MPS-GNSs PEG-

Dox-MPS-GNSs

-30-20-10

0102030

Zeta

pot

entia

l (m

V)

MPS-GNSs (d) (a), and the average value of the zeta potential for the prepared

(b)

400 500 600 700 800 9000.0

0.2

0.4

0.6

0.8

1.0 MPS-GNSs Dox-MPS-GNSs Dox

Abs

orba

nce

(a.u

)

Wavelength (nm)

ds, DOX-MPS seeds (a), GNSs, DOX-MPS-GNSs, and free DOX (b).

0 10 20 30 40 50 6002468

1012141618

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Dox

-rel

ease

d pe

rcen

tage

(%)

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1

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3

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5

10

15

20

25

30

35

40

45

Dox

-rel

ease

d pe

rcen

tage

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Time (hr)

Fig. 7. The release percentage of doxorubicin from PEG-DOX-MPS-GNSs as a function of the NIR irradiation time (a). The release of doxorubicin from the MPS seeds at pH7.4(d), pH 5.8 (N) and from PEG-DOX-MPS-GNSs at pH 5.8 (j).

Fig. 8. The change in the Ehrlich tumor interstitial temperature during andfollowing NIR laser irradiation for Case I, Case II and Case III.

N. Elbialy et al. / Microporous and Mesoporous Materials 190 (2014) 197–207 203

rapid release of the DOX molecules from the surface or near-surface regions was clearly observed from the release profile atpH 5.8. This was attributed to the degradation of the gold shellafter the exposure to NIR laser, where irradiation of the goldnanoshells can induce melting, evaporation, and fragmentation ofnanoshells [38]. This breakdown of gold shells makes the surfaceof the mesoporous silica exfoliated, which in turn enables thesustain release of the chemotherapeutic loaded drug. This explana-tion can be emphasized by the UV–VIS absorption spectrum of NIR

A B C D E F0

20

40

60

80

100

Cel

l via

bilit

y (%

)

(a)

Fig. 9. Cytotoxicity of PEG-DOX-MPS-GNSs (A), PEG-MPS-GNSs (C) against MCF-7 cells iPEG-DOX-MPS-GNSs (B), PEG-MPS-GNSs (D) without NIR irradiation. Cells irradiated with(a). Cytotoxicity of PEG-DOX-MPS-GNSs and free DOX against human amnion wish cells

irradiated gold nanoshells which show the loss of the characteristicNIR absorption band of PEG-DOX-MPS-GNSs (Supporting information).As the pH decrease to 5.8 the electrostatic attraction between theDOX molecules and MPS pores decreases which lead to the sustainrelease of DOX in this pH. Such marked variation in the releaseprofile at different pH substantiates the pH responsiveness ofPEG-DOX-MPS-GNSs and indicated that this novel formulationcould release doxorubicin specifically at the tumor sites. The releaseprofile of the drug loaded MPS seeds were higher compared to thePEG-DOX-MPS-GNSs, indicating an important role of the formed goldshell in controlling the amount of released drug (Fig. 7b).

The ability of the prepared formulations to convert light intoheat was assessed in vivo by measuring the tumor temperatureas a function of the NIR exposure time (Fig. 8). Thermal transientcurves showed the successful photo-heat conversion of NIR-irradi-ated GNSs, which accumulated in the tumor tissues. The markedincrease in tumor tissue temperature, with an average of 13 �C,indicated that efficient photo-heat conversion was induced by boththe PEG-MPS-GNSs and PEG-DOX-MPS-GNSs. As the laser beamwas switched off, the temperature decreased to normal tissuetemperatures. At temperatures greater than 43 �C, protein denatur-ation and disruption of the cellular membrane are known to occurand ablation of tumor tissues has been shown in numerous cases[39]. In contrast, mice/tumors injected with PBS and treated withNIR laser exposure showed an average increase in tumor tempera-ture up to 4 �C.

