Journal of Photochemistry and Photobiology B: Biology 130 (2014) 40–46

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Gold nanorods-bombesin conjugate as a potential targeted imaging agent for detection of breast cancer Zahra Heidari a, Reyhaneh Sariri a,⇑, Mojtaba Salouti b,⇑ a b

Department of Biology, University of Guilan, Rasht, Iran Biology Research Center, Zanjan Branch, Islamic Azad University, Zanjan, Iran

a r t i c l e

i n f o

Article history: Received 20 July 2013 Received in revised form 26 October 2013 Accepted 31 October 2013 Available online 15 November 2013 Keywords: Breast cancer Photoacoustic imaging agent Targeting GRP receptor Bombesin

a b s t r a c t Photoacoustic imaging (PAI) is a hybrid biomedical imaging modality that offers both strong optical absorption contrast and high ultrasonic resolution. PAI is capable of in vivo molecular imaging, thus facilitating further molecular and cellular characterization of cancer. In this study, Gold nanorods (GNRs) were synthesized and coated with polyethyleneglycol (PEG). Then, the PEG-GNRs were conjugated with bombesin (BBN), a cancer seeking peptide, for production of a potential photoacoustic targeting imaging agent for detection of breast cancer. The optical property, biocompatibility, stability and in vitro/in vivo binding affinities of GNR-PEG-BBN for breast cancer cells were investigated. UV–vis spectroscopy confirmed the conjugation of bombesin with PEG-coated GNRs. The stability assessment proved high optical stability of GNR-PEG-BBN in human blood serum up to 12 h. Cytotoxicity study showed biocompatibility of GNR–PEG–BBN conjugate. Molecular targeting ability was approved in cells over expressing gastrinreleasing peptide (GRP) receptor (breast cancer cell line) in comparison with cells that do not express GRP receptor (skin fibroblast cell line). The selective accumulation of GNR-PEG-BBN was demonstrated in breast tumor in comparison with unconjugated gold nanorods, following the intravenous administration of GNR-PEG-BBN to breast tumor-bearing mice. This study demonstrated the potential of GNR-PEG-BBN as a photoacoustic imaging agent that can provide improved specificity and sensitivity for breast cancer detection. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Breast cancer has become one of the leading causes of death in women today [1]. Many researches are ongoing to develop more effective imaging methods for early diagnosis of breast cancer [2]. Molecular imaging refers to remote sensing the characteristics of biological process and interactions at the molecular level [2–4]. It has a great potential for early detection, because aberrations at the cellular and molecular levels occur much earlier than anatomic changes [4]. However, high resolution molecular imaging has been limited to relatively shallow penetration depth that can be accessed with microscopy [5]. The need for an imaging technique that can provide high optical contrast image in microscale resolution and at a reasonable penetration depth has now been filled with photoacoustic imaging (PAI) [5–7]. In this technique, non-ionizing laser pulses are delivered into the biological tissues. Some ⇑ Corresponding authors. Address: Namjoo St., Department of Biology, University of Guilan, Rasht, Iran. Tel./fax: +98 131 3233647, mobile: +98 9111325824 (R. Sariri). Address: Moallem Street, Etemadieh, Zanjan Branch, Islamic Azad University, Iran. Tel./fax: +98 241 4224024, mobile: +98 9121454954 (M. Salouti). E-mail addresses: [email protected] (R. Sariri), [email protected] (M. Salouti). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.10.019

delivered energy is absorbed and converted into heat, leading to transient thermoelastic expansion and thus wideband ultrasonic emission [5–7]. The generated ultrasonic waves are then detected by ultrasonic transducers to form the image [5–7]. The recent studies are focused on utilizing gold nanorods (GNRs) as a contrast agent to enhance the image contrast in PAI [7–9]. GNRs can absorb light about one thousand times more strongly than an equivalent volume of an organic dye and their plasmon resonance absorption and scatter in the near infrared region makes them suitable for in vivo imaging applications [7–9]. Jokerst and co-workers reported (2012) that gold nanorods, under in vivo conditions, exhibit significant photoacoustic contrast and increase the diagnostic power of photoacoustic imaging modality [9]. Advances in the field of molecular biology (i.e., Discovery of various disease specific biomarkers) have prompted the development of site-specific contrast agents (active targeting) that can provide molecular information in the context of a high resolution anatomical map of the body [4,10]. Active targeting takes advantage of the fact that cancer cells over-express certain receptors on the cell surface [4]. Many active targeting strategies use the enhanced permeability and retention (EPR) effect, so that active and passive targeting mechanisms act synergistically that lead to

