Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo–photothermal therapy

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International Journal of Pharmaceutics xxx (2014) xxx–xxx

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Mesoporous silica coated gold nanorods loaded doxorubicin forcombined chemo–photothermal therapy

A. Soltan Monem*, Nihal Elbialy, Noha MohamedBiophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt

A R T I C L E I N F O

Article history:Received 23 March 2014Received in revised form 24 April 2014Accepted 29 April 2014Available online xxx

Keywords:Gold nanorodsMesoporous silicaCombined chemo–photothermal therapyDrug deliverypH responsive drug releaseMCF-7 cell lineHuman amnion wish cellsEhrlich tumor

A B S T R A C T

The efficacy of the combined chemo–photothermal therapy, using a mesoporous silica-coated goldnanorods loaded DOX (pGNRs@mSiO2-DOX), was consistently tested both in vitro and in vivo. Theprepared nanoparticles that were characterized using transmission electron microscopy (TEM), UV–visabsorption spectroscopy and zeta potential showed high doxorubicin loading capacity in addition to itspH-responsive release. The pGNRs@mSiO2-DOX photo-heat conversion characteristic found to be stablefor several repeated NIR irradiated doses was tested in simulated body fluid. In vitro results showed thatpGNRs@mSiO2-DOX causes a significant damage in breast cancer cell line MCF-7 compared to free DOX.Contrary to this, it showed low toxicity to human amnion wish cells compared to CTAB coated GNRs andfree DOX. In vivo results showed that intravenous administration of pGNRs@mSiO2-DOX (1.7 mg/kg)markedly suppresses the growth of subcutaneous Ehrlich carcinoma in female Balb mice (p < 0.0001).Consistently, histopathological examination revealed a complete loss of tumor cellular details for micethat received the combined treatment. Based on the obtained results, this passively targetedpGNRs@mSiO2-DOX could specifically deliver drug and excessive local heat to tumor sites achievinghigh combined therapeutic efficacy.

ã 2014 Published by Elsevier B.V.

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsev ier .com/locate / i jpharm

1. Introduction

Noble metal nanoparticles have been widely studied in the pastdecades because of their high potential applications in many areasespecially medical therapy and diagnosis (Moores and Goettmann,2006; Huang et al., 2007; Zhang et al., 2012). They provideremarkable opportunities due to their inherently low toxicity(Connor et al., 2005a; Khan et al., 2007; Shukla et al., 2005) andstrong enhanced optical properties associated with localizedsurface plasmon resonance (El-Sayed, 2001; Link and El-Sayed,2000; Mie, 1976). Recently extensive studies have been focused ongold nanorods (GNRs) for cancer therapy because of theircharacteristic surface plasmon resonance (Dickerson et al.,2008; Shen et al., 2013; Alkilany et al., 2009; Connor et al.,2005b; Wang et al., 2011). However, the traditional preparation ofcetyltrimethyl ammonium bromide (CTAB) with bilayer coatingGNRs display significant cytotoxicity to human cells in its free form(Alkilany et al., 2009; Connor et al., 2005b). Additionally, CTABinduces GNRs aggregation which leads to the loss of their unique

* Corresponding author at: Biophysics Department, Faculty of Science, CairoUniversity, Giza 12613, Egypt. Tel.: +20 1224340195.

E-mail address: asoltanh@yahoo.com (A. S. Monem).

http://dx.doi.org/10.1016/j.ijpharm.2014.04.0670378-5173/ã 2014 Published by Elsevier B.V.

Please cite this article in press as: Monem, A.S., et al., Mesoporous silicphotothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.101

optical properties and minimizes its cellular uptake. Thus the usesof CTAB-coated GNRs hinder its biomedical applications. On theother hand, mesoporous silica was found to be suitable for beingused as a coatingmaterial for GNRs because of its high drug loadingcapacity and non-toxic biodegradable content. It has beenextensively highlighted for many biomedical applications asnanocarriers for anticancer drugs, DNA and proteins. They alsopossess large surface area, tunable size, high accessible porevolume and well-defined surface properties capable for modifica-tion (Yang et al., 2012; Tang et al., 2012; Knezevic and Lin, 2013; Al-Kady et al., 2011; He et al., 2011; Tan et al., 2011). They arecharacterized by a pH responsive drug delivery that provides highdrug release profile in the acidic tumor environment (Falk andIssels, 2001; Chen et al., 2007; Zhang et al., 2011; Park et al., 2009).

