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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo–photothermal therapy
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A. Soltan Monem *, Nihal Elbialy, Noha Mohamed
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Biophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 March 2014 Received in revised form 24 April 2014 Accepted 29 April 2014 Available online xxx
The efficacy of the combined chemo–photothermal therapy, using a mesoporous silica-coated gold nanorods loaded DOX (pGNRs@mSiO2-DOX), was consistently tested both in vitro and in vivo. The prepared nanoparticles that were characterized using transmission electron microscopy (TEM), UV–vis absorption spectroscopy and zeta potential showed high doxorubicin loading capacity in addition to its pH-responsive release. The pGNRs@mSiO2-DOX photo-heat conversion characteristic found to be stable for several repeated NIR irradiated doses was tested in simulated body fluid. In vitro results showed that pGNRs@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 and free 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 mice that received the combined treatment. Based on the obtained results, this passively targeted pGNRs@mSiO2-DOX could specifically deliver drug and excessive local heat to tumor sites achieving high combined therapeutic efficacy. ã 2014 Published by Elsevier B.V.
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Keywords: Gold nanorods Mesoporous silica Combined chemo–photothermal therapy Drug delivery pH responsive drug release MCF-7 cell line Human amnion wish cells Ehrlich tumor
1. Introduction Noble metal nanoparticles have been widely studied in the past decades because of their high potential applications in many areas especially medical therapy and diagnosis (Moores and Goettmann, 2006; Huang et al., 2007; Zhang et al., 2012). They provide remarkable opportunities due to their inherently low toxicity (Connor et al., 2005a; Khan et al., 2007; Shukla et al., 2005) and strong enhanced optical properties associated with localized surface plasmon resonance (El-Sayed, 2001; Link and El-Sayed, 2000; Mie, 1976). Recently extensive studies have been focused on gold nanorods (GNRs) for cancer therapy because of their characteristic 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 of cetyltrimethyl ammonium bromide (CTAB) with bilayer coating GNRs display significant cytotoxicity to human cells in its free form (Alkilany et al., 2009; Connor et al., 2005b). Additionally, CTAB induces GNRs aggregation which leads to the loss of their unique
* Corresponding author at: Biophysics Department, Faculty of Science, Cairo University, Giza 12613, Egypt. Tel.: +20 1224340195. E-mail address: [email protected] (A. S. Monem).
optical properties and minimizes its cellular uptake. Thus the uses of CTAB-coated GNRs hinder its biomedical applications. On the other hand, mesoporous silica was found to be suitable for being used as a coating material for GNRs because of its high drug loading Q6 capacity and non-toxic biodegradable content. It has been extensively highlighted for many biomedical applications as nanocarriers for anticancer drugs, DNA and proteins. They also possess large surface area, tunable size, high accessible pore volume and well-defined surface properties capable for modification (Yang et al., 2012; Tang et al., 2012; Knezevic and Lin, 2013; AlKady et al., 2011; He et al., 2011; Tan et al., 2011). They are characterized by a pH responsive drug delivery that provides high drug release profile in the acidic tumor environment (Falk and Issels, 2001; Chen et al., 2007; Zhang et al., 2011; Park et al., 2009). In this study, we developed a preparative method for multifunctional nanoparticles (pGNRs@mSiO2-DOX) which are suitable for a combined chemo–photothermal cancer therapy. These nanoparticles were tested for pH responsive delivery of chemotherapeutic drug (DOX) to tumor cites as well as their unique simultaneous NIR light-based induction of hyperthermia. The physical and the chemical properties of these nanoparticles were investigated in a buffer as well as in a simulated body fluid solutions. The therapeutic efficacy of the prepared pGNRs@mSiO2DOX was tested in both in vitro and in vivo using a single dose
http://dx.doi.org/10.1016/j.ijpharm.2014.04.067 0378-5173/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: Monem, A.S., et al., Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo– photothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.067
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protocol at a very low DOX concentrations aiming to reduce drug toxicity to critical tissues.
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2. Materials and methods
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Doxorubicin (DOX), chloroauric acid (HAuCl43H2O), cetyltrimethyl ammonium bromide (CTAB), tetraethyoxysilane (TEOS) and L-ascorbic acid (AA) were purchased from Sigma–Aldrich. Silver nitrate (AgNO3), tris buffer (CH2OH)3CNH2 and sodium borohydride (NaBH4) were purchased from Merck. Ammonia hydroxide (NH4OH, 28%) was purchased from Fluka.
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2.1. The preparation of pGNRs@mSiO2-DOX
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2.1.1. The preparation of GNRS The gold nanorods were prepared according to previously 59 reported silver ion-assisted seed mediated method using CTAB as a 60 template (Huang et al., 2008). Briefly, 1.5 ml of 0.1 M CTAB solution 61 was mixed with 100 ml of 0.02 M HAuCl4. Then 100 ml ice-cold of 62 0.01 M NaBH4 was added forming a brownish yellow seed solution. 63 The solution was vigorously stirred for 2 min and kept in a water 64 bath at 25 C for 2 h. 65 The gold nanorods growth solution was prepared by mixing 66 1.5 ml of 0.02 M HAuCl4 and 1.0 ml of 0.01 M AgNO3 with 30 ml of 67 0.1 M CTAB. Then 0.8 ml of 0.08 M ascorbic acid solution was added to 68 the growth solution changing its color from dark yellow to colorless. 69 Then 70 ml seed solution was added to the total volume of the growth 70 solution at 25 C. The color of the solution gradually changed until 71 finally it became purple. The obtained GNRs were centrifuged at 72 4472 g for 30 min (Sigma 202, refrigerated centrifuge; Germany). 73 The billet was then washed twice in deionized water to remove 74Q8 excess CTAB. It was finely dispersed in 40 ml deionized water.
The concentration of the sample was adjusted by measuring its optical density at 805 nm until it reads 2.
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2.1.3. The preparation of GNRs@mSiO2-DOX Two milliliters of GNRs@mSiO2 nanoparticles was added to 250 mg of doxorubicin hydrochloride (DOX) at pH 8. The mixture was stirred at room temperature for 24 h. The GNRs@mSiO2-DOX was then centrifuged at 4472 g for 30 min, and the billet washed with PBS several times. The free DOX contents of the supernatant were determined from the calibration curve of DOX concentration and emission intensity at an excitation l of 480 nm and emission l of 585 nm using a spectrofluorometer (Shimadzu, RF 5301pc, Japan). The drug loading efficiency was then calculated from the relation
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Loading efficiencyð%Þ
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2.1.2. The preparation of GNRs@mSiO2 The mesoporous silica coating was achieved by modified Stober method (Shen et al., 2013). Briefly, 50 ml aqueous ammonia was added 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 GNRs solution at a rate of 3.5 ml/h, and the mixture was kept at 40 C under gentle stirring for 24 h. The synthesized product GNRs@mSiO2 was centrifuged and washed in deionized water and ethanol several times. The billet of GNRs@mSiO2 was then dispersed in ethanol solution (60 ml) containing concentrated HCl (120 ml) and stirred at 30 C for 3 h to remove the template (CTAB). This surfactant removal process was repeated twice to ensure complete sample clearance of CTAB. The sample was then centrifuged and washed in deionized water three times in order to remove the traces of HCl and ammonia.
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Initial amount of DOX Supernant free amount of DOX Initial amount of drug
2.1.4. Coating GNRs@mSiO2 with polyethylene glycol The GNRs@mSiO2-DOX surfaces were coated with polyethylene glycol for intravenous injection, by adding 10 ml of 25 mM PEG-SH to each 1 ml of GNRs@mSiO2-DOX solution and incubating the mixture for 12 h at 4 C. The suspension was then centrifuged at 4472 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.
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2.2. Sample characterization
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The morphology and size of the GNRs and GNRs@mSiO2 were determined using transmission electron microscopy (TEM) (FEI Tecnai G20, Super twin, Double tilt, LaB6 Gun) operating at 200 kV. The absorption spectra of GNRs, GNRs@mSiO2, GNRs@mSiO2-DOX and free DOX were measured, using a UV–vis spectrophotometer (Jenway UV-6420; Barloworld Scientific, Essex, UK), at the wavelength range 400–900 nm. Furthermore, zeta potential of the GNRs and pGNRs@mSiO2 was measured in deionized distilled water using a dynamic light scattering apparatus (zeta potential/ particle sizer NICOMPTM 380 ZLS, USA).
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2.3. Measurement of the pGNRs@mSiO2-DOX responsive release
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The sterilized dialysis bags with a dialyzer molecular-weight cut-off of 12,000 Da (Cellulose Dialysis Tubing, Fisherbrand, USA) were used to perform the drug release experiments. Two phosphate buffered saline (PBS) solutions of pH 7.4 and pH 5.6
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Fig. 1. TEM image of gold nanorods (a) and gold nanorods coated mesoporous silica (b).
Please cite this article in press as: Monem, A.S., et al., Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo– photothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.067
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were used as the drug release media to simulate normal blood/ tissues and tumor environments, respectively. The used dialysis bags were soaked overnight in the release media. One milliliter of pGNRs@mSiO2-DOX (190 mg/ml) was centrifuged, and the billet was dispersed in 1 ml of the release media which was then placed into the dialysis bags. The sealed dialysis bags were placed into brown glass bottles; then 20 ml of release media was added to each bottle. These bottles were shaken at a speed of 105 rpm at 37 C under a light-sealed condition. At successive time intervals, 3 ml of the release media were used to quantify the concentration of the released drug using a spectrofluorometer. Then, it was refilled to the original release media. The concentrations of the released drug were determined from the calibration curve at an excitation l of 480 nm and emission l of 585 nm. Amount of DOX released Amount of DOX in the nanoparticles 100%
Cumulative releaseð%Þ ¼
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2.4. In vitro cytotoxicity of pGNRs@mSiO2-DOX
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The breast cancer cell line MCF-7 was cultured in RPMI 1640 containing 10% fetal bovine serum (FBS). THe cells were maintained at 37 C in a humidified incubator containing 5% CO2. For all the experiments, the cells were harvested using 0.25% trypsin in EDTA which was then suspended in fresh medium prior to plating. In vitro cytotoxicity against MCF-7 cells was determined using the WST-1 cell viability and proliferation assay. The MCF-7 cells were seeded into 96-well plates at a density of 100 cells per well (100 ml of the medium solution). After incubation for 24 h at 37 C in 100 ml of RPMI 1640 medium containing 10% FBS, 50 ml of the culture medium was discarded and replaced by various concentrations of free DOX and pGNRs@mSiO2-DOX. After 24 h of cell incubation in different concentrations of pGNRs@mSiO2-DOX, the cells were then exposed to NIR laser for 60 min. Post-treatment, the digital microscopic images of the wells were taken using an inverted light microscope (Leica) at a magnification of 20. The cells viability was then counted as a function of the drugs concentrations. The human amnion wish cells were seeded into 96-well plates at a density of 55,000 cell per well. After incubation for 24 h at 37 C in 200 ml of RPMI 1640 medium containing 10% FBS, 50 ml culture medium was discarded and the cells were treated with 50 ml of CTAB coated GNRs, pGNRs@mSiO2-DOX (of DOX concentration
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Fig. 2. The absorption spectra of GNRs, GNRs@mSiO2, GNRs@mSiO2-DOX and free DOX.
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190 mg/ml) and free DOX at a concentration of 120 mg/m. After 24 h cells incubation, 10 ml of the WST-1 solution was added into each well. The cells were incubated for another 4 h, and its absorbance was monitored at 450 nm on an Elisa micro-plate reader (TECAN). The culture medium without nanoparticles was used as the blank control. The cytotoxicity was then expressed as the percentage of the cell viability compared with the blank control.
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2.5. Inoculation of the mice with tumor cells
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The Ehrlich ascites tumor was chosen as a rapidly growing experimental tumor model where various experimental designs for anticancer agents can be applied (Elbialy et al., 2010; Dasyukevich and Solyanikn, 2007). The Ehrlich ascites carcinomas cells were obtained from National Cancer Institute “NCI” – Cairo University and were intraperitoneally injected into female Balb mice. The ascites fluid was collected after 7 days post injection. The Ehrlich cells were washed twice and then suspended in 5 ml saline solution. The female Balb mice (20–25 g weight and 6–8 week old) were obtained from the animal house of NCI and were injected subcutaneously in their right flanks, where the tumors had developed into a single solid form. The tumor growth was monitored post-inoculation as soon as the desired volume was approximately 0.3–0.6 cm3. All animal procedures and care were performed using the guidelines for the Care and Use of Laboratory Animals and was approved by the Animal Ethics Committee at Cairo University (National Research Council, 1996).
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2.6. In vivo NIR laser photothermal therapy
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In this study sixty mice were divided into four groups: A, B, C and D. The mice were anesthetized via an intraperitoneal injection with thiopental (48 mg/kg). The mice of group A were intravenously injected with 200 ml pGNRs@mSiO2-DOX (equivalent 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
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Fig. 3. Zeta potential of CTAB coated gold nanorods (a), and pGNRs@mSiO2 (b).
Please cite this article in press as: Monem, A.S., et al., Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo– photothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.067
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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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60 min. The mice of group B were intravenously injected with 40 ml of free DOX (equivalent to 4 mg DOX/kg body weight) which is the typical routine therapeutic dose of free doxorubicin used in treating human. The mice of group C (positive control) were intravenously injected with 200 ml PBS at pH 7.4 and followed the same irradiation conditions as group A. The skin at the tumor site for groups A and C was shaved to maximize the radiation transmittance to the target area. The mice of group D (negative control) received neither the injections nor the subsequent laser irradiation.
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2.7. Tumor size measurements
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As Ehrlich tumor model is characterized by its high growth rate, the change in the tumor volume (DV) was measured every three days over a period of eighteen days for the four groups (A, B, C and D). The ellipsoidal tumor volume (V) was calculated using the
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formula V = (p/6)(d)2(D), 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 performed using Fisher’s LSD (least significance difference) multiple-comparison test. The p-values less than 0.05 were considered statistically significant. Each data point was presented as the mean standard error (SE) of at least 7–10 measurements. In addition, SPSS version 17 was used for the statistical analyses.
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2.8. Histopathological examination
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The treatment groups A and C were sacrificed immediately after laser exposure, and the necrotic percentage of tumor cells was determined. The tumors were excised, fixed in 10% neutral formalin, embedded in paraffin blocks and then sectioned. The tissue sections were obtained directly after treatment and stained with hematoxylin and eosin (H&E). The previous procedures were repeated for group B (3 days post injection) and the control group D. All the tissue sections were examined using a light microscope (CX31 Olympus microscope) that was connected with a digital camera (Canon).
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3. Results and discussion
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The size and shape of the prepared CTAB-coated GNRs and GNRs@mSiO4 were determined using TEM images, Fig. 1a and b respectively. The average GNRs length and width were 40 nm and 10 nm, respectively, equivalent to a size of 3000 nm3. The mesoporous silica coat thickness found to vary from 10 nm to 13 nm giving rise to GNRs@mSiO4 volume to be 42,000 nm3 (Fig 1b). The relatively large volume of such pours material allows high DOX loading per particle and efficient delivery upon NIR radiation. As shown in Fig. 2, the prepared GNRs have a weak transverse plasmon band at 530 nm and a strong longitudinal plasmon band at 820 nm in agreement with the reported results (Orendorff et al.,
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Fig. 5. Inverted microscope images of control MCF7 cells (a), cells incubated with GNRs@mSiO2-DOX (b), cells incubated with free DOX (c), percentage of cell viability for the MCF-7 cellline (d) and percentage of cell viability forhuman amnion wish cells (e).gr5
Please cite this article in press as: Monem, A.S., et al., Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo– photothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.067
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2006). The sample GNRs@mSiO2 showed about 20 nm shifts in its longitudinal peak compared to that of GNRs, and this could be due to the change in the local refractive index of the medium surrounding GNRs. An additional peak at 490 nm appears in the absorption spectrum of GNRs@mSiO2-DOX, which is attributed to the absorbance band of DOX indicating its high incorporation within the silica shell pours. The relatively high amplitude of this band insures the successful doping of the drug, at adequate concentration, 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 negative potential of 22.5 mV confirming the complete removal of CTAB and the formation of firm stable pGNRs@mSiO2 samples (Fig. 3). The DOX responsive release of GNRs@mSiO2-DOX was carried out in PBS at pH values 7.4 and 5.6 at 3 C for a period of 120 h. The DOX release rate was obviously pH dependent that increases at relatively low acidic media (Fig. 4). The marked variations in the release profile at different pH confirm the pH responsiveness of GNRs@mSiO2-DOX. Also it indicates that this formulation could selectively release doxorubicin specifically at the tumor sites. The results of DOX loading and its responsive release motivated us to further investigate the in vitro cellular cytotoxicity for 4 h post MCF-7 cells incubation with pGNRs@mSiO2-DOX followed by 1 h NIR exposure; the morphology of the cells was completely changed and became spherical rather than its normal spindle
267 shape (Fig. 5a and b). The observed dark aggregates in cells treated 268 with pGNRs@mSiO2-DOX revealed the accumulation of the nano269 particles inside MCF-7 cells (Fig. 5b). It seems that treated MCF-7 270 cells suffer high apoptosis rates in agreement with the in vitro 271 cytotoxicity results using WST-1 assay (Fig. 5d). In case of treating 272 the cells with free DOX, a remarkable percentage of them appeared 273 intact with their normal spindle shape (Fig. 5c). The breast cancer 274 cell line MCF-7 treated with pGNRs@mSiO2-DOX showed high 275 cytotoxic effects, four times greater, than that cells treated with 276 free DOX at the three different concentrations used (Fig. 5d). Q11 277 The human amnion wish cells are characterized by its high 278 productivity, easy accessibility, free from contamination with non279 fibroblastoid cells and ethically acceptable source of cells for 280 biomedical applications (Hu et al., 2009). The cytotoxicity of 281 pGNRS@mSiO2-DOX on normal cells and human amnion wish cells 282 were incubated with CTAB coated GNRs, pGNRS@mSiO2-DOX and 283 free DOX for 24 h, and cell viability was then measured. The cells 284 incubated with pGNRS@mSiO2-DOX show cell viability of 87% 285 which is very high compared to CTAB coated GNR 29% and free DOX 35% (Fig. 5e). The results indicated that the normal cells up take of Q12 286 287 pGNRS@mSiO2-DOX suffer a neglected percentage of apoptosis 288 and/or necrosis as long as they are screened from NIR irradiation. 289 The cytotoxicity of CTAB coated GNRs showed a relatively high 290 cytotoxic effect on normal cells even though the cells were not 291 exposed to NIR irradiation. The toxicity of CTAB was obviously high 292 and may lead to lethal side effects; thus any therapeutic 293 formulation containing CTAB as a coating material must be used 294 with great caution. The human amnion wish cells incubated with 295 free DOX also showed a low cell viability percentage which would 296 be attributed to its high toxicity to normal cell. 297 The temperature of Ehrlich tumor tissue was measured as a 298 function of NIR exposure time in order to assess the photo-heat 299 conversion characteristics of the prepared pGNRs@mSiO2-DOX 300 (Fig. 6). An elevation in tissue temperature of about 14 C and 4 C 301 were recorded throughout the time of NIR exposure for group A and C 302 , respectively. The temperature drops to its initial values shortly after 303 switching off laser exposure. These results were similar to our recent 304 work using mesoporous silica gold nanoshells (Elbialy and 305 Mohamed, 2014). The advantage of using the prepared 306 pGNRs@mSiO2-DOX in this combined chemo–photothermal thera307 py is that it keeps its major characteristic of photo-heat conversion 308 upon repeated exposure to NIR radiation. The photo-heat conversion 309 of the sample was tested in vitro using simulated body fluid (Fig. 7). 310 Also the absorption spectra of samples repeatedly exposed to NIR
Fig. 7. The change in temperature of pGNRs@mSiO2 suspended in a simulated body fluid throughout NIR irradiation.
Fig. 8. The absorption spectra of pGNRs@mSiO2 before and after repeated NIR irradiation.
Fig. 6. The change in the Ehrlich tumor interstitial temperature during NIR laser irradiation for group A, group C. 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 Q10 264 265 266
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Please cite this article in press as: Monem, A.S., et al., Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo– photothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.067
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Fig. 9. The average changes in the Ehrlich tumor volume as a function of time for the treatment groups A, B, C, and D. 311 312 313 314 315 316 317 318 319 320 321 322 323 324
radiation suffer no changes (Fig. 8). According to the obtained data, pGNRs@mSiO2-DOX can keep its rod structure as well as its physical identity after exposure to the excessive local heat generated upon NIR radiation. It is important to note that a few micrograms of GNRs can deliver such a quantity of heat, to about 1 g of cancerous tissue that rises its temperature to 10 C throughout NIR exposure time. The change in temperature of GNRs times its specific heat would be surprisingly very high depending on GNRs/tissue mass ratio and other minor insulating conditions. The volume change of the implanted Ehrlich tumor in the right flank of the mice of the four groups A, B, C and D were measured over a period of 18 days (Fig. 9). It is clear from the figure that treated group A showed a pronounced inhibition of tumor volume growth for a period of 4–5 days (compared to group D, p < 0.0001)
followed by a very slow growth rate up to 18 days. The results showed that the cancerous cells suffer a high necrotic percentage, and the remaining viable tumor cells have shown such a slow growth up to the end of the measuring period. Group B, treated with DOX alone, showed a similar volume inhibition behavior as that of group A followed by a much faster growth after 3 days compared to group A. Because of the natural high growth rate of Ehrlich tumor, untreated group D showed an enhanced tumor volume growth rates of about 10% per day. While group C, which was 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 histopathological examination (Fig. 10). The negative control, group D, showed a normal necrosis percentage of focal and diffuse necrosis. The former appears as scattered necro-apoptotic bodies within the groups of viable cells while the latter appears as islands of coagulative necrosis (geographic distribution) showing the ghosts of the cells. The hemorrhagic necrosis was also observed (lower Rt, “encircled" (Fig. 10a)). While the positive control, group C, showed a diffuse cellular affection and geographic appearance in addition to necrotic regions (Fig. 10b). This mild cell coagulative necrosis could be attributed to a deep penetrative power of the NIR laser beneath the skin. The treated group B showed a remarkable amount of necrotic fields (encircled) with the appearance of many apoptotic and karyorrhechtic bodies (Fig. 10c). The treated group A showed an area of total tumor necrosis with the appearance of nuclear debris. Also some tumor cells appeared with apoptotic bodies, pointed at by arrows (Fig. 10d). It is clear that treated group A suffers the most of necrosis as well as apoptosis compared to the controls and even more than treated group B with DOX alone. Consistently, histopathological examinations have confirmed the observed inhibition of tumor growth rate of the treated groups compared to the controls.
Fig. 10. Sections of Ehrlich tumor cells excised from group D (a), C (b), B (c) and A (d) tumor tissues stained with H&E.
Please cite this article in press as: Monem, A.S., et al., Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo– photothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.067
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So far we have characterized a new formulation of nanoparticles pGNRs@mSiO2-DOX suitable for a combined chemo– photothermal treatment of cancer cells and tumors. It has an adequate drug loading capacity and a responsive drug release in tumor sites. They have been found to have a high effective photoheat conversion and may possess a supper specific heat capacity characteristics. The samples also tested for a repeated NIR exposure for 1 h and showed no changes in its physical properties, its absorption spectra and photo-heat conversion. These results confirm its stability against excessive heat produced during NIR exposure and its ability to deliver that heat for a repeated exposure within the tumor tissue. The pGNRs@SiO4-DOX used in this work has shown a great therapeutic efficacy using only a single chemotherapeutic dose protocol followed by a single exposure to NIR irradiation. Although they have the ability for a repeated photo-thermal ablation, its single irradiation protocol results in effective damage to tumor cells both in vivo and in vitro. The tumor cell membrane permeation and retention allows its accumulation in tumor cells at a suitable mass concentration as well as its responsive The DOX-release effectively enhances its therapeutic efficacy. These characteristics gave them the priority over other delivery systems such as gold nanoshells @SiO4 loaded DOX. These gold shells may be debrided post NIR irradiation (Elbialy et al., 2014; Lee et al., 2013).
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4. Conclusion
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The preparation method used in this work was found to reproduce pGNRs@mSiO2-DOX capable to incorporate chemotherapeutic drug, DOX, and have a high quality repeated photo-heat conversion properties. The size and shape of the nanoparticles were determined using TEM. Its absorption spectra were also determined to identify its effective photo-heat conversion band. The samples zeta potentials were determined which were found to be an important factor for both DOX loading and its pH responsive release. The therapeutic efficacy of pGNRs@mSiO2-DOX was tested both in vitro and in vivo. The formulation used in this work was found to possess a very low toxicity effects to normal tissue and can target its full damage to cancerous tissues. This study demonstrated that the combined chemo–photothermal therapy accomplished by pGNRs@mSiO2-DOX has an enhanced potential to kill cancer cells compared to both photothermal therapy and chemotherapy alone.
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Acknowledgments
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Authors gratefully acknowledge Dr. Tark El-Bolkini, and Veterinary Heba M., Cancer National Institute, Cairo University, for his help in examining Ehrlich tumor tissue and animal care and treatment. We also like to thank Prof. Dr. M. Aman, Faculty of Science, Ein Shams University for the TEM imaging.