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higher concentration of nanostructures in the cancer region than that in healthy tissues [4,10]. Motamedi and co-workers showed that Herceptin-gold nanorods conjugate, under in vivo conditions, exhibit significant optoacoustic contrast and increase the diagnostic power of optoacoustic imaging modality [11]. The nature of peptide itself makes it an attractive compound to act as a bullet targeted at the corresponding peptide receptors. Peptides used for tumor targeting, show some advantages over antibodies: peptides are small, high affinity and show rapid diffusion into (target) tissues resulting in rapid pharmacokinetics. The fast blood clearance could lead to a high tumor to background ratio shortly after administration [12,13]. In addition, the peptides can tolerate harsh chemical conditions and are easy to purify and modify peptide receptors [12,13]. Bombesin (BBN) and its human counterpart gastrin-releasing peptide (GRP) belong to a family of brain-gut peptides, has shown, in addition to the physiological effects, to play an important role in cancer growth [13]. Peptides GRP receptors are over expressed in many cancers including breast, prostate and small cell lung cancer [12–17]. The low expression of BBN/GRP receptors in normal tissues and relatively high expression in a variety of human tumors can, therefore, be of biological importance and form a molecular basis for BBN/GRP receptor mediated PAI imaging [12–17]. In this study, we used a BBN analog derived from the universal binding sequence (KGGCDFQWAV-bAla-HF-NIe), which was known to bind to all four receptor subtypes [13]. This analog was previously used only in a nuclear medicine study and there is not any report about its using in a nano-medicine study [13]. So, the objective of present study was the conjugation of bombesin with PEG-coated GNRs as a specific GRP receptor contrast agent for subsequent application in photoacoustic imaging. 2. Experimental 2.1. Material Chloroauric acid (HAuCl4.3H2O), cetyltrimethylammonium bromide (CTAB), ascorbic acid, fetal bovine serum (FBS), RPMI 1640, trypan blue, streptomycin, penicillin and trypsin–EDTA, analytical grade nitric and hydrocholoric acid were purchased from Sigma (St. Louis, MO, USA). Sodium borohydride (NaBH4), silver nitrate (AgNO3) and dithiothreitol (DTT) were purchased from Merck, Germany. Thiol-PEG-carboxylate-5000 (COOH-PEG-SH) was purchased from Nanocs, USA. The human breast cancer cell line (T47D) and the human skin cancer cell line (E8 (AG01522) were purchased from Pasteur Institute, Tehran, Iran. Bombesin (BBN) peptide synthesis was ordered to GenScript Company (USA). 2.2. Synthesis of gold nanorods GNRs with a peak absorption wavelength of 780 nm were prepared via the seeded-growth mechanism previously described by Eghtedari and co workers [5,11]. Briefly, gold seed was prepared by the addition of 7.5 ml of 0.1 M CTAB to 0.25 ml of 0.01 M gold chloride. Then, 0.6 ml of 0.01 M NaBH4 (prechilled for 10 min in an ice water bath) was quickly added and the mixture was shaken for 2 min. The growth mixture was prepared with the following: 4.7 ml of 0.1 M CTAB, 0.2 ml of 0.01 M gold chloride and 45 ll 0.01 M AgNO3. The solution was yellow/brown, but became translucent upon the addition of 32 ll 0.1 M ascorbic acid. Then, the seed solution (21 ll) was added. The solution color was changed to purple/brownish over 60 min. The size and morphology of GNRs were analyzed by transmission electron microscopy (TEM) using a Hitachi HF-2000 field emission high-resolution TEM operating at 200 kV. The particles were then characterized for zeta-potential

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and UV–vis light absorption. Measurements were made in triplicate for each sample. 2.3. Biofunctionalization of gold nanorods Five ml of CTAB-stabilized GNRs was centrifuged twice in a 1.5 ml eppendorf tube at 10,000 rpm for 15 min and resuspended in 5 ml of deionized water [11]. An amount of DTT was added in the solution with molar ratio DTT/HS-PEG-COOH about 1/1 [18,19]. The solution DTT/HS-PEG-COOH then was added into 5 ml of GNRs and the mixture was incubated at room temperature for 24 h and dialyzed using cellulose ester membrane dialysis (Spectrapor) for 12 h against PBS buffer [11–19]. The final solution was again centrifuged at 5500 rpm for 15 min to remove any excess HOOC-PEG-SH [11–19]. Next, bombesin immobilized covalently onto the PEG coated GNRs surfaces [11]. EDC and sulfo-NHS was added to 5 ml of PEG coated GNRs at a final concentration of 4 mM and 1 mM, respectively [11]. The mixture was sonicated for 25 min at 4 °C to produce activated GNRs (GNRs that are capable of binding to the amine side chain of bombesin). Bombesin was added to a final concentration of 500 mg/ml to 5 ml of activated GNRs. The mixture was sonicated at room temperature for 2 h [11]. After successful conjugation, the unbound peptide was separated by centrifugation at 12,000 rpm for 10 min [14]. The final solution was diluted by adding PBS to achieve an optical density of 1.0 at 780 nm. A schematic is given in Fig. 1 to simply illustrate the synthesis process of the functionalized GNRs. 2.4. GNR–PEG–BBN characterization The physical chemistry property of the conjugates was monitored through optical and zeta potential measurements to confirm surface chemistry [11,13,18–20]. UV–vis spectrophotometer (CARY-100 BIO, Varian, USA) was used for investigating the interaction of bombesin with gold nanorods. The spectrum ranged from 200 to 900 nm at the resolution of 1 nm [11,13,18–20]. As a means to confirm the altered nanorod surface chemistry at various stages, zeta potential measurements (Malvern Zetasizer Nano, UK) were performed on gold nanorods in the original CTAB, after stabilization with HOOC-PEG-SH, and finally after bombesin conjugation [14,18]. 2.5. Optical stability of the bioconjugate in human serum To verify the stability of GNR-PEG-BBN in human blood serum, the conjugate was added to 1/1 ratio of human blood serum/PBS (total volume 2 ml, GNR concentration 0.02 lM) [21]. The optical absorbance of solution at 800 nm was monitored at multiple time points for 12 h. The control solution was included human blood serum and PBS only (1/1 ratio) [21]. Subtracting the ‘‘serum’’ absorbance from ‘‘the conjugate in serum’’ absorbance shows GNR-PEG-BBN absorbance. If the conjugate absorbance remained intact around 800 nm, it is indicated that the conjugate is stable in human blood serum [21]. 2.6. Cytotoxicity study Cell viability of T47D cell line (human breast cancer cell line) with GNR-PEG-BBN, GNR-PEG and GNR-CTAB was determined using trypan blue test [11]. T47D cells were harvested and resuspended at 1  104 cells/200 ll in RPMI-1640 medium and cultured in 24-well plate. After 24 h, 100 ll (100 lg/ml) of each sample (GNR-PEG-BBN, GNR-PEG and GNR-CTAB) was added to the wells [11]. 100 ll PBS buffer was added to 6 wells of plate as control too. After 24 h incubation, the living/dead cells were visualized

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Fig. 1. Schematic of GNR–PEG–BBN conjugation. PEG molecules are attached to gold nanorods through their thiol functional group. Then, the bombesin molecules are attached covalently to PEG-coated GNR through amid bound between COOH–PEG and NH2–BBN.

under a bright-field microscope by using trypan blue staining [11]. All experiments were subjected to five repetitive evaluations. 2.7. Binding study This experiment was performed to illustrate that the GRP receptor targeted GNR-PEG-BBN do not bind to irrelevant cells that are not expressing the receptor. The selectivity of GNRPEG-BBN was investigated using T47D cells (human breast cancer cell line) and E8 (AG01522) cells (human skin cancer cell line). T47D cells over express gasterin releasing peptide receptor (GRP-R) and E8 (AG01522) cells do not express the GRP receptor [11,13,22]. Both cell lines were cultivated in RPMI-1640 with 10% (w/v) fetal bovine serum (FBS) and 1% (w/v) penicillin [17,22]. GNR-PEG-BBN (as an active targeting agent) and GNRPEG (as a passive targeting agent) with concentration of 0.02 lM/ml were added to the cell plates separately and the cell incubation process lasted for 1 h at 37°C with 5% CO2. Then, the cell plates were rinsed with PBS twice to remove the unattached conjugate molecules [11,22]. The samples were finally stained using silver enhancement kit (Life Technologies, L-24919, USA) to the visualize conjugate using a Micros Model IX70 microscope equipped with a Lumenera digital camera [11,22]. 2.8. Internalization study To measure the intracellular concentration of GNR-PEG-BBN conjugates, 1  105 T47D cells were overnight in 24-plated and incubated with the GNR-PEG-BBN or GNR-PEG [23]. At various times (1, 2, 4 and 6 h), RPMI medium in wells were removed and the cells, half of wells, were then exposed to 1 ml solution of 1.0 M NaOH and half of wells were exposed to 1 ml solution of acetic acid pH 4.0 [23]. NaOH solution destructed whole of T47D cells and this solution was considered as a whole conjugate (internalized, bound and dissociated) [23]. Acetic acid dissociates interaction between GNR-PEG-BBN and GNR-PEG with GRP receptor in the surface of T47D cells and the conjugate concentration of this solution was considered as a bound and dissociated GNRPEG-BBN and GNR-PEG [23]. Differences between the two concentrations were considered as an internalized GNR-PEG-BBN and GNR-PEG. The solutions were digested in 500 ll of aqua regia (HCl: HNO3 = 3: 1 v/v) at 60°C for 5 h [23]. The concentration of GNRs was determined using atomic absorbance spectroscopy. In addition, one other 24-plate prepared according to above mentioned method and cellular uptake of GNR-PEG-BBN and GNRPEG in T47D cells was imaged by silver enhanced staining too [11,23]. All the experiments were subjected to five repetitive evaluations.

2.9. Biodistribution study Breast tumor was established by subcutaneous implantation of spontaneous tumor fragments (3  3  3 mm) in the left side of abdominal region of normal inbred female BALB/c mice (20–30 g and 6–7 weeks old) [24,25]. Biodistribution studies were performed when the xenograft volume reached 5  5  5 mm. Twenty-five mice with subcutaneous tumor xenograft were injected via tail vein with 200 ll of 0.02 lM GNR-PEG-BBN (active targeting) and sacrificed at 2, 4, 8, 12 and 24 h time points The blood was collected by cardiac puncture and after euthanasia, the liver, spleen, lungs, kidneys, stomach, pancreas, muscle and tumor were collected, weighted and dissolved completely by adding 9 ml of HCl and 3 ml of HNO3 at 70 °C for 30 min [24,25]. The solution was diluted with deionized water and filtered with 0.45 lm Teflon filter [24,25]. The solution was evaporated to be dried, a necessary amount of 0.5 N hydrochloric acid was added and the sample was analyzed for gold by an atomic absorption spectroscopy (AAS) (VARIAN, model AA240FS) [24,25]. All the samples were analyzed by AAS using a standard curve spanning 0–100 lg/L. The uptakes of the GNR-PEG-BBN conjugate in the organs were calculated as a mean percentage of injected doses per gram of organ tissues (%ID/g) [24,25]. For comparison study, twenty-five BALB/c mice with subcutaneous tumor xenograft were injected via tail vein with 200 ll of 0.02 lM GNR-PEG (passive targeting) for analyzing gold concentration according to the above mentioned method. In addition, a similar protocol was performed for determining the biodistribution of GNR-PEG-BBN in normal BALB/c mice. Each sample and time point included five independent repetitions [24,25]. All the animal experiments were approved by the Animal Care Committee of Tarbiat Modares University. 2.10. Statistical methods All the experiments were repeated 3 times for each test and the results were expressed as mean ± SD. Anova test was used to compare the biodistribution data of GNR-PEG-BBN and GNR-PEG in mouse model (p-value < 0.05). 3. Results 3.1. Synthesis and characterization of gold nanorods The optical absorption of the sample was measured using a spectrophotometer. According to the UV–visble absorbance spectrum (Fig. 2), the synthesized GNRs have two SPR peaks: a strong long-wavelength band due to the longitudinal oscillation of electrons around 780 nm and a weak short-wavelength band around

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Fig. 2. UV–vis spectra of GNR–CTAB, GNR–PEG and GNR–PEG–BBN conjugate. The red shifting occurring during each step of the synthesis is further indication that the fabrication of GNRs had been successful. Inset: TEM image of synthesized gold nanorods.