In this study, we developed a preparative method formultifunctional nanoparticles (pGNRs@mSiO2-DOX) which aresuitable for a combined chemo–photothermal cancer therapy.

These nanoparticles were tested for pH responsive delivery ofchemotherapeutic drug (DOX) to tumor cites as well as theirunique simultaneous NIR light-based induction of hyperthermia.The physical and the chemical properties of these nanoparticleswere investigated in a buffer as well as in a simulated body fluidsolutions. The therapeutic efficacy of the prepared pGNRs@mSiO2-DOX was tested in both in vitro and in vivo using a single dose

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otocol at a very low DOX concentrations aiming to reduce drugxicity to critical tissues.

Materials and methods

Doxorubicin (DOX), chloroauric acid (HAuCl4�3H2O), cetyltri-ethyl ammonium bromide (CTAB), tetraethyoxysilane (TEOS)d L-ascorbic acid (AA) were purchased from Sigma–Aldrich.lver nitrate (AgNO3), tris buffer (CH2OH)3CNH2 and sodiumrohydride (NaBH4) were purchased from Merck. Ammoniadroxide (NH4OH, 28%) was purchased from Fluka.

. The preparation of pGNRs@mSiO2-DOX

.1. The preparation of GNRSThe gold nanorods were prepared according to previously

ported silver ion-assisted seed mediated method using CTAB as amplate (Huang et al., 2008). Briefly, 1.5 ml of 0.1 M CTAB solutionas mixed with 100 ml of 0.02 M HAuCl4. Then 100 ml ice-cold of01 M NaBH4 was added forming a brownish yellow seed solution.e solution was vigorously stirred for 2 min and kept in a waterth at 25 �C for 2 h.The gold nanorods growth solution was prepared by mixing

ml of 0.02 M HAuCl4 and 1.0 ml of 0.01 M AgNO3 with 30 ml of M CTAB. Then 0.8 ml of 0.08 M ascorbic acid solutionwas added toe growth solution changing its color from dark yellow to colorless.en 70 ml seed solutionwas added to the total volume of the growthlution at 25 �C. The color of the solution gradually changed untilally it became purple. The obtained GNRs were centrifuged at72 g for 30 min (Sigma 202, refrigerated centrifuge; Germany).e billet was then washed twice in deionized water to removecess CTAB. It was finely dispersed in 40 ml deionized water.

.2. The preparation of GNRs@mSiO2

The mesoporous silica coating was achieved by modified Stoberethod (Shen et al., 2013). Briefly, 50 ml aqueous ammonia wasded to GNRs solution in order to adjust its pH value to 10. Then,.5 ml of 10 mM TEOS/ethanol solution was added to the GNRslution at a rate of 3.5 ml/h, and the mixture was kept at 40 �C underntle stirring for 24 h. The synthesized product GNRs@mSiO2 wasntrifuged and washed in deionized water and ethanol severales. The billet of GNRs@mSiO2 was then dispersed in ethanol

lution (60 ml) containing concentrated HCl (120 ml) and stirred at�C for 3 h to remove the template (CTAB). This surfactant removalocess was repeated twice to ensure complete sample clearance ofAB. The sample was then centrifuged and washed in deionizedater three times in order to remove the traces of HCl and ammonia.

Fig. 1. TEM image of gold nanorods (a) and g

Please cite this article in press as: Monem, A.S., et al., Mesoporous silphotothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.10

The concentration of the sample was adjusted by measuring itsoptical density at 805 nm until it reads 2.

2.1.3. The preparation of GNRs@mSiO2-DOXTwo milliliters of GNRs@mSiO2 nanoparticles was added to

250 mg of doxorubicin hydrochloride (DOX) at pH 8. The mixturewas stirred at room temperature for 24 h. The GNRs@mSiO2-DOXwas then centrifuged at 4472 g for 30 min, and the billet washedwith PBS several times. The free DOX contents of the supernatantwere determined from the calibration curve of DOX concentrationand emission intensity at an excitation l of 480 nm and emission lof 585 nm using a spectrofluorometer (Shimadzu, RF 5301pc,Japan). The drug loading efficiency was then calculated from therelation

Loading efficiencyð%Þ

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

2.1.4. Coating GNRs@mSiO2 with polyethylene glycolThe GNRs@mSiO2-DOX surfaces were coated with polyethylene

glycol for intravenous injection, by adding 10 ml of 25 mM PEG-SHto each 1 ml of GNRs@mSiO2-DOX solution and incubating themixture for 12 h at 4 �C. The suspension was then centrifuged at4472 g for 30 min to remove residual PEG-SH from the formulation.The pGNRs@mSiO2-DOX billet was suspended in a sterile 0.9%saline solution shortly before in vivo application.