To evaluate and compare the cytotoxicity of the PEG-DOX-MPS-GNSs and free DOX, the WST-1 assay was used. Cells ofgroups A and C were incubated with PEG-DOX-MPS-GNSs, andPEG-MPS-GNSs, followed by NIR exposure for 1 h. As observed in

6 7 8 9 10 11 120

20

40

60

80

100

Cel

l via

bilit

y (%

)

DOX concentration (μM)

Free DOX PEG-DOX-MPS-GNSs

(b)

ncubated for 24 h followed by NIR irradiation for 1 h. Cells incubated for 24 h withNIR laser irradiation (F), and cells incubated with free DOX (120 mg/ml) for 24 h (E)(b).

(a)

(e)

(d)(c)

(b)

(f)

Fig. 10. Confocal scanning microscopy images for the MCF7 cell line, control MCF7 cells (a), high magnification of the control cells (b), cells treated with the NIR laser for 1 h(c), cells incubated with PEG-MPS-GNSs for 24 h followed by NIR exposure for 1 h (d) , cells incubated with PEG-DOX-MPS-GNSs for 24 h followed by NIR exposure for 1 h (e)and the fluorescence field of ‘‘e’’ (f).

204 N. Elbialy et al. / Microporous and Mesoporous Materials 190 (2014) 197–207

Fig. 8, PEG-DOX-MPS-GNSs showed the highest cytotoxicity (cellviability percent of 2.53) against MCF7, compared to either PEG-MPS-GNSs (cell viability percent of 33.8) or free DOX (group F) (cellviability percent of 80.15). Also, cells incubated with PEG-DOX-MPS-GNSs (group B), and PEG-MPS-GNSs (group D) without NIRirradiation show cell viability percent of 68, 97.6, respectively.Moreover, the positive control cells (group E) exposed to the NIRlaser for 1 h showed a cell viability of 94%. This rate might beattributed to the therapeutic effect of the released anticancer drug(doxorubicin) out of PEG-DOX-MPS-GNSs during laser exposurewhich in turn decreased the surviving fraction to 2.53% (Fig. 9a).

To assess the cytotoxicity of PEG-DOX-MPS-GNSs on normalcells, human amnion wish cells were exposed to differentconcentrations of doxorubicin (6–10–12 lM) as free drug andPEG-DOX-MPS-GNSs for 24 h and cell viability were measured.PEG-DOX-MPS-GNSs have no effect on the viability compared tofree drug with the same concentration. These results confirm theselectivity of the prepared nanoparticles (Fig. 9b).

Confocal laser scanning microscopy images of MCF7 cellsshowed the rod like shape of the intact cells with the appearance

of few dead cells (Fig. 10a and b). Similarly, MCF7 cells that wereexposed to the NIR laser for 60 min showed few dead cells(Fig. 10c). Meanwhile, MCF7 cells incubated with PEG-MPS-GNSsfor 24 h showed high cellular uptake leading to severe damageupon NIR laser exposure for 1 h (Fig. 10d). Fig. 10e and f showedMCF7 cells incubated with 120 lg/ml PEG-DOX-MPS-GNSs for24 h and treated with the NIR laser for 60 min. The observed redfluorescence indicated the accumulation of PEG-DOX-MPS-GNSsin the cytoplasm without an evidence of entering into the nucleus(Fig. 10f). This confirmed that PEG-DOX-MPS-GNSs have beenentered into the cell by endocytosis through the plasma membraneof MCF7 cells. As a consequence, these accumulated nanoparticlesconvert the NIR light into heat, which not only induced thermaldamage of tumor cells but also triggered the release of doxorubicinfrom PEG-DOX-MPS-GNSs.

Next, we assessed the effects of the PEG-DOX-MPS-GNSs on cellcycle progression and cell death by the analysis of DNA contentusing flow cytometry (Fig. 11). Free DOX arrested MCF7 cells inthe S phase of the cell cycle. Cell cycle arrest can trigger specificcellular responses, resulting in apoptotic cell death (Fig. 11b).

Fig. 11. Changes of DNA content in MCF7 cells, (a) control cells, (b) and (c) cell treated with free DOX and PEG-DOX-MPS-GNSs for 24 h, respectively. Then cells wereharvested, stained with propidium iodide, and analyzed on FACScan (Becton Dickinson). The percentage of cells in each phase of the cell cycle was evaluated using the ModFitsoftware.