520 nm due to the transverse electronic oscillation [11,26]. The transmission electron microscopy (TEM) image of the gold nanorods with longitudinal plasmon band maximum at 780 nm is shown in Fig. 2. Gold nanorods have diameter and longitudinal size of 10 ± 5 nm and 37 ± 5 nm, respectively. The zeta potential of the GNRs was + 60 mV due to the existence of the cationic surfactant (i.e., CTAB) which was used for capping and stabilizing the GNRs during synthesis [11,20]. 3.2. Characterization of GNR–PEG–BBN Upon surface modification, the optical resonance peak of the GNRs was slightly red-shifted due to a minor change in refractive index on the surface [1,24,26,27]. So, the red shift in the spectrum of GNR-CTAB indicated that the PEG molecules have successfully bound to the GNRs surface (Fig. 2). The UV–vis spectrum of GNRPEG-BBN conjugate showed a red shift of 10 nm in the absorbance peaks between the GNR-PEG and GNR-PEG-BBN conjugate too [1,24,26,27]. Bombesin binding changed the local environment and thus the surface plasmon absorption of the PEG-coated GNR was changed [1,24,26,27]. As a means to confirm the altered nanorod surface chemistry at various stages, zeta potential measurements were performed on gold nanorods in the original CTAB, after stabilization with HOOC-PEGSH, and finally after peptide conjugation [11,14,18,21]. The zeta potential of the GNR-CTAB complex was highly positive (+60 ± 12 mV) due to the presence of the positively charged CTAB molecules [11,14,18,21]. The GNR-PEG and GNR-PEG-BBN complexes solution showed a zeta potential which is slightly negative (-30 mV and 20 mV, respectively) [11,14,18,21]. The results are consistent with the cationic and anionic surface charges, respectively, associated with these three states of nanorods.

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Fig. 3. Optical serum stability of GNR–PEG-BBN. The GNR–PEG–BBN showed a high level of optical stability when exposed to serum.

3.4. Cytotoxicity study Trypan blue dye exclusion test were performed to examine the effect of GNR-PEG-BBN, GNR-PEG and GNR-CTAB on cell cytotoxicity [11]. The dead cells were stained with trypan blue while live cells remained unstained [11]. The number of dead cells in each treatment group was counted after staining cells with trypan blue. GNR-CTAB increased the number of dead cells in vitro while GNRPEG-BBN and GNR-PEG did not increase the number of dead cells under similar conditions (Fig. 4). This means that while CTABcoated GNRs are toxic to the cells, functionalized GNRs had good biocompatibility [11]. 3.5. Binding study When the GNR-PEG-BBN conjugate were incubated with T47D and E8 (AG01522) cells for the same time, the T47D cell surfaces were heavily labeled with gold nanorods due to the specific binding of bombesin to the over expressed GRP receptor on the surface of the carcinoma cells. This specific interaction between the bombesin functionalized nanoparticles and the cells were stained using a silver staining kit to visualize gold nanorods as dark spots under conventional bright field microscopy [11,21]. The results indicated that highly sensitive and selective binding of cancer cells can be achieved by utilizing the bombesin targeted gold nanorods (Fig. 5A). The receptor specific interaction of GNR-PEG-BBN conjugate provides realistic opportunity in design and development of in vivo molecular imaging for breast cancer. The E8 (AG01522) cells that do not express GRP receptors when incubated with GNRPEG-BBN conjugate do not show any dark spot signal (Fig. 5C) as the bombesin has no receptor to attach. Thus, the negative controls show no binding of gold to cells. The negative controls used in the study was the PEGylated gold nanorods (no peptide bound) incubated with T47D and E8 (AG01522) cells as shown in Fig. 5B and D.

3.3. Optical stability of the bioconjugate in human blood serum 3.6. Internalization study Over the course of 12 h, the absorbance of serum (Fig. 3, blue1 curve) and serum plus the conjugate (Fig. 3, red curve) were monitored. The blue and red curves show a slight increase of absorbance over time, an effect which is likely due to the evaporation of water from the samples, thereby increasing the concentration of serum in the sample [20]. The green curve represents the subtraction of ‘‘serum’’ from ‘‘GNR-PEG-BBN’’ that is a stable and consistent absorbance curve. This result indicated the GNR-PEG-BBN had a high level of optical stability when exposed to serum [20]. 1 For interpretation of color in Fig. 3, the reader is referred to the web version of this article.

The cellular uptake of GNR-PEG-BBN complex was estimated by measuring GNRs concentration within T47D cells using atomic absorption spectroscopy [11,14,21,22]. It could be seen that, GNR-PEG-BBN complex exhibited time-dependent taken-up by T47D cells. The results showed that 50% of GNR-PEG-BBN was internalized by the cells in comparison with 10% of GNR-PEG up to 6 h (Fig. 6A). Furthermore, after being decorated with bombesin, GNR concentration increased significantly in relation to GNR–PEG in T47D cells implied that peptide–receptor-mediated endocytosis was helpful for cellular uptake of functionalized gold nanorods [11,14,21,22]. Light microscopy studies also showed high and

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Fig. 4. Viability of T47D cells after 24 h incubation with GNR–PEG–BBN (A), GNR–PEG (B), GNR–CTAB (C) and PBS buffer (D). Cancer cells were stained with a live/dead cell viability assay to visualize live (unstained) and dead (blue stained) cells, respectively.

Fig. 5. Selective binding of GNR–PEG–BBN to T47D cells. T47D and E8 (AG01522) cells were seeded onto a 96-well plate and incubated with GNR–PEG–BBN and GNR–PEG for 30 min at 37 °C. Silver staining was done to enable visualization during bright field microscopy to study the fate of the gold nanorods in their interaction with the T47D and E8 (AG01522) cells. (A) T47D (breast cancer with overexpression of GRP receptor) incubated with GNR–PEG–BBN (A) and GNR–PEG (B); E8 (AG01522) cells (skin cancer cell, no GRP receptor expression) incubated with GNR–PEG–BBN (C) and GNR–PEG (D).

time-dependent taken-up GNR-PEG-BBN by T47D cells in comparison with GNR-PEG (Fig. 6B).

compared with tumor tissue (p value < 0.05) [13–17,23,25,28]. As shown in Fig. 7 A, the amount of conjugate in the liver and kidney was decreased in a time-dependent manner. Furthermore, GNR-BBN-PEG could be eliminated by kidney and liver in the experimental model which may reduce the unpredictable side effect for the long term treatment [13–17,23,25,28]. To examine the role of the GNR-BBN-PEG conjugate as an active targeting agent, GNR-PEG as a passive targeting agent, were injected in breast-tumor-bearing mice. For the passive targeting group, small PEG-GNR were observed to be accumulated in tumor sites, indicating PEG-GNRs can preferentially accumulate in the tumor tissues due to the EPR (enhanced permeability and retention) effects (Fig. 7B). However, the amount of PEG-GNR in tumor locations was less than active targeting group (p value < 0.05), which fully suggests that the prepared GNR-BBN-PEG conjugate has ability to target tumor tissues [13–17,23,25,28]. The biodistribution of GNR-PEG-BBN conjugate was calculated in normal BALB/c mice at 2, 4, 8, 12 and 24 h after IV administration expressed as percentage of injected dose per gram of each organ (%ID/g). The predominant uptake of GNR-PEG-BBN conjugate was observed in GRP-receptor–expressing pancreatic acini [34% injected dose/gram (ID/g) after 8 h]. A recent study has quantified that more than 300  103 bombesin receptor sites per cell are available in mouse pancreas [13,14]. The selective uptake of GNR-PEG-BBN within GRP receptors in pancreas are consistent with the GRP-receptor uptake and pharmacokinetics data previously reported for a number of bombesin analogs labeled with 99m-Tc in mouse model [13,14]. 4. Discussion

3.7. Biodistribution study Fig. 7A shows the biodistribution of GNR-PEG-BBN conjugate in BALB/c mice bearing breast cancer at 2, 4, 8, 12 and 24 h after IV administration expressed as percentage of injected dose per gram of each organ (%ID/g). Biodistribution study revealed the specific localization of GNR-BBN-PEG at the site of tumors [13–17, 23,25,28]. The uptake of GNR-BBN-PEG in the tumor tissue increased gradually as time increased, reaching a maximum at 8 h. The accumulation of GNR-BBN-PEG was higher in pancreas than in tumors due to higher GRP-receptor density in the pancreas as

The development of ligand-targeting contrast agents has resulted in the ability to image molecular distributions in vivo with enhanced contrast, potentially improving the ability of early cancer detection [29,30]. In this study, the synthesis of a GNR-PEG-BBN conjugate and it’s in vitro and in vivo properties was performed. This study takes the advantage of strong near-infrared absorption (peak at 780 nm) of GNRs and high cellular uptake of nanoparticles results in their conjugation with bombesin. We used a BBN analog derived from the universal binding sequence [13]. This analog has several novelties such as [12–17]: (a) with using of this

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Fig. 6. (A) Quantification of gold nanorod uptake by T47D cells using AAS. The results showed that, within 6 h, the intracellular uptake of GNR–BBN–PEG conjugate increased significantly in comparison with GNR–PEG. Data are representative of five independent experiments. Error bars represent standard deviation. (B) Bright field microscopic images of T47D cells that were incubated with GNR–PEG–BBN (top row) and GNR–PEG (bottom row). Samples were silver-stained to reveal GNR as dark spots. It could be seen that, GNR–PEG–BBN complex exhibited time-dependent taken-up by T47D cells.

Fig. 7. Biodistribution data of GNR–BBN–PEG (A) and PEG–GNR (B) in breast tumor bearing BALB/c mice at different time intervals measured by atomic adsorption spectroscopy (n = 5).

analog, some of the problems related to the receptor heterogeneity in tumors may be overcome. (b) Kidney retentation is an important factor when considering the development of a therapeutic or an imaging agent. This analog has a hydrophilic and negatively charged aspartic acid residue to enhance renal clearance of the produced conjugate. First, gold nanorods were synthesized according to the seed mediated protocol [11]. Formation of the gold nanorods and its size were characterized by UV–vis spectroscopy and transmission electron microscopy. Although many fruitful features of GNRs have been introduced so far, the GNRs itself is believed to show strong cytotoxicity; since it has been synthesized in the presence of CTAB [31,32]. The cationic surfactant is also known to play important role in the stabilization of GNRs and maintenance of rod morphology [31,32]. Herein, CTAB was removed by two round of centrifugation at 10,000 rpm for 15 min and replaced with polyethyleneglycol [11]. Covalent conjugation of gold nanorods to bombesin without PEGylation has been reported in the literature [14,17]. PEGylation of nanoparticles has several advantages. PEG can inhibit both aggregation and the adsorption of blood serum proteins on its sur-

face, reduce the uptake by liver and consequently extend the circulation time, in that more GNRs can have a chance to accumulate in the tumor [33]. PEGylation of nanoparticles also helps in providing a stealth character when administered in the blood stream of living organisms that allows them to evade the immune response and so that they do not interact with cells of the reticuloendothelial system (RES) [33]. Green and co workers demonstrated that the binding efficiency of the antibodies conjugated to the PEGylated GNRs is comparable to the binding efficiency of the unmodified antibodies and 33.9% greater than PEGylated antibody-GNR conjugates [11]. In the current work, to improve the targeting efficiency of conjugate to breast cancer cells, we modify conjugating procedure of gold nanorods with bombesin according the reported protocol by Green and co workers [34]. So, gold nanorods (GNRs) were PEGylated firstly and then GNR-PEG was conjugated with bombesin peptide [34]. It has been shown that the C-terminal amino acid sequence BBN is necessary for retaining receptor binding affinity and preserving the biological activity of BBN-like peptide [12–17]. Hence, the Nterminal region of the peptide used for conjugation to PEG-coated GNR [12–17]. This interaction confirmed by UV–vis spectroscopy. The plasmon resonance is kept in the near infrared area, and changes from strong positive charge for GNR-CTAB to slightly negative for GNR-PEG-BBN conjugates are observed. The results were also compatible with the literature results [11,14,18,21,24,26,27]. These results (UV–vis spectroscopy and zeta potential measurements) suggested that this complex is a non precipitated stable complex. Furthermore in order to check the characteristics of new contrast agent, the stability of conjugate in human blood serum, its biocompatibility and its binding ability to T47D cells were studied. The results of serum stability of conjugate indicated that PEG molecules on the surface of the GNR-PEG-BBN were enough to prevent interaction between serum proteins and the GNRs, which is particularly important for achieving lengthy blood-circulation of the GNRs in vivo [20]. So, GNR-PEG-BBN conjugate have optimum kinetic stability for use in subsequent receptor-targeting applications under in vitro profiles. Biocompatibility studies are necessary to determine if the use of the nanoparticles is safe for medical applications. The particles biocompatibility was tested using tryban blue test [11]. After 24 h incubation with GNR-PEG-BBN, the results showed no significant difference in cellular viability [11]. Nonspecific binding to nontargeting cells can diminish the effectiveness of targeting molecules [11,21]. In vitro study indicated the highly selective binding of targeted contrast agent towards breast cancer cells. It is important to note here that GNR-PEG-BBN conjugate has a negative zeta-potential of -20 mV [14]. As a negative zeta potential candidate, GNR-PEG-BBN conjugate is expected to have very minimal or no interaction with negatively charged cell surface

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[14]. In addition, GNR-PEG-BBN conjugate does not have any positively charged ions on the surface, thus possesses no ability to trigger charge-mediated endocytosis [14]. These results indirectly suggest that internalization of GNR-PEG-BBN conjugate would be possible only by means of specific internalization events [14]. This behavior is also expected because the bombesin sequence chosen in the present study has agonist properties, which means they have specific receptor (in this case, GRP receptor) triggering characteristics to internalize within the cells [14]. The results of biodistribution study after the injection of GNR-PEG-BBN conjugate (high uptake) and GNR-PEG conjugate (low uptake) approved the targeting ability of GNR-PEG-BBN conjugate to breast cancer cells [14]. Recently, Chanda and co-workers have demonstrated the feasibility of using 198AuNP–BBN as a contrast agent in CT imaging [14]. Our results clearly demonstrated that the biodistribution of GNRBBN-PEG follows a similar trend with 198AuNP–BBN-3 in his study [14,17]. In addition, according to the available information in the literature on the bombesin-like peptides, labeled with radionuclides, indicate that the accumulation of peptide is always higher in pancreas than in tumors due to the higher GRP receptors in the pancreas as compared with that of tumor tissue [13,14,17]. Okarvi and co workers (2010) demonstrated that the introduction of a hydrophilic spacer (aspartic acid) considerably improved the renal excretion of the pe [13]. According to the Fig. 7A and B, the higher uptake of GNR-BBN-PEG in kidney in comparison with GNR-PEG suggests that introduction of aspartic acid considerably improves the renal excretion of the conjugate. These results provide unequivocal evidence that the significant uptake of GNR-PEG-BBN (Fig. 7A) in pancreatic acini and within the tumors in breast tumor bearing BALB/c mice are indeed mediated through GRP receptors, and therefore GNR-PEG-BBN is a reliable vector for targeting GRP receptors that are over expressed in a host of cancer types including prostate cancer, HER + breast cancer and small-cell lung carcinoma. 5. Conclusion The findings demonstrated that GNR-PEG-BBN conjugate has some favorable properties, which may make it an attractive candidate for photoacoustic imaging of breast cancer. References [1] R.J. Lee, A.C. Armstrong, A.M. Wardley, Emerging targeted combinations in the management of breast cancer, Breast Cancer: Targets Ther. 5 (2013) 61–72. [2] H.S. Choi, W. Liu, F. Liu, K. Nasr, P. Misra, M.G. Bawendi, J.V. Frangion, Design considerations for tumor-targeted nanoparticles, Nat Nanotechnol 5 (1) (2010) 42–47. [3] C. Kim, E.C. Cho, J. Chen, K.H. Song, L. Au, C. Favazza, Q. Zhang, C.M. Cobley, F. Gao, Y. Xia, L.V. Wang, In vivo molecular photoacoustic tomography of melanomas targeted by bio-conjugated gold nanocages, ACS Nano 4 (8) (2010) 4559–4564. [4] P.F. Jiao, H.Y. Zhou, L.X. Chen, B. Yan, Cancer-targeting multifunctionalized gold nanoparticles in imaging and therapy, Curr. Med. Chem 18 (2011) 2086–2102. [5] M. Eghtedari, A. Oraevsky, J.A. Copland, N.A. Kotov, A. Conjusteau, M. Motamedi, High sensitivity of in vivo detection of gold nanorods using a laser optoacoustic imaging system, Nano Lett. 7 (7) (2007) 1914–1918. [6] J. Hu, M. Yu, F. Ye, D. Xing, In vivo photoacoustic imaging of osteosarcoma in a rat model, JBO Lett. 16 (2) (2011) 020503-1–020503-3. [7] S. Yang, F. Ye, D. Xing, Intracellular label-free gold nanorods imaging with photoacoustic microscopy, Opt. Express 9 (2010) 10370–10376. [8] F. Cai, J. Qian, L. Jiang, S. Hea, Multifunctional optical imaging using dye-coated gold nanorods in a turbid medium, J. Biomed. Opt. 16 (1) (2011). 0160021-8. [9] J.V. Jokerst, A.J. Cole, D.V. Sompel, S.S. Gambhir, Gold nanorods for ovarian cancer detection with photoacoustic imaging and resection guidance via raman imaging in living mice, ACS Nano 6 (11) (2012) 10366–10377. [10] H. Ju, R.A. Roy, T.W. Murray, Gold nanoparticle targeted photoacoustic cavitation for potential deep tissue imaging and therapy, Biomed. Opt. Express 4 (1) (2013) 1–11.

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Gold nanorods-bombesin conjugate as a potential targeted imaging agent for detection of breast cancer.

Photoacoustic imaging (PAI) is a hybrid biomedical imaging modality that offers both strong optical absorption contrast and high ultrasonic resolution...
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