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Sci. 19, 1047–1052. 438 Huang, X., Jain, P.K., El-Sayed, I.H., El-Sayed, M.A., 2007. Gold nanoparticles: 439 interesting optical properties and recent applications in cancer diagnostics and 440 therapy. Nanomedicine (London, U.K.) 2, . 441 Huang, H., He, C., Zeng, Y., Xia, X., Yu, X., Yi, P., Chen, Z., 2008. Preparation and optical 442 properties of worm-like gold nanorods. J. Colloid Interf. Sci. 322, 136–142. 443 Khan, J.A., Pillai, B., Das, T.K., Singh, Y., Maiti, S., 2007. Molecular effects of uptake of 444 gold nanoparticles in HeLa cells. ChemBioChem 8, 1237–1240. 445 Knezevic, N., Lin, V.S.-Y., 2013. A magnetic mesoporous silica nanoparticle-based 446 drug delivery system for photosensitive cooperative treatment of cancer with a 447 mesopore-capping agent and mesopore-loaded drug. Nanoscale 5, 1544–1551. 448 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. 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ACS 3, 463 2919–2926. 464 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– 465 photothermal therapy with near-infrared radiation. Biomaterials 34, 3150– 466 3158. 467 Shukla, R., Bansal, V., Chaudhary, M., Basu, A., Bhonde, R.R., Sastry, M., 2005. 468 Biocompatibility of gold nanoparticles and their endocytotic fate inside the 469 cellular compartment: amicroscopic overview. Langmuir 21, 10644–10654. 470 Tan, S., Wu, Q., Wang, J., Wang, Y., Liu, X., Sui, K., Deng, X., Wang, H., Wu, M., 2011. 471 Dynamic self-assembly synthesis and controlled release as drug vehicles of 472 porous hollow silica nanoparticles. Micropor. Mesopor. Mat. 142, 601–608. 473 Tang, F., Li, L., Chen, D., 2012. Mesoporous silica nanoparticles: synthesis, 474 biocompatibility and drug delivery. Adv. Mater. 24, 1504–1534. 475 Wang, Z., Zong, S., Yang, J., Li, J., Cu, Y., 2011. Dual-mode probe based on mesoporous 476 silica coated gold nanorods for targeting cancer cells. Biosens. Bioelectron. 26, 477 2883–2889. 478 Yang, P., Gaib, S., Lin, J., 2012. Functionalized mesoporous silica materials for 479 controlled drug delivery. Chem. Soc. Rev. 41, 3679–3698. 480 Zhang, W., Guo, Z.Y., Huang, D.Q., Liu, Z.M., Guo, X., Zhong, H.Q., 2011. Synergistic 481 effect of chemo–photothermal therapy using PEGylated graphene oxide. 482 Biomaterials 32, 8555–8561. 483 Zhang, Y., Huang, R., Zhu, X., Wang, L.Z., Wu, C.X., 2012. Synthesis, properties, and 484 optical applications of noble metal nanoparticle-biomolecule conjugates. Chin. 485 Sci. Bull. 57, 238–246.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo–photothermal therapy
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A. Soltan Monem *, Nihal Elbialy, Noha Mohamed
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Biophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 March 2014 Received in revised form 24 April 2014 Accepted 29 April 2014 Available online xxx
The efficacy of the combined chemo–photothermal therapy, using a mesoporous silica-coated gold nanorods loaded DOX (pGNRs@mSiO2-DOX), was consistently tested both in vitro and in vivo. The prepared nanoparticles that were characterized using transmission electron microscopy (TEM), UV–vis absorption spectroscopy and zeta potential showed high doxorubicin loading capacity in addition to its pH-responsive release. The pGNRs@mSiO2-DOX photo-heat conversion characteristic found to be stable for several repeated NIR irradiated doses was tested in simulated body fluid. In vitro results showed that pGNRs@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 and free 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 mice that received the combined treatment. Based on the obtained results, this passively targeted pGNRs@mSiO2-DOX could specifically deliver drug and excessive local heat to tumor sites achieving high combined therapeutic efficacy. ã 2014 Published by Elsevier B.V.
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Keywords: Gold nanorods Mesoporous silica Combined chemo–photothermal therapy Drug delivery pH responsive drug release MCF-7 cell line Human amnion wish cells Ehrlich tumor
1. Introduction Noble metal nanoparticles have been widely studied in the past decades because of their high potential applications in many areas especially medical therapy and diagnosis (Moores and Goettmann, 2006; Huang et al., 2007; Zhang et al., 2012). They provide remarkable opportunities due to their inherently low toxicity (Connor et al., 2005a; Khan et al., 2007; Shukla et al., 2005) and strong enhanced optical properties associated with localized surface plasmon resonance (El-Sayed, 2001; Link and El-Sayed, 2000; Mie, 1976). Recently extensive studies have been focused on gold nanorods (GNRs) for cancer therapy because of their characteristic 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 of cetyltrimethyl ammonium bromide (CTAB) with bilayer coating GNRs display significant cytotoxicity to human cells in its free form (Alkilany et al., 2009; Connor et al., 2005b). Additionally, CTAB induces GNRs aggregation which leads to the loss of their unique
* Corresponding author at: Biophysics Department, Faculty of Science, Cairo University, Giza 12613, Egypt. Tel.: +20 1224340195. E-mail address: [email protected] (A. S. Monem).
optical properties and minimizes its cellular uptake. Thus the uses of CTAB-coated GNRs hinder its biomedical applications. On the other hand, mesoporous silica was found to be suitable for being used as a coating material for GNRs because of its high drug loading Q6 capacity and non-toxic biodegradable content. It has been extensively highlighted for many biomedical applications as nanocarriers for anticancer drugs, DNA and proteins. They also possess large surface area, tunable size, high accessible pore volume and well-defined surface properties capable for modification (Yang et al., 2012; Tang et al., 2012; Knezevic and Lin, 2013; AlKady et al., 2011; He et al., 2011; Tan et al., 2011). They are characterized by a pH responsive drug delivery that provides high drug release profile in the acidic tumor environment (Falk and Issels, 2001; Chen et al., 2007; Zhang et al., 2011; Park et al., 2009). In this study, we developed a preparative method for multifunctional nanoparticles (pGNRs@mSiO2-DOX) which are suitable for a combined chemo–photothermal cancer therapy. These nanoparticles were tested for pH responsive delivery of chemotherapeutic drug (DOX) to tumor cites as well as their unique simultaneous NIR light-based induction of hyperthermia. The physical and the chemical properties of these nanoparticles were investigated in a buffer as well as in a simulated body fluid solutions. The therapeutic efficacy of the prepared pGNRs@mSiO2DOX was tested in both in vitro and in vivo using a single dose
http://dx.doi.org/10.1016/j.ijpharm.2014.04.067 0378-5173/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: Monem, A.S., et al., Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo– photothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.067
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protocol at a very low DOX concentrations aiming to reduce drug toxicity to critical tissues.
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2. Materials and methods
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Doxorubicin (DOX), chloroauric acid (HAuCl43H2O), cetyltrimethyl ammonium bromide (CTAB), tetraethyoxysilane (TEOS) and L-ascorbic acid (AA) were purchased from Sigma–Aldrich. Silver nitrate (AgNO3), tris buffer (CH2OH)3CNH2 and sodium borohydride (NaBH4) were purchased from Merck. Ammonia hydroxide (NH4OH, 28%) was purchased from Fluka.
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2.1. The preparation of pGNRs@mSiO2-DOX
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2.1.1. The preparation of GNRS The gold nanorods were prepared according to previously 59 reported silver ion-assisted seed mediated method using CTAB as a 60 template (Huang et al., 2008). Briefly, 1.5 ml of 0.1 M CTAB solution 61 was mixed with 100 ml of 0.02 M HAuCl4. Then 100 ml ice-cold of 62 0.01 M NaBH4 was added forming a brownish yellow seed solution. 63 The solution was vigorously stirred for 2 min and kept in a water 64 bath at 25 C for 2 h. 65 The gold nanorods growth solution was prepared by mixing 66 1.5 ml of 0.02 M HAuCl4 and 1.0 ml of 0.01 M AgNO3 with 30 ml of 67 0.1 M CTAB. Then 0.8 ml of 0.08 M ascorbic acid solution was added to 68 the growth solution changing its color from dark yellow to colorless. 69 Then 70 ml seed solution was added to the total volume of the growth 70 solution at 25 C. The color of the solution gradually changed until 71 finally it became purple. The obtained GNRs were centrifuged at 72 4472 g for 30 min (Sigma 202, refrigerated centrifuge; Germany). 73 The billet was then washed twice in deionized water to remove 74Q8 excess CTAB. It was finely dispersed in 40 ml deionized water.
The concentration of the sample was adjusted by measuring its optical density at 805 nm until it reads 2.
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2.1.3. The preparation of GNRs@mSiO2-DOX Two milliliters of GNRs@mSiO2 nanoparticles was added to 250 mg of doxorubicin hydrochloride (DOX) at pH 8. The mixture was stirred at room temperature for 24 h. The GNRs@mSiO2-DOX was then centrifuged at 4472 g for 30 min, and the billet washed with PBS several times. The free DOX contents of the supernatant were determined from the calibration curve of DOX concentration and emission intensity at an excitation l of 480 nm and emission l of 585 nm using a spectrofluorometer (Shimadzu, RF 5301pc, Japan). The drug loading efficiency was then calculated from the relation
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Loading efficiencyð%Þ
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2.1.2. The preparation of GNRs@mSiO2 The mesoporous silica coating was achieved by modified Stober method (Shen et al., 2013). Briefly, 50 ml aqueous ammonia was added 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 GNRs solution at a rate of 3.5 ml/h, and the mixture was kept at 40 C under gentle stirring for 24 h. The synthesized product GNRs@mSiO2 was centrifuged and washed in deionized water and ethanol several times. The billet of GNRs@mSiO2 was then dispersed in ethanol solution (60 ml) containing concentrated HCl (120 ml) and stirred at 30 C for 3 h to remove the template (CTAB). This surfactant removal process was repeated twice to ensure complete sample clearance of CTAB. The sample was then centrifuged and washed in deionized water three times in order to remove the traces of HCl and ammonia.
¼
Initial amount of DOX Supernant free amount of DOX Initial amount of drug
2.1.4. Coating GNRs@mSiO2 with polyethylene glycol The GNRs@mSiO2-DOX surfaces were coated with polyethylene glycol for intravenous injection, by adding 10 ml of 25 mM PEG-SH to each 1 ml of GNRs@mSiO2-DOX solution and incubating the mixture for 12 h at 4 C. The suspension was then centrifuged at 4472 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.
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2.2. Sample characterization
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The morphology and size of the GNRs and GNRs@mSiO2 were determined using transmission electron microscopy (TEM) (FEI Tecnai G20, Super twin, Double tilt, LaB6 Gun) operating at 200 kV. The absorption spectra of GNRs, GNRs@mSiO2, GNRs@mSiO2-DOX and free DOX were measured, using a UV–vis spectrophotometer (Jenway UV-6420; Barloworld Scientific, Essex, UK), at the wavelength range 400–900 nm. Furthermore, zeta potential of the GNRs and pGNRs@mSiO2 was measured in deionized distilled water using a dynamic light scattering apparatus (zeta potential/ particle sizer NICOMPTM 380 ZLS, USA).
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2.3. Measurement of the pGNRs@mSiO2-DOX responsive release
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The sterilized dialysis bags with a dialyzer molecular-weight cut-off of 12,000 Da (Cellulose Dialysis Tubing, Fisherbrand, USA) were used to perform the drug release experiments. Two phosphate buffered saline (PBS) solutions of pH 7.4 and pH 5.6
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Fig. 1. TEM image of gold nanorods (a) and gold nanorods coated mesoporous silica (b).
Please cite this article in press as: Monem, A.S., et al., Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo– photothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.067
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were used as the drug release media to simulate normal blood/ tissues and tumor environments, respectively. The used dialysis bags were soaked overnight in the release media. One milliliter of pGNRs@mSiO2-DOX (190 mg/ml) was centrifuged, and the billet was dispersed in 1 ml of the release media which was then placed into the dialysis bags. The sealed dialysis bags were placed into brown glass bottles; then 20 ml of release media was added to each bottle. These bottles were shaken at a speed of 105 rpm at 37 C under a light-sealed condition. At successive time intervals, 3 ml of the release media were used to quantify the concentration of the released drug using a spectrofluorometer. Then, it was refilled to the original release media. The concentrations of the released drug were determined from the calibration curve at an excitation l of 480 nm and emission l of 585 nm. Amount of DOX released Amount of DOX in the nanoparticles 100%
Cumulative releaseð%Þ ¼
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2.4. In vitro cytotoxicity of pGNRs@mSiO2-DOX
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The breast cancer cell line MCF-7 was cultured in RPMI 1640 containing 10% fetal bovine serum (FBS). THe cells were maintained at 37 C in a humidified incubator containing 5% CO2. For all the experiments, the cells were harvested using 0.25% trypsin in EDTA which was then suspended in fresh medium prior to plating. In vitro cytotoxicity against MCF-7 cells was determined using the WST-1 cell viability and proliferation assay. The MCF-7 cells were seeded into 96-well plates at a density of 100 cells per well (100 ml of the medium solution). After incubation for 24 h at 37 C in 100 ml of RPMI 1640 medium containing 10% FBS, 50 ml of the culture medium was discarded and replaced by various concentrations of free DOX and pGNRs@mSiO2-DOX. After 24 h of cell incubation in different concentrations of pGNRs@mSiO2-DOX, the cells were then exposed to NIR laser for 60 min. Post-treatment, the digital microscopic images of the wells were taken using an inverted light microscope (Leica) at a magnification of 20. The cells viability was then counted as a function of the drugs concentrations. The human amnion wish cells were seeded into 96-well plates at a density of 55,000 cell per well. After incubation for 24 h at 37 C in 200 ml of RPMI 1640 medium containing 10% FBS, 50 ml culture medium was discarded and the cells were treated with 50 ml of CTAB coated GNRs, pGNRs@mSiO2-DOX (of DOX concentration
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Fig. 2. The absorption spectra of GNRs, GNRs@mSiO2, GNRs@mSiO2-DOX and free DOX.
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190 mg/ml) and free DOX at a concentration of 120 mg/m. After 24 h cells incubation, 10 ml of the WST-1 solution was added into each well. The cells were incubated for another 4 h, and its absorbance was monitored at 450 nm on an Elisa micro-plate reader (TECAN). The culture medium without nanoparticles was used as the blank control. The cytotoxicity was then expressed as the percentage of the cell viability compared with the blank control.
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2.5. Inoculation of the mice with tumor cells
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The Ehrlich ascites tumor was chosen as a rapidly growing experimental tumor model where various experimental designs for anticancer agents can be applied (Elbialy et al., 2010; Dasyukevich and Solyanikn, 2007). The Ehrlich ascites carcinomas cells were obtained from National Cancer Institute “NCI” – Cairo University and were intraperitoneally injected into female Balb mice. The ascites fluid was collected after 7 days post injection. The Ehrlich cells were washed twice and then suspended in 5 ml saline solution. The female Balb mice (20–25 g weight and 6–8 week old) were obtained from the animal house of NCI and were injected subcutaneously in their right flanks, where the tumors had developed into a single solid form. The tumor growth was monitored post-inoculation as soon as the desired volume was approximately 0.3–0.6 cm3. All animal procedures and care were performed using the guidelines for the Care and Use of Laboratory Animals and was approved by the Animal Ethics Committee at Cairo University (National Research Council, 1996).
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2.6. In vivo NIR laser photothermal therapy
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In this study sixty mice were divided into four groups: A, B, C and D. The mice were anesthetized via an intraperitoneal injection with thiopental (48 mg/kg). The mice of group A were intravenously injected with 200 ml pGNRs@mSiO2-DOX (equivalent 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
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Fig. 3. Zeta potential of CTAB coated gold nanorods (a), and pGNRs@mSiO2 (b).
Please cite this article in press as: Monem, A.S., et al., Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo– photothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.067
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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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60 min. The mice of group B were intravenously injected with 40 ml of free DOX (equivalent to 4 mg DOX/kg body weight) which is the typical routine therapeutic dose of free doxorubicin used in treating human. The mice of group C (positive control) were intravenously injected with 200 ml PBS at pH 7.4 and followed the same irradiation conditions as group A. The skin at the tumor site for groups A and C was shaved to maximize the radiation transmittance to the target area. The mice of group D (negative control) received neither the injections nor the subsequent laser irradiation.
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2.7. Tumor size measurements
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As Ehrlich tumor model is characterized by its high growth rate, the change in the tumor volume (DV) was measured every three days over a period of eighteen days for the four groups (A, B, C and D). The ellipsoidal tumor volume (V) was calculated using the
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formula V = (p/6)(d)2(D), 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 performed using Fisher’s LSD (least significance difference) multiple-comparison test. The p-values less than 0.05 were considered statistically significant. Each data point was presented as the mean standard error (SE) of at least 7–10 measurements. In addition, SPSS version 17 was used for the statistical analyses.
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2.8. Histopathological examination
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The treatment groups A and C were sacrificed immediately after laser exposure, and the necrotic percentage of tumor cells was determined. The tumors were excised, fixed in 10% neutral formalin, embedded in paraffin blocks and then sectioned. The tissue sections were obtained directly after treatment and stained with hematoxylin and eosin (H&E). The previous procedures were repeated for group B (3 days post injection) and the control group D. All the tissue sections were examined using a light microscope (CX31 Olympus microscope) that was connected with a digital camera (Canon).
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3. Results and discussion
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The size and shape of the prepared CTAB-coated GNRs and GNRs@mSiO4 were determined using TEM images, Fig. 1a and b respectively. The average GNRs length and width were 40 nm and 10 nm, respectively, equivalent to a size of 3000 nm3. The mesoporous silica coat thickness found to vary from 10 nm to 13 nm giving rise to GNRs@mSiO4 volume to be 42,000 nm3 (Fig 1b). The relatively large volume of such pours material allows high DOX loading per particle and efficient delivery upon NIR radiation. As shown in Fig. 2, the prepared GNRs have a weak transverse plasmon band at 530 nm and a strong longitudinal plasmon band at 820 nm in agreement with the reported results (Orendorff et al.,
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Fig. 5. Inverted microscope images of control MCF7 cells (a), cells incubated with GNRs@mSiO2-DOX (b), cells incubated with free DOX (c), percentage of cell viability for the MCF-7 cellline (d) and percentage of cell viability forhuman amnion wish cells (e).gr5
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2006). The sample GNRs@mSiO2 showed about 20 nm shifts in its longitudinal peak compared to that of GNRs, and this could be due to the change in the local refractive index of the medium surrounding GNRs. An additional peak at 490 nm appears in the absorption spectrum of GNRs@mSiO2-DOX, which is attributed to the absorbance band of DOX indicating its high incorporation within the silica shell pours. The relatively high amplitude of this band insures the successful doping of the drug, at adequate concentration, 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 negative potential of 22.5 mV confirming the complete removal of CTAB and the formation of firm stable pGNRs@mSiO2 samples (Fig. 3). The DOX responsive release of GNRs@mSiO2-DOX was carried out in PBS at pH values 7.4 and 5.6 at 3 C for a period of 120 h. The DOX release rate was obviously pH dependent that increases at relatively low acidic media (Fig. 4). The marked variations in the release profile at different pH confirm the pH responsiveness of GNRs@mSiO2-DOX. Also it indicates that this formulation could selectively release doxorubicin specifically at the tumor sites. The results of DOX loading and its responsive release motivated us to further investigate the in vitro cellular cytotoxicity for 4 h post MCF-7 cells incubation with pGNRs@mSiO2-DOX followed by 1 h NIR exposure; the morphology of the cells was completely changed and became spherical rather than its normal spindle
267 shape (Fig. 5a and b). The observed dark aggregates in cells treated 268 with pGNRs@mSiO2-DOX revealed the accumulation of the nano269 particles inside MCF-7 cells (Fig. 5b). It seems that treated MCF-7 270 cells suffer high apoptosis rates in agreement with the in vitro 271 cytotoxicity results using WST-1 assay (Fig. 5d). In case of treating 272 the cells with free DOX, a remarkable percentage of them appeared 273 intact with their normal spindle shape (Fig. 5c). The breast cancer 274 cell line MCF-7 treated with pGNRs@mSiO2-DOX showed high 275 cytotoxic effects, four times greater, than that cells treated with 276 free DOX at the three different concentrations used (Fig. 5d). Q11 277 The human amnion wish cells are characterized by its high 278 productivity, easy accessibility, free from contamination with non279 fibroblastoid cells and ethically acceptable source of cells for 280 biomedical applications (Hu et al., 2009). The cytotoxicity of 281 pGNRS@mSiO2-DOX on normal cells and human amnion wish cells 282 were incubated with CTAB coated GNRs, pGNRS@mSiO2-DOX and 283 free DOX for 24 h, and cell viability was then measured. The cells 284 incubated with pGNRS@mSiO2-DOX show cell viability of 87% 285 which is very high compared to CTAB coated GNR 29% and free DOX 35% (Fig. 5e). The results indicated that the normal cells up take of Q12 286 287 pGNRS@mSiO2-DOX suffer a neglected percentage of apoptosis 288 and/or necrosis as long as they are screened from NIR irradiation. 289 The cytotoxicity of CTAB coated GNRs showed a relatively high 290 cytotoxic effect on normal cells even though the cells were not 291 exposed to NIR irradiation. The toxicity of CTAB was obviously high 292 and may lead to lethal side effects; thus any therapeutic 293 formulation containing CTAB as a coating material must be used 294 with great caution. The human amnion wish cells incubated with 295 free DOX also showed a low cell viability percentage which would 296 be attributed to its high toxicity to normal cell. 297 The temperature of Ehrlich tumor tissue was measured as a 298 function of NIR exposure time in order to assess the photo-heat 299 conversion characteristics of the prepared pGNRs@mSiO2-DOX 300 (Fig. 6). An elevation in tissue temperature of about 14 C and 4 C 301 were recorded throughout the time of NIR exposure for group A and C 302 , respectively. The temperature drops to its initial values shortly after 303 switching off laser exposure. These results were similar to our recent 304 work using mesoporous silica gold nanoshells (Elbialy and 305 Mohamed, 2014). The advantage of using the prepared 306 pGNRs@mSiO2-DOX in this combined chemo–photothermal thera307 py is that it keeps its major characteristic of photo-heat conversion 308 upon repeated exposure to NIR radiation. The photo-heat conversion 309 of the sample was tested in vitro using simulated body fluid (Fig. 7). 310 Also the absorption spectra of samples repeatedly exposed to NIR
Fig. 7. The change in temperature of pGNRs@mSiO2 suspended in a simulated body fluid throughout NIR irradiation.
Fig. 8. The absorption spectra of pGNRs@mSiO2 before and after repeated NIR irradiation.
Fig. 6. The change in the Ehrlich tumor interstitial temperature during NIR laser irradiation for group A, group C. 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 Q10 264 265 266
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Fig. 9. The average changes in the Ehrlich tumor volume as a function of time for the treatment groups A, B, C, and D. 311 312 313 314 315 316 317 318 319 320 321 322 323 324
radiation suffer no changes (Fig. 8). According to the obtained data, pGNRs@mSiO2-DOX can keep its rod structure as well as its physical identity after exposure to the excessive local heat generated upon NIR radiation. It is important to note that a few micrograms of GNRs can deliver such a quantity of heat, to about 1 g of cancerous tissue that rises its temperature to 10 C throughout NIR exposure time. The change in temperature of GNRs times its specific heat would be surprisingly very high depending on GNRs/tissue mass ratio and other minor insulating conditions. The volume change of the implanted Ehrlich tumor in the right flank of the mice of the four groups A, B, C and D were measured over a period of 18 days (Fig. 9). It is clear from the figure that treated group A showed a pronounced inhibition of tumor volume growth for a period of 4–5 days (compared to group D, p < 0.0001)
followed by a very slow growth rate up to 18 days. The results showed that the cancerous cells suffer a high necrotic percentage, and the remaining viable tumor cells have shown such a slow growth up to the end of the measuring period. Group B, treated with DOX alone, showed a similar volume inhibition behavior as that of group A followed by a much faster growth after 3 days compared to group A. Because of the natural high growth rate of Ehrlich tumor, untreated group D showed an enhanced tumor volume growth rates of about 10% per day. While group C, which was 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 histopathological examination (Fig. 10). The negative control, group D, showed a normal necrosis percentage of focal and diffuse necrosis. The former appears as scattered necro-apoptotic bodies within the groups of viable cells while the latter appears as islands of coagulative necrosis (geographic distribution) showing the ghosts of the cells. The hemorrhagic necrosis was also observed (lower Rt, “encircled" (Fig. 10a)). While the positive control, group C, showed a diffuse cellular affection and geographic appearance in addition to necrotic regions (Fig. 10b). This mild cell coagulative necrosis could be attributed to a deep penetrative power of the NIR laser beneath the skin. The treated group B showed a remarkable amount of necrotic fields (encircled) with the appearance of many apoptotic and karyorrhechtic bodies (Fig. 10c). The treated group A showed an area of total tumor necrosis with the appearance of nuclear debris. Also some tumor cells appeared with apoptotic bodies, pointed at by arrows (Fig. 10d). It is clear that treated group A suffers the most of necrosis as well as apoptosis compared to the controls and even more than treated group B with DOX alone. Consistently, histopathological examinations have confirmed the observed inhibition of tumor growth rate of the treated groups compared to the controls.
Fig. 10. Sections of Ehrlich tumor cells excised from group D (a), C (b), B (c) and A (d) tumor tissues stained with H&E.
Please cite this article in press as: Monem, A.S., et al., Mesoporous silica coated gold nanorods loaded doxorubicin for combined chemo– photothermal therapy, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.067
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So far we have characterized a new formulation of nanoparticles pGNRs@mSiO2-DOX suitable for a combined chemo– photothermal treatment of cancer cells and tumors. It has an adequate drug loading capacity and a responsive drug release in tumor sites. They have been found to have a high effective photoheat conversion and may possess a supper specific heat capacity characteristics. The samples also tested for a repeated NIR exposure for 1 h and showed no changes in its physical properties, its absorption spectra and photo-heat conversion. These results confirm its stability against excessive heat produced during NIR exposure and its ability to deliver that heat for a repeated exposure within the tumor tissue. The pGNRs@SiO4-DOX used in this work has shown a great therapeutic efficacy using only a single chemotherapeutic dose protocol followed by a single exposure to NIR irradiation. Although they have the ability for a repeated photo-thermal ablation, its single irradiation protocol results in effective damage to tumor cells both in vivo and in vitro. The tumor cell membrane permeation and retention allows its accumulation in tumor cells at a suitable mass concentration as well as its responsive The DOX-release effectively enhances its therapeutic efficacy. These characteristics gave them the priority over other delivery systems such as gold nanoshells @SiO4 loaded DOX. These gold shells may be debrided post NIR irradiation (Elbialy et al., 2014; Lee et al., 2013).
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The preparation method used in this work was found to reproduce pGNRs@mSiO2-DOX capable to incorporate chemotherapeutic drug, DOX, and have a high quality repeated photo-heat conversion properties. The size and shape of the nanoparticles were determined using TEM. Its absorption spectra were also determined to identify its effective photo-heat conversion band. The samples zeta potentials were determined which were found to be an important factor for both DOX loading and its pH responsive release. The therapeutic efficacy of pGNRs@mSiO2-DOX was tested both in vitro and in vivo. The formulation used in this work was found to possess a very low toxicity effects to normal tissue and can target its full damage to cancerous tissues. This study demonstrated that the combined chemo–photothermal therapy accomplished by pGNRs@mSiO2-DOX has an enhanced potential to kill cancer cells compared to both photothermal therapy and chemotherapy alone.
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Acknowledgments
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Authors gratefully acknowledge Dr. Tark El-Bolkini, and Veterinary Heba M., Cancer National Institute, Cairo University, for his help in examining Ehrlich tumor tissue and animal care and treatment. We also like to thank Prof. Dr. M. Aman, Faculty of Science, Ein Shams University for the TEM imaging.
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