2.2. Sample characterization

The morphology and size of the GNRs and GNRs@mSiO2 weredetermined using transmission electron microscopy (TEM) (FEITecnai G20, Super twin, Double tilt, LaB6 Gun) operating at 200 kV.The absorption spectra of GNRs, GNRs@mSiO2, GNRs@mSiO2-DOXand free DOX were measured, using a UV–vis spectrophotometer(Jenway UV-6420; Barloworld Scientific, Essex, UK), at thewavelength range 400–900 nm. Furthermore, zeta potential ofthe GNRs and pGNRs@mSiO2 was measured in deionized distilledwater using a dynamic light scattering apparatus (zeta potential/particle sizer NICOMPTM 380 ZLS, USA).

2.3. Measurement of the pGNRs@mSiO2-DOX responsive release

The sterilized dialysis bags with a dialyzer molecular-weightcut-off of 12,000 Da (Cellulose Dialysis Tubing, Fisherbrand, USA)were used to perform the drug release experiments. Twophosphate buffered saline (PBS) solutions of pH 7.4 and pH 5.6

old nanorods coated mesoporous silica (b).

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were used as the drug release media to simulate normal blood/tissues and tumor environments, respectively. The used dialysisbags were soaked overnight in the release media. One milliliter ofpGNRs@mSiO2-DOX (190 mg/ml) was centrifuged, and the billetwas dispersed in 1 ml of the release media which was then placedinto the dialysis bags. The sealed dialysis bags were placed intobrown glass bottles; then 20 ml of release media was added to eachbottle. These bottles were shaken at a speed of 105 rpm at 37 �Cunder a light-sealed condition. At successive time intervals, 3 ml ofthe release media were used to quantify the concentration of thereleased drug using a spectrofluorometer. Then, it was refilled tothe original release media. The concentrations of the released drugwere determined from the calibration curve at an excitation l of480 nm and emission l of 585 nm.

Cumulative releaseð%Þ ¼ Amount of DOX releasedAmount of DOX in the nanoparticles

� 100%

2.4. In vitro cytotoxicity of pGNRs@mSiO2-DOX

The breast cancer cell line MCF-7 was cultured in RPMI 1640containing 10% fetal bovine serum (FBS). THe cells were main-tained at 37 �C in a humidified incubator containing 5% CO2. For allthe experiments, the cells were harvested using 0.25% trypsin inEDTA which was then suspended in fresh medium prior to plating.In vitro cytotoxicity against MCF-7 cells was determined using theWST-1 cell viability and proliferation assay. The MCF-7 cells wereseeded into 96-well plates at a density of 100 cells per well (100 mlof the medium solution). After incubation for 24 h at 37 �C in 100 mlof RPMI 1640 medium containing 10% FBS, 50 ml of the culturemedium was discarded and replaced by various concentrations offree DOX and pGNRs@mSiO2-DOX. After 24 h of cell incubation indifferent concentrations of pGNRs@mSiO2-DOX, the cells werethen exposed to NIR laser for 60 min. Post-treatment, the digitalmicroscopic images of the wells were taken using an inverted lightmicroscope (Leica) at a magnification of 20�. The cells viabilitywas then counted as a function of the drugs concentrations.

The human amnion wish cells were seeded into 96-well platesat a density of 55,000 cell per well. After incubation for 24 h at 37 �Cin 200 ml of RPMI 1640 medium containing 10% FBS, 50 ml culturemedium was discarded and the cells were treated with 50 ml ofCTAB coated GNRs, pGNRs@mSiO2-DOX (of DOX concentration

Fig. 2. The absorption spectra of GNRs, GNRs@mSiO2, GNRs@mSiO2-DOX and freeDOX.

190 mg/ml) and free DOX at a concentration of 120 mg/m. After 24 hcells incubation, 10 ml of the WST-1 solution was added into eachwell. The cells were incubated for another 4 h, and its absorbancewas monitored at 450 nm on an Elisa micro-plate reader (TECAN).The culture medium without nanoparticles was used as the blankcontrol. The cytotoxicity was then expressed as the percentage ofthe cell viability compared with the blank control.

2.5. Inoculation of the mice with tumor cells

The Ehrlich ascites tumor was chosen as a rapidly growingexperimental tumor model where various experimental designsfor anticancer agents can be applied (Elbialy et al., 2010;Dasyukevich and Solyanikn, 2007). The Ehrlich ascites carcinomascells were obtained from National Cancer Institute “NCI” – CairoUniversity and were intraperitoneally injected into female Balbmice. The ascites fluid was collected after 7 days post injection. TheEhrlich cells were washed twice and then suspended in 5 ml salinesolution. The female Balb mice (20–25 g weight and 6–8 week old)were obtained from the animal house of NCI and were injectedsubcutaneously in their right flanks, where the tumors haddeveloped into a single solid form. The tumor growth wasmonitored post-inoculation as soon as the desired volume wasapproximately 0.3–0.6 cm3. All animal procedures and care wereperformed using the guidelines for the Care and Use of LaboratoryAnimals and was approved by the Animal Ethics Committee atCairo University (National Research Council, 1996).

2.6. In vivo NIR laser photothermal therapy

In this study sixty mice were divided into four groups: A, B, Cand D. The mice were anesthetized via an intraperitonealinjection with thiopental (48 mg/kg). The mice of group A wereintravenously injected with 200 ml pGNRs@mSiO2-DOX (equiva-lent to 1.7 mg DOX/kg body weight) via the tail vein. After 24 h,the tumors were exposed extra corporeally to the NIR laser for

Fig. 3. Zeta potential of CTAB coated gold nanorods (a), and pGNRs@mSiO2 (b).

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Fig. 4. Drug release profile of pGNRs@mSiO2-DOX at pH 7.4 and pH 5.6.

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min. The mice of group B were intravenously injected with ml of free DOX (equivalent to 4 mg DOX/kg body weight) whichthe typical routine therapeutic dose of free doxorubicin used ineating human. The mice of group C (positive control) weretravenously injected with 200 ml PBS at pH 7.4 and followed theme irradiation conditions as group A. The skin at the tumorte for groups A and C was shaved to maximize the radiationansmittance to the target area. The mice of group D (negativentrol) received neither the injections nor the subsequent laseradiation.

7. Tumor size measurements

As Ehrlich tumor model is characterized by its high growth rate,e change in the tumor volume (DV) was measured every threeys over a period of eighteen days for the four groups (A, B, C and. The ellipsoidal tumor volume (V) was calculated using the

. 5. Inverted microscope images of control MCF7 cells (a), cells incubated with GNRsF-7 cellline (d) and percentage of cell viability forhuman amnion wish cells (e).gr5

formula V = (p/6)(d)2(D), where D and d are the long and shortaxes, respectively, measured using a digital caliper.

The statistical evaluation of the tumor size data was performedusing Fisher’s LSD (least significance difference) multiple-compar-ison test. The p-values less than 0.05 were considered statisticallysignificant. Each data point was presented as the mean � standarderror (SE) of at least 7–10 measurements. In addition, SPSS version17 was used for the statistical analyses.

2.8. Histopathological examination

The treatment groups A and C were sacrificed immediately afterlaser exposure, and the necrotic percentage of tumor cells wasdetermined. The tumors were excised, fixed in 10% neutralformalin, embedded in paraffin blocks and then sectioned. Thetissue sections were obtained directly after treatment and stainedwith hematoxylin and eosin (H&E). The previous procedures wererepeated for group B (3 days post injection) and the control groupD. All the tissue sections were examined using a light microscope(CX31 Olympus microscope) that was connected with a digitalcamera (Canon).

3. Results and discussion

The size and shape of the prepared CTAB-coated GNRs andGNRs@mSiO4 were determined using TEM images, Fig. 1a and brespectively. The average GNRs length and width were 40 nm and10 nm, respectively, equivalent to a size of 3000 nm3. Themesoporous silica coat thickness found to vary from 10 nm to13 nm giving rise to GNRs@mSiO4 volume to be �42,000 nm3

(Fig 1b). The relatively large volume of such pours material allowshigh DOX loading per particle and efficient delivery upon NIRradiation.

As shown in Fig. 2, the prepared GNRs have a weak transverseplasmon band at 530 nm and a strong longitudinal plasmon bandat 820 nm in agreement with the reported results (Orendorff et al.,

@mSiO2-DOX (b), cells incubated with free DOX (c), percentage of cell viability for the

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Fig. 6. The change in the Ehrlich tumor interstitial temperature during NIR laserirradiation for group A, group C.

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2006). The sample GNRs@mSiO2 showed about 20 nm shifts in itslongitudinal peak compared to that of GNRs, and this could be dueto the change in the local refractive index of the mediumsurrounding GNRs. An additional peak at 490 nm appears in theabsorption spectrum of GNRs@mSiO2-DOX, which is attributed tothe absorbance band of DOX indicating its high incorporationwithin the silica shell pours. The relatively high amplitude of thisband insures the successful doping of the drug, at adequateconcentration, inside the silica shell (Fig. 2).

The average zeta potential of GNRs coated CTAB was 18.76 mV.Contrary to this, pGNRs@mSiO2 showed a significant negativepotential of �22.5 mV confirming the complete removal of CTABand the formation of firm stable pGNRs@mSiO2 samples (Fig. 3).

The DOX responsive release of GNRs@mSiO2-DOX was carriedout in PBS at pH values 7.4 and 5.6 at 3 �C for a period of 120 h. TheDOX release rate was obviously pH dependent that increases atrelatively low acidic media (Fig. 4). The marked variations in therelease profile at different pH confirm the pH responsiveness ofGNRs@mSiO2-DOX. Also it indicates that this formulation couldselectively release doxorubicin specifically at the tumor sites.

The results of DOX loading and its responsive release motivatedus to further investigate the in vitro cellular cytotoxicity for 4 hpost MCF-7 cells incubation with pGNRs@mSiO2-DOX followed by1 h NIR exposure; the morphology of the cells was completelychanged and became spherical rather than its normal spindle

Fig. 7. The change in temperature of pGNRs@mSiO2 suspended in a simulated bodyfluid throughout NIR irradiation.

shape (Fig. 5a and b). The observed dark aggregates in cells treatedwith pGNRs@mSiO2-DOX revealed the accumulation of the nano-particles inside MCF-7 cells (Fig. 5b). It seems that treated MCF-7cells suffer high apoptosis rates in agreement with the in vitrocytotoxicity results using WST-1 assay (Fig. 5d). In case of treatingthe cells with free DOX, a remarkable percentage of them appearedintact with their normal spindle shape (Fig. 5c). The breast cancercell line MCF-7 treated with pGNRs@mSiO2-DOX showed highcytotoxic effects, four times greater, than that cells treated withfree DOX at the three different concentrations used (Fig. 5d).

The human amnion wish cells are characterized by its highproductivity, easy accessibility, free from contamination with non-fibroblastoid cells and ethically acceptable source of cells forbiomedical applications (Hu et al., 2009). The cytotoxicity ofpGNRS@mSiO2-DOX on normal cells and human amnion wish cellswere incubated with CTAB coated GNRs, pGNRS@mSiO2-DOX andfree DOX for 24 h, and cell viability was then measured. The cellsincubated with pGNRS@mSiO2-DOX show cell viability of 87%which is very high compared to CTAB coated GNR 29% and free DOX35% (Fig. 5e). The results indicated that the normal cells up take ofpGNRS@mSiO2-DOX suffer a neglected percentage of apoptosisand/or necrosis as long as they are screened from NIR irradiation.The cytotoxicity of CTAB coated GNRs showed a relatively highcytotoxic effect on normal cells even though the cells were notexposed to NIR irradiation. The toxicity of CTAB was obviously highand may lead to lethal side effects; thus any therapeuticformulation containing CTAB as a coating material must be usedwith great caution. The human amnion wish cells incubated withfree DOX also showed a low cell viability percentage which wouldbe attributed to its high toxicity to normal cell.

The temperature of Ehrlich tumor tissue was measured as afunction of NIR exposure time in order to assess the photo-heatconversion characteristics of the prepared pGNRs@mSiO2-DOX(Fig. 6). An elevation in tissue temperature of about 14 �C and 4 �Cwere recordedthroughout the time of NIRexposureforgroup A andC, respectively. The temperature drops to its initial values shortly afterswitching off laser exposure. These results were similar to our recentwork using mesoporous silica gold nanoshells (Elbialy andMohamed, 2014). The advantage of using the preparedpGNRs@mSiO2-DOX in this combined chemo–photothermal thera-py is that it keeps its major characteristic of photo-heat conversionupon repeated exposure to NIR radiation. The photo-heat conversionof the sample was tested in vitro using simulated body fluid (Fig. 7).Also the absorption spectra of samples repeatedly exposed to NIR

Fig. 8. The absorption spectra of pGNRs@mSiO2 before and after repeated NIRirradiation.

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Fig. 9. The average changes in the Ehrlich tumor volume as a function of time forthe treatment groups A, B, C, and D.

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diation suffer no changes (Fig. 8). According to the obtained data,NRs@mSiO2-DOX can keep its rod structure as well as its physicalentity after exposure to the excessive local heat generated uponR radiation. It is important to note that a few micrograms of GNRsn deliver such a quantity of heat, to about 1 g of cancerous tissueat rises its temperature to �10 �C throughout NIR exposure time.e change in temperature of GNRs times its specific heat would berprisingly very high depending on GNRs/tissue mass ratio andher minor insulating conditions.The volume change of the implanted Ehrlich tumor in the rightnk of the mice of the four groups A, B, C and D were measureder a period of 18 days (Fig. 9). It is clear from the figure thateated group A showed a pronounced inhibition of tumor volumeowth for a period of 4–5 days (compared to group D, p < 0.0001)

Fig. 10. Sections of Ehrlich tumor cells excised from group D

followed by a very slow growth rate up to 18 days. The resultsshowed that the cancerous cells suffer a high necrotic percentage,and the remaining viable tumor cells have shown such a slowgrowth up to the end of the measuring period. Group B, treatedwith DOX alone, showed a similar volume inhibition behavior asthat of group A followed by a much faster growth after 3 dayscompared to group A. Because of the natural high growth rate ofEhrlich tumor, untreated group D showed an enhanced tumorvolume growth rates of about 10% per day. While group C, whichwas exposed to NIR, only showed a growth rate of about 6% per day.

The Ehrlich tumor sections were excised from mice of group D(a), group C (b), group B (c) and group A (d) for a histopathologicalexamination (Fig. 10). The negative control, group D, showed anormal necrosis percentage of focal and diffuse necrosis. Theformer appears as scattered necro-apoptotic bodies within thegroups of viable cells while the latter appears as islands ofcoagulative necrosis (geographic distribution) showing the ghostsof the cells. The hemorrhagic necrosis was also observed (lower Rt,“encircled" (Fig. 10a)). While the positive control, group C, showeda diffuse cellular affection and geographic appearance in additionto necrotic regions (Fig. 10b). This mild cell coagulative necrosiscould be attributed to a deep penetrative power of the NIR laserbeneath the skin. The treated group B showed a remarkableamount of necrotic fields (encircled) with the appearance of manyapoptotic and karyorrhechtic bodies (Fig. 10c). The treated group Ashowed an area of total tumor necrosis with the appearance ofnuclear debris. Also some tumor cells appeared with apoptoticbodies, pointed at by arrows (Fig. 10d). It is clear that treated groupA suffers the most of necrosis as well as apoptosis compared to thecontrols and even more than treated group B with DOX alone.Consistently, histopathological examinations have confirmed theobserved inhibition of tumor growth rate of the treated groupscompared to the controls.

(a), C (b), B (c) and A (d) tumor tissues stained with H&E.

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So far we have characterized a new formulation of nano-particles pGNRs@mSiO2-DOX suitable for a combined chemo–photothermal treatment of cancer cells and tumors. It has anadequate drug loading capacity and a responsive drug release intumor sites. They have been found to have a high effective photo-heat conversion and may possess a supper specific heat capacitycharacteristics. The samples also tested for a repeated NIRexposure for 1 h and showed no changes in its physical properties,its absorption spectra and photo-heat conversion. These resultsconfirm its stability against excessive heat produced during NIRexposure and its ability to deliver that heat for a repeated exposurewithin the tumor tissue.

The pGNRs@SiO4-DOX used in this work has shown a greattherapeutic efficacy using only a single chemotherapeutic doseprotocol followed by a single exposure to NIR irradiation. Althoughthey have the ability for a repeated photo-thermal ablation, its singleirradiationprotocol results in effective damage to tumorcells both invivo and in vitro. The tumor cell membrane permeation andretention allows its accumulation in tumor cells at a suitable massconcentration as well as its responsive The DOX-release effectivelyenhances its therapeutic efficacy. These characteristics gave themthe priority over other delivery systems such as gold nanoshells@SiO4 loaded DOX. These gold shells may be debrided post NIRirradiation (Elbialy et al., 2014; Lee et al., 2013).

4. Conclusion

The preparation method used in this work was found toreproduce pGNRs@mSiO2-DOX capable to incorporate chemother-apeutic drug, DOX, and have a high quality repeated photo-heatconversion properties. The size and shape of the nanoparticleswere determined using TEM. Its absorption spectra were alsodetermined to identify its effective photo-heat conversion band.The samples zeta potentials were determined which were found tobe an important factor for both DOX loading and its pH responsiverelease. The therapeutic efficacy of pGNRs@mSiO2-DOX was testedboth in vitro and in vivo. The formulation used in this work wasfound to possess a very low toxicity effects to normal tissue and cantarget its full damage to cancerous tissues. This study demonstrat-ed that the combined chemo–photothermal therapy accomplishedby pGNRs@mSiO2-DOX has an enhanced potential to kill cancercells compared to both photothermal therapy and chemotherapyalone.

Acknowledgments

Authors gratefully acknowledge Dr. Tark El-Bolkini, andVeterinary Heba M., Cancer National Institute, Cairo University,for his help in examining Ehrlich tumor tissue and animal care andtreatment. We also like to thank Prof. Dr. M. Aman, Faculty ofScience, Ein Shams University for the TEM imaging.

References

Al-Kady, A.S., Gaber, M., Hussein, M.M., Ebeid, E.M., 2011. Nanostructure-loadedmesoporous silica for controlled release of coumarinderivatives: a novel testingof the hyperthermia effect. Euro. J. Pharm. Biopharm. 7766–7774.

Alkilany, A.M., Nagaria, P.K., Hexel, C.R., Shaw, T.J., Murphy, C.J., Wyatt, M.D., 2009.Cellular uptake and cytotoxicity of gold nanorods molecular origin ofcytotoxicity and surface effects. Small 5.

Chen, J.Y., Wang, D.L., Xi, J.F., Au, L., Siekkinen, A., Warsen, A., Li, Z.-Y., Zhang, H., Xia,Y., Xi, Li, 2007. Immuno gold nanocages with tailored optical properties fortargeted photothermal destruction of cancer cells. Nano Lett. 7, 1318–1322.

Connor, E.E., Mwamuka, J., Gole, A., Murphy, C.J., Wyatt, M.D., 2005a. Goldnanoparticles are taken up by human cells but do not cause acute cytotoxicitySmall 1, 325–327.

Connor, E.E., Mwamuka, J., Wyatt, C.J., Gole, A., Murphy, M.D., 2005b. Goldnanoparticles are taken up by human cells but do not cause acute cytotoxicity.Small 1.

Dasyukevich, O.I., Solyanikn, G.I., 2007. Comparative study of anticancer efficacy ofaconitine-containing agent BC1 against ascite and solid tumors of Ehrlich’scarcinoma. Exp. Oncol. 29, 317–319.

Dickerson, E.B., Dreaden, E.C., Huang, X., El-Sayed, I.H., Chu, H., Pushpanketh, S.,McDonald, J.F., El-Sayed, M.A., 2008. Gold nanorod assisted near-infraredplasmonic photothermal therapy (PPTT) of squamous cell carcinoma in mice.Cancer Lett. 28, 57–66.

El-Sayed, M.A., 2001. Some interesting properties of metals confined in time andnanometer space of different shapes. Acc. Chem. Res. 34, 257–264.

Elbialy, N., Abdelhamid, M., Youssef, T., 2010. Low power argon laser-inducedthermal therapy for subcutaneous Ehrlich carcinoma in mice using sphericalgold nanoparticles. J. Biomed. Nanotechnol. 6, 687–693.

Elbialy, N., Mohamed, N., Monem, A.S., 2014. Micopor. Mesopor. Mat. 190, 197–207.Falk, M.H., Issels, R.D., 2001. Hyperthermia in oncology. Int. J. Hyperther. 17, 1–18.He, Q., Gao, Y., Zhang, L., Zhang, Z., Gao, F., Ji, X., Li, Y., Shi, J., 2011. A pH-responsive

mesoporous silica nanoparticles-based multi-drug delivery system for over-coming multi-drug resistance. Biomaterials 32, 7711–7720.

Hu, J., Cai, Z., Zhou, Z., 2009. Prog. Nat. Sci. 19, 1047–1052.Huang, X., Jain, P.K., El-Sayed, I.H., El-Sayed, M.A., 2007. Gold nanoparticles:

interesting optical properties and recent applications in cancer diagnostics andtherapy. Nanomedicine (London, U.K.) 2, .

Huang, H., He, C., Zeng, Y., Xia, X., Yu, X., Yi, P., Chen, Z., 2008. Preparation and opticalproperties of worm-like gold nanorods. J. Colloid Interf. Sci. 322, 136–142.

Khan, J.A., Pillai, B., Das, T.K., Singh, Y., Maiti, S., 2007. Molecular effects of uptake ofgold nanoparticles in HeLa cells. ChemBioChem 8, 1237–1240.

Knezevic, N., Lin, V.S.-Y., 2013. A magnetic mesoporous silica nanoparticle-baseddrug delivery system for photosensitive cooperative treatment of cancer with amesopore-capping agent and mesopore-loaded drug. Nanoscale 5, 1544–1551.

Lee, H.J., Liu, Y., Zhao, J., Zhou, M., Bouchard, R.R., Mitcham, T., Wallace, M., Stafford,R.J., Li, C., Gupta, S., Melancon, M.P., 2013. J. Control. Release 172, 152–158.

Link, S., El-Sayed, M.A., 2000. Shape and size dependence of radiative, non-radiativeand photothermal properties of gold nanocrystals. Int. Rev. Phys. Chem.19, 409–453.

Mie, G., 1976. Contribution to the optics of turbid media, especially colloidal metalsuspensions. Ann. Phys. 25, 377–445.

Moores, A., Goettmann, F., 2006. Metallic nanoparticles hosted in mesoporous oxidethin films for catalytic applications. New J. Chem. 30, 1121–1132.

National Research Council, 1996. Guide for the Care and Use of Laboratory Animals.National Academy Press, Washington, DC.

Orendorff, C., Gearheart, L., Jand, N., Murphy, C., 2006. Aspect ratio dependence onsurface enhanced Raman scattering using silver and gold nanorod substrates.Phys. Chem.Chem. Phys. 8, 165–170.

Park, H.Y., Yang, J., Jaemin, L., Haam, S., Choi, I.H., Yoo, K.H., 2009. Multifunctionalnanoparticles for combined doxorubicin and photothermal treatments. ACS 3,2919–2926.

Shen, S., Tang, H., Zhang, X., Ren, Z., Wang, D., Gao, H., Qian, Y., Jiang, X., Yang, W.,2013. Targeting mesoporous silica-encapsulated gold nanorods for chemo–photothermal therapy with near-infrared radiation. Biomaterials 34, 3150–3158.

Shukla, R., Bansal, V., Chaudhary, M., Basu, A., Bhonde, R.R., Sastry, M., 2005.Biocompatibility of gold nanoparticles and their endocytotic fate inside thecellular compartment: amicroscopic overview. Langmuir 21, 10644–10654.

Tan, S., Wu, Q., Wang, J., Wang, Y., Liu, X., Sui, K., Deng, X., Wang, H., Wu, M., 2011.Dynamic self-assembly synthesis and controlled release as drug vehicles ofporous hollow silica nanoparticles. Micropor. Mesopor. Mat. 142, 601–608.

Tang, F., Li, L., Chen, D., 2012. Mesoporous silica nanoparticles: synthesis,biocompatibility and drug delivery. Adv. Mater. 24, 1504–1534.

Wang, Z., Zong, S., Yang, J., Li, J., Cu, Y., 2011. Dual-mode probe based on mesoporoussilica coated gold nanorods for targeting cancer cells. Biosens. Bioelectron. 26,2883–2889.

Yang, P., Gaib, S., Lin, J., 2012. Functionalized mesoporous silica materials forcontrolled drug delivery. Chem. Soc. Rev. 41, 3679–3698.

Zhang, W., Guo, Z.Y., Huang, D.Q., Liu, Z.M., Guo, X., Zhong, H.Q., 2011. Synergisticeffect of chemo–photothermal therapy using PEGylated graphene oxide.Biomaterials 32, 8555–8561.

Zhang, Y., Huang, R., Zhu, X., Wang, L.Z., Wu, C.X., 2012. Synthesis, properties, andoptical applications of noble metal nanoparticle-biomolecule conjugates. Chin.Sci. Bull. 57, 238–246.

Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo–photothermal therapy

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