Fig. 12. The average changes in the Ehrlich tumor volume as a function of time forthe treatment groups A, B, C, and D as well as the control group E.

N. Elbialy et al. / Microporous and Mesoporous Materials 190 (2014) 197–207 205

Twenty-four hour post treatment with PEG-DOX-MPS-GNS, allcells appear in sub G1 phase of the cell cycle (Fig. 11c). Apoptoticcells are often distinguished on frequency histograms by their frac-tional DNA content in sub-G1 phase [40]. So, PEG-DOX-MPS-GNSsinduce direct cell cycle blocking effect and cell death. These resultsemphasize that released DOX was the agent responsible for cellcycle arrest and subsequent apoptosis.

Therapeutic efficacy of the developed formulation has beenassessed by following up the change in tumor volume over a 21-dayperiod in the five groups. Under our experimental conditions, pro-nounced inhibition in tumor growth was demonstrated in the twoanimal groups, A and B, compared with the control group E(p < 0.0001 and p < 0.0001, respectively). Group C, administratedwith the therapeutic dose of doxorubicin, showed a slight decrease

in tumor volume at day 3, followed by a rapid growth throughoutthe 19-day period. In addition, group D showed a slight delay in thetumor growth rate compared with the control group (group E)(Fig. 12). These marked decreases in the Ehrlich tumor volumefor groups A and B, treated with the suggested protocol, wereattributed to selective hyperthermia of tumor tissues injectedintravenously with PEG-DOX-MPS-GNSs followed by NIR exposure.Moreover, group A, which was irradiated with NIR laser, triggeredthe release of doxorubicin markedly from PEG-DOX-MPS-GNSs.We observed that, after laser irradiation was switched off, DOXrelease extended over at least five days period which is sufficientto induce a high therapeutic efficacy.

Histopathological examination of entire tumor sections for thetreated experimental groups revealed marked differences in thecellular features accompanied by varying degrees in the necrosispercentage when compared with the control sections. Tumor sec-tions for the Ehrlich tumor cells were excised from the mice ofgroup E (a), group C (b), group B (c) and group A (d) (Fig. 13).The calculated percentages of necrosis for experimental groups A,B, C, and E were 97%, 69%, 34% and 18%, respectively. The negativecontrol (group E), in which the tumors received neither laser treat-ment nor GNSs injection, showed a normal necrosis percentage offocal and diffuse necrosis (thin & bold arrows, respectively). Theformer appears as scattered necro-apoptotic bodies within thegroups of viable cells, while the latter appears as islands of coagu-lative necrosis (geographic distribution) showing the ghosts ofcells. Hemorrhagic necrosis was also observed (Lower Rt, ‘‘encir-cled’’), where the mean field count was approximately 18(Fig. 13a). For the positive control (group C), microscopic examina-tion revealed an increase in the necrosis percentage by up to 34%,with diffuse cellular affection and geographic appearance. Thenecrotic regions exhibited scattered nuclear karyorrhectic debrisand apoptotic bodies (Fig. 13b). This mild cell coagulative necrosiscould be attributed to a deep penetrative power of the NIR laser

(a) (b)

(c) (d)

Fig. 13. Sections of Ehrlich tumor cells excised from group E (a), e (b), B (c) and A (d) tumor tissues stained with H&E and quantification of the percentage of necrosis. Themean necrosis field counts were 18%, 34%, �69%, �97% for groups E, C, B and A, respectively.

Free DOX PEG-DOX-MPS-GNSs0

100020003000400050006000700080009000

Dox

orub

icin

con

cent

ratio

n (n

g/g)

3 hr 6 hr 24 hr 48 hr 72 hr

Fig. 14. The concentration of DOX in tumor tissue after intravenous administrationof free DOX and PEG-DOX-MPS-GNSs.

206 N. Elbialy et al. / Microporous and Mesoporous Materials 190 (2014) 197–207

beneath the skin. Treatment group B exhibited a tumor field ofmoderate necrosis with a mean field count of approximately 69%,with viable cells (arrows), pre-necrotic degenerative changes inthe vacuolated tumor cells (hydropic change) and indistinct nuclei(encircled). The apoptotic and karyorrhechtic bodies were alsoincreased (Fig. 13c). Treatment group A showed an area of totaltumor necrosis (99%). The viable forms show the end stage degen-eration of cellular shrinkage and nuclear pycnosis (arrows). Thefocal groups of neoplastic cells revealed evidence of cytolysis andmicrocystic change (encircled). Nuclear debris was observed at alower Rt. zone (Fig. 13d). The upper left island of viable tumor cellsshowed apoptotic bodies. Histopathological examinationsconfirmed the observed inhibition of tumor growth rate in thetreatment groups compared with the control groups. It can beemphasized from the above results that the doxorubicin releasedinside the cell lead to apoptotic cell death in addition to heatreleased induces necrotic cell death.

To quantify and compare the amount of DOX in tumor afterintravenous administration of free DOX and PEG-DOX-MPS-GNSs,the concentration of DOX in tumor tissue lysate was measured.Six hours post injection high accumulation of DOX for PEG-DOX-MPS-GNSs was maintained, in tumor tissues, over a period of72 h compared to free DOX. This marked retention of DOX is dueto the lake of lymphatic drainage in tumor and the ability of tumortissue to retain the accumulated particles (EPR effect [35] (Fig. 14).

PEG-DOX-MPS-GNSs is an excellent photothermal agent candi-date that initiates sustained release. Using this drug carrier, theoverall drug consumption and side effects could be significantlyreduced. Importantly, this nanocarrier has the ability to be localizedat the tumor site to release the loaded drug in a controlled manner.

Both the in vitro and in vivo studies suggested that this dualfunction of gold nanoshells demonstrate an enhanced potentialto kill cancer cells compared to both photothermal therapy andchemotherapy alone.

4. Conclusions

This study reports a simple method for the preparation of doxo-rubicin-loaded mesoporous silica. This formulation provides twooncological modalities: photothermal therapy and chemotherapy.The promising DOX-PEG-MPS-GNSs displayed a high potential fortherapeutic treatment against MCF7 (in vitro) and Ehrlich carci-noma (in vivo). Furthermore, DOX-PEG-MPS-GNSs also showedan enhanced cellular uptake.

Interestingly, this passively targeted nanocarrier was capable ofconverting the NIR laser into heat, which not only induced tumorcell damage but also triggered drug release with high therapeuticefficacy.

Acknowledgments

The authors gratefully acknowledge Dr. Tarek El-Bolkini,National Cancer Institute (NCI) – Cairo University for his help in

N. Elbialy et al. / Microporous and Mesoporous Materials 190 (2014) 197–207 207

the histopathological examination. In addition, we thank Dr. TaherSalah, at the Nanotechnology Characterization Center at theAgriculture Research Center for his help with the confocal laserscanning microscope imaging.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.micromeso.2014.02.003.

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Synthesis, characterization and application of gold nanoshells using mesoporous silica core1 Introduction2 Experimental2.1 Materials2.2 Methods2.2.1 Preparation of gold nanoshells loaded with doxorubicin (PEG-DOX-MPS-GNSs)2.2.1.1 Preparation of mesoporous silica nanoparticles2.2.1.2 Amino functionalization of mesoporous silica nanoparticles2.2.1.3 Preparation of seed solution and Doxorubicin loading2.2.1.4 Preparation of pegylated gold nanoshells loaded with doxorubicin (PEG-DOX-MPS-GNSs)

2.2.2 Sample characterization2.2.3 Drug release from DOX-MPS seeds and PEG-DOX-MPS-GNSs2.2.4 Inoculation of the mice with tumor cells2.2.5 Thermal transient measurements2.2.6 In vitro cytotoxicity of PEG-DOX-MPS-GNSs2.2.7 Laser scanning confocal microscopy2.2.8 In vivo NIR laser photo-thermal therapy2.2.9 Tumor size measurements2.2.10 Histopathological examination2.2.11 Quantitative determination of the amount of doxorubicin in tumor tissue

3 Results and discussion4 ConclusionsAcknowledgmentsAppendix A Supplementary dataReferences