Nuclear Medicine and Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Nuclear Medicine and Biology journal homepage: www.elsevier.com/locate/nucmedbio
Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles Hamidreza Zolata a, Fereydoun Abbasi Davani b,⁎, Hossein Afarideh a a b
AmirKabir University of Technology, Energy Engineering and Physics Faculty Shahid Beheshti university of Tehran, Radiation Application Department
a r t i c l e
i n f o
Article history: Received 23 July 2014 Received in revised form 19 September 2014 Accepted 24 September 2014 Available online xxxx Keywords: Superparamagnetic iron oxide nanoparticles Doxorubicin Trastuzumab Indium-111 SPECT/MR imaging
a b s t r a c t Indium-111 labeled, Trastuzumab-Doxorubicin Conjugated, and APTES-PEG coated magnetic nanoparticles were designed for tumor targeting, drug delivery, controlled drug release, and dual-modal tumor imaging. Superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by thermal decomposition method to obtain narrow size particles. To increase SPIONs circulation time in blood and decrease its cytotoxicity in healthy tissues, SPIONs surface was modified with 3-Aminopropyltriethoxy Silane (APTES) and then were functionalized with N-Hydroxysuccinimide (NHS) ester of Polyethylene Glycol Maleimide (NHS-PEG-Mal) to conjugate with thiolated 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid (PCTA) bifunctional chelator (BFC) and Trastuzumab antibody. In order to tumor SPECT/MR imaging, SPIONs were labeled with Indium-111 (T1/2 = 2.80d). NHS ester of monoethyl malonate (MEM-NHS) was used for conjugation of Doxorubicin (DOX) chemotherapeutic agent onto SPIONs surface. Mono-Ethyl Malonate allows DOX molecules to be attached to SPIONs via pH-sensitive hydrazone bonds which lead to controlled drug release in tumor region. Active and passive tumor targeting were achieved through incorporated anti-HER2 (Trastuzumab) antibody and EPR effect of solid tumors for nanoparticles respectively. In addition to in vitro assessments of modified SPIONs in SKBR3 cell lines, their theranostic effects were evaluated in HER2 + breast tumor bearing BALB/c mice via biodistribution study, dual-modal molecular imaging and tumor diameter measurements. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Nanotechnology takes advantage of the special properties of various materials when they are in the scale of a few nanometers. Since most of potential therapeutics has poor pharmacokinetics and biopharmaceutical properties, there is a need to develop suitable drug delivery systems that distribute the therapeutically active drug molecule only to the site of action, without affecting healthy organs and tissues [1,2]. Superparamagnetic iron oxide nanoparticles (SPIONs) with appropriate surface modification have been widely used for numerous in vivo applications, such as magnetic resonance imaging (MRI) contrast enhancement, immunoassay, and drug delivery [3–7]. These areas include delivery by avoiding the reticuloendothelial system (RES), utilizing the enhanced permeability and retention effect (EPR) and tumor targeting. Nanoparticles will usually be taken up by the liver, spleen and other parts of the RES depending on their surface characteristics. Particles with more hydrophobic surfaces will preferentially be taken up by the liver, followed by the spleen and lungs. Particles with longer circulation times, and hence greater ability to target to the site of interest, should be 100 nm or less in diameter and have a hydrophilic surface in order to reduce clearance
⁎ Corresponding author. E-mail addresses: [email protected] (H. Zolata), [email protected] (F. Abbasi Davani).
by macro-phages [8]. Tumor vessels are known to have large fenestrations which result in preferential accumulation of nanomaterials within tumors [9]. One well-known strategy to reduce proteins binding to nanoparticles is attaching PEG chains; this is also thought to prevent NP uptake by cells of the RES [10–13]. Most conventional chemotherapeutic agents, for example, doxorubicin, cisplatin or paclitaxel, elicit mitochondrial permeabilization in an indirect fashion by triggering perturbations of intermediate metabolism or by increasing the concentration of proapoptotic second messengers [14]. Design and synthesis of non-targeted or antibody targeted biodegradable PEG coupled with doxorubicin attached through acidsensitive hydrazone bond was reported [15,16]. Targeted drug therapy, potentially combined with simultaneous imaging modalities to monitor the delivery of drugs to specific tissues. The potential to deliver active chemotherapeutic drugs in the vicinity or directly within specific tumors via receptor mediated pathways, and to image tumors through the use of nanoparticles has been conceptually and experimentally shown for several classes of nanoparticles [10]. Anti-HER2 antibody can induce antitumor responses, and can be used in delivering drugs to HER2-overexressing cancer [17,18]. Furthermore, dual-modality SPECT/MR imaging bioprobes would allow for simultaneous dynamic imaging of structure and function, facilitate noninvasive monitoring of treatment, and directly provide information on pharmacokinetics and metabolism of drugs [19].
http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007 0969-8051/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
2
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx
2. Experiment 2.1. Materials Iron (III) acetylacetonate, benzyl ether (99%), oleylamine (N 70%), 3-aminopropyltriethoxysilane (APTES), mono-ethyl malonate (MEM), and anhydrous hydrazine were provided by Sigma Aldrich, USA. Trastuzumab (Herceptin) from Roche (Germany) and p-SCN-Bn-PCTA from Macrocyclics (USA). All other chemicals were of analytical reagent grade. 2.2. Preparation of bare SPIONs Hydrophobic SPIONPs were prepared by thermal decomposition method to obtain uniform and narrow-sized SPIONs [20–23]. Briefly, 1.4 gr Fe (acac) 3 was dissolved in a mixture of 20 mL benzyl ether and 20 mL oleylamine that was mingled by a magnetic stirrer. The solution was dehydrated at 120 °C for 1 hr and then quickly heated up to 280 °C for 2 h. After cooling, 65 mL ethanol was added to the mixture and precipitated by centrifuge at 6000 rpm. Attained mixture was eluted with ethanol for three times. The product was re-dispersed in 20 ml hexane and stored at 4 °C. 2.3. SPIONs treatment with amino-silane 5 ml of (3-aminopropyl) triethoxysilane (APTES) is placed into an aqueous solution of an acid (pH = 4) that acts as a catalyst [24]. APTES was hydrolyzed, and then a condensation reaction takes place to form a silane polymer. In the hydrolysis reaction, alkoxide groups are replaced by hydroxyl groups to form reactive silanol groups, which condense with other silanol groups to produce siloxane bonds. The solution of silane polymer was added to 30 ml of the SPIONs in a flask. The mixture was stirred for 4 h at 65 °C under argon protection. After cooling, the product was eluted with DI water and ethanol, pursued by drying in vacuum at room temperature [24].
Afterward, Trastuzumab was dissolved at a concentration of 1 mg/mL in phosphate buffer (pH 8.0). Trastuzumab solution (1 mL) was incubated with 40.2 μL (50-fold molar excess) of 2-iminothiolane (Traut's reagent) solution (5.7 mg in 5.0 mL phosphate buffer, pH 8.0) [26,27]. The thiolation reaction was stirred gently for 5 hrs at room temperature. Unreacted Traut's reagent was excluded by centrifugation three times with 30 kDa cutoff centrifugal ultrafilters (Millipore Corporation) at 4000 g and 10 °C for 15 minutes. The amount of thiol groups within the antibody were quantified through disulfide building with 5, 50dithio-bis-2 (nitrobenzoic acid) (Ellman's reagent) which 2-nitro-5thiobenzoic acid (TNB) was released stoichiometrically and could be quantified photometrically at 412 nm [27,28]. 2.7. Thiolated Trastuzumab and PCTA-SH conjugation onto the SPIONs Thiolated Trastuzumab and PCTA conjugation onto the SPIONs was performed by a reaction between the maleimide groups of the PEG conjugated on the surface of SPIONs and the attached SH group of the Trastuzumab and PCTA. The molar ratio for thiolated Trastuzumab: PCTA was set at 2:1, and total molar values of Trastuzumab and PCTA were equal to molar value (0.0042 mM) of PEG-Mal. Thiolated trastuzumab (0.0028 mM) and PCTA-SH solution (0.0014 mM) were added into the 10 mL water solution containing 10 mg modified SPIONs at room temperature for 6 h under a N2 atmosphere. 2.8.
111
In-labeling of SPIONs and radiolabeling assessment
111 InCl3 was obtained from Radiation Application School cyclotron (Cyclone-30, IBA). 111InCl3 (74 MBq) was diluted in 400 μL of sodium acetate buffer (pH 6.5, 0.1 M) and added to the PCTA-conjugated SPIONs solution. The mixture was incubated at 40 °C for 90 min followed by testing the radiochemical purity by RTLC (Whatman No. 2) using a RTLC scanner (Bioscan Inc.). For to animal studies, 111In-labeled SPIONs were purified using PD10 column with PBS. Exerted procedures to prepare multifunctional SPIONs are depicted in Fig. 1. Obtained specific activity was 7.18 MBq/mg of SPIONs.
2.4. SPIONs conjugation with Mal-PEG-NHS and MEM-NHS 3. In vitro and in vivo assay Synthesis of MEM-NHS and its conjugation with Mal-PEG-NHS altogether onto SPIONs surface were performed according to previous experiments [21,25]. Briefly, 1.70 mg MEM-NHS and 20 mg Mal-PEGNHS (Mw: 5000) were added to 10 mL DI water containing 10 mg SPIONs under a N2 atmosphere. After 30 min of N2 bubbling, the mixture was allowed to react for 5 h at room temperature. After the reaction was complete, the solution was purified by dialysis against DI water for 2 days. The solution was then filtered through a 0.22 μm membrane followed by freeze-drying. 2.5. Conjugation of the DOX molecules onto the SPIONPs DOX molecules were conjugated onto SPIONs through a two-phase reaction [21]. Briefly, MEM methoxy group was substituted with hydrazide in anhydrous DMSO at 40 °C for 24 h. Obtained SPIONs were purified by dialysis using an ammonia solution (0.25%) for 48 h before freeze-drying. Then DOX (0.006 mM) was bound to hydrazide group of SPIONs (10 mL) through an acid-sensitive hydrazone linkage. The reaction was performed in DMSO solution at room temperature for 24 h with an excess amount of DOX which was removed by dialysis using DI water and then purified by Sephadex gel column. 2.6. Thiolation of Trastuzumab and p-SCN-Bn-PCTA The PCTA-SH was prepared by a reaction between the NCS group of p-SCN-Bn-PCTA and the amino group of 2-aminoethanethiol hydrochloride in the presence of triethanol-amine. The reaction was allowed to stay in DI water at room temperature for 3 h under a N2 atmosphere.
The SKBr3 cell line (1.0 × 10 6cells/well) and mouse fibroblasts (L929, adhesive) (1.0 × 104cells/well) were obtained from the National Cell Bank of Iran (NCBI), Pasteur Institute (Tehran, Iran). Mouse fibroblasts were seeded on glass cover slips in 96 well plates and incubated for 24 h. After the 24 h incubation period, medium containing control cells, bare-SPIONs, APTES and APTES-PEG Coated SPIONs (0.2 mM iron) were added to the wells, and cells were incubated at 37 °C for 1 h. The medium was then extracted, and the cells were allowed to rest. At 24 h post-treatment, the cell viability was assessed by the MTT assay using an ELISA reader (microreader, Hyperion) at 540 nm. In order to in vivo assay of complex, female BALB/c mice (20–25 g, 6–8 weeks old) were subcutaneously injected with 3 × 105 SKBR3 cells (expressing high levels of HER2) in the flank. The organ distribution experiments were performed when the tumor volumes reached 0.5 cm in diameter. For tumor imaging, mice (n = 5) were injected intravenously with Trastuzumab-Functionalized, DOX-Conjugated, Indium111labeled, APTES-PEG Coated SPIONs (100 μL, 3.6 MBq, 20 μgm Ab, and 0.5 mg Fe) via the tail vein. At 12, 24 and 48 h after injection, the animals were anesthetized with ether. Images were recorded using a clinical dual-head coincidence gamma camera (GE, USA). Moreover, a clinical 1.5 T MRI scanner (SIEMENS) was used to measure T2-weighted signal intensities (SI) for in vivo MRI studies. For biodistribution study, animals were sacrificed by ether; their organs were removed and weighed. Amount of incorporated 111In was measured with a HPGe gamma detector (CANBERRA). Specificity of Trastuzumab modified SPIONs was determined through HER2 receptors blocking by Trastuzumab (20 μg) injection. For therapeutic evaluation, solid tumors were allowed to
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx
3
Fig. 1. Exerted procedures to prepare multifunctional SPIONs.
develop over a period of 3 weeks, and animals were randomized into control and treatment groups (n = 3) having no significant difference in tumor volumes or body weights. On day 8 animals in the treatment group received injections of radiolabeled modified SPIONs (3.6 MBq). Tumor volumes were determined each week until on day 28. All animal experiments were performed in compliance with the regulations of our institution.
shown in Fig. 2a–b. Moreover, the magnetization of the SPIONs in a variable magnetic field was measured using a vibrating sample magnetometer (VSM) with a sensitivity of 10 − 3 emu and magnetic field up to 8 kOe. Bare and surface modified SPIONs show magnetic behavior. Obtained magnetization values for bare SPIONs, SPIONs treated by APTES and PEG coated SPIONs were 65 emu/g, 58 emu/g and 52 emu/g respectively as shown in Fig. 2c which indicates their potential as a contrast agent for MR imaging.
4. Results 4.1. SPIONs characterization
4.2. SPIONs radiolabeling and antibody conjugation assay
The particle size and morphology of core and amino-silane treated and PEG coated SPIONs were investigated by TEM (ZEISS Model EM10C) and SEM (FEG LECO). APTES-PEG coated SPIONs with spherical shape and an average diameter of 16 ± 0.27 nm were observed, as
Using RTLC, SPIONs radiolabeling efficiency was measured about 97.6% (± 1.2). The highest number of 0.74 ± 0.52 sulfhydryl groups per trastuzumab was introduced. The amount of antibody was determined by UV spectrometry using Warburg's formula [3,29]. We
Fig. 2. TEM (A) and SEM (B) micrographs of APTES-PEG coated SPIONs, (C) magnetization curves obtained by VSM at room temperature of (a) Fe3O4 MNPs; (b) Fe3O4 MNPs treated by APTES; (c) PEG coated Fe3O4 MNPs.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
4
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx
determined that 63.79 ± 2.60 % of thiolated trastuzumab was conjugated to SPIONs surface via maleimide-thiol coupling reaction. 4.3. Drug release study Amount of released DOX was analyzed photometrically at 485 nm [18]. Fig. 3a shows DOX release profiles of modified SPIