International Journal of Pharmaceutics 470 (2014) 1–7

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 2 May 2014

Keywords:Gold nanorodsMesoporous silicaCombined chemo–photothermal therapyDrug deliverypH responsive drug releaseMCF-7 cell line

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., 2005; 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.,2005; 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., 2005). 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.

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 coating material 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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protocol at a very low DOX concentrations aiming to reduce drugtoxicity to critical tissues.

2. Materials and methods

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

2.1. The preparation of pGNRs@mSiO2-DOX

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

reported silver ion-assisted seed mediated method using CTAB as atemplate (Huang et al., 2008). Briefly, 1.5 ml of 0.1 M CTAB solutionwas mixed with 100 ml of 0.02 M HAuCl4. Then 100 ml ice-cold of0.01 M NaBH4 was added forming a brownish yellow seed solution.The solution was vigorously stirred for 2 min and kept in a waterbath at 25 �C for 2 h.

The gold nanorods growth solution was prepared by mixing1.5 ml of 0.02 M HAuCl4 and 1.0 ml of 0.01 M AgNO3 with 30 ml of0.1 M CTAB. Then 0.8 ml of 0.08 M ascorbic acid solution was addedto the growth solution changing its color from dark yellow tocolorless. Then 70 ml seed solution was added to the total volumeof the growth solution at 25 �C. The color of the solution graduallychanged until finally it became purple. The obtained GNRs werecentrifuged at 4472 g for 30 min (Sigma 202, refrigerated centri-fuge; Germany). The pellet was then washed twice in deionizedwater to remove excess CTAB. It was finely dispersed in 40 mldeionized water.

2.1.2. The preparation of GNRs@mSiO2

The mesoporous silica coating was achieved by modified Stobermethod (Shen et al., 2013). Briefly, 50 ml aqueous ammonia wasadded to GNRs solution in order to adjust its pH value to 10. Then,10.5 ml of 10 mM TEOS/ethanol solution was added to the GNRssolution at a rate of 3.5 ml/h, and the mixture was kept at 40 �C undergentle stirring for 24 h. The synthesized product GNRs@mSiO2 wascentrifuged and washed in deionized water and ethanol severaltimes. The pellet of GNRs@mSiO2 was then dispersed in ethanolsolution (60 ml) containing concentrated HCl (120 ml) and stirred at30 �C for 3 h to remove the template (CTAB). This surfactant removalprocess was repeated twice to ensure complete sample clearance ofCTAB. The sample was then centrifuged and washed in deionizedwater three times in order to remove the traces of HCl and ammonia.

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

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 pellet 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

ld nanorods coated mesoporous silica (b).

A.S. Monem et al. / International Journal of Pharmaceutics 470 (2014) 1–7 3

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 pelletwas 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 release ð%Þ ¼ Amount of DOX releasedAmount of DOX inthe 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 (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/ml. After24 h cells incubation, 10 ml of the WST-1 solution was added intoeach well. The cells were incubated for another 4 h, and itsabsorbance was monitored at 450 nm on an Elisa micro-platereader (TECAN). The culture medium without nanoparticles wasused as the blank control. The cytotoxicity was then expressed asthe percentage of the cell viability compared with the blankcontrol.

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,

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

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

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the tumors were exposed extra corporeally to the NIR laser for60 min. The mice of group B were intravenously injected with40 ml of free DOX (equivalent to 4 mg DOX/kg body weight) whichis the typical routine therapeutic dose of free doxorubicin used intreating human. The mice of group C (positive control) wereintravenously injected with 200 ml PBS at pH 7.4 and followed thesame irradiation conditions as group A. The skin at the tumor sitefor groups A and C was shaved to maximize the radiationtransmittance to the target area. The mice of group D (negativecontrol) received neither the injections nor the subsequent laserirradiation.

2.7. Tumor size measurements

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

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

formula V = (d2D), where D and d are the long and short axes,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 (Fig1b). 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.,2006). The sample GNRs@mSiO2 showed about 20 nm shifts in its

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

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

A.S. Monem et al. / International Journal of Pharmaceutics 470 (2014) 1–7 5

longitudinal 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 37 �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 spindleshape (Fig. 5a and b). The observed dark aggregates in cells treated

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

with 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 human amnion wish cells were incubatedwith CTAB coated GNRs, pGNRs@mSiO2-DOX and free DOX for 24 h,and cell viability was then measured. The cells incubated withpGNRs@mSiO2-DOX show cell viability of 87% which is very highcompared to CTAB coated GNR 29% and free DOX 35% (Fig. 5e). Theresults indicated that the normal cells up take of pGNRs@mSiO2-DOX suffer a neglected percentage of apoptosis and necrosis aslong as they are screened from NIR irradiation. The cytotoxicity ofCTAB coated GNRs showed a relatively high cytotoxic effect onnormal cells even though the cells were not exposed to NIRirradiation. The toxicity of CTAB was obviously high and may leadto lethal side effects; thus any therapeutic formulation containingCTAB as a coating material must be used with great caution. Thehuman amnion wish cells incubated with free DOX also showed alow cell viability percentage which would be attributed to its hightoxicity 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 recorded throughout the time of NIR exposure for group Aand C, respectively. The temperature drops to its initial valuesshortly after switching off laser exposure. These results weresimilar to our recent work using mesoporous silica gold nanoshells(Elbialy and Mohamed, 2014). The advantage of using the preparedpGNRs@mSiO2-DOX in this combined chemo–photothermal ther-apy is that it keeps its major characteristic of photo-heatconversion upon repeated exposure to NIR radiation. The photo-heat conversion of the sample was tested in vitro using simulatedbody fluid (Fig. 7). Also the absorption spectra of samplesrepeatedly exposed to NIR radiation suffer no changes (Fig. 8).

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

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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According to the obtained data, pGNRs@mSiO2-DOX can keep itsrod structure as well as its physical identity after exposure to theexcessive local heat generated upon NIR radiation. It is importantto note that a few micrograms of GNRs can deliver such a quantityof heat, to about 1 g of cancerous tissue that rises its temperature to�10 �C throughout NIR exposure time. The change in temperatureof GNRs times its specific heat would be surprisingly very highdepending on GNRs/tissue mass ratio and other minor insulatingconditions.

The volume change of the implanted Ehrlich tumor in the rightflank of the mice of the four groups A, B, C and D were measuredover a period of 18 days (Fig. 9). It is clear from the figure thattreated group A showed a pronounced inhibition of tumor volumegrowth 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 increased 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.

A.S. Monem et al. / International Journal of Pharmaceutics 470 (2014) 1–7 7

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@SiO22-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 singleirradiation protocol results in effective damage to tumor cells 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@SiO2 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 chemothera-peutic drug, DOX, and have a high quality repeated photo-heatconversion properties. The size and shape of the nanoparticles weredetermined using TEM. Its absorption spectra were also determinedto identify its effective photo-heat conversion band. The sampleszeta potentials were determined which were found to be animportant factor for both DOX loading and its pH responsive release.The therapeutic efficacy of pGNRs@mSiO2-DOX was tested both invitro and in vivo. The formulation used in this work was found topossess a very low toxicity effects to normal tissue and can target itsfull damage to cancerous tissues. This study demonstrated that thecombined chemo–photothermal therapy accomplished bypGNRs@mSiO2-DOX has an enhanced potential to kill cancer cellscompared to both photothermal therapy and chemotherapy alone.

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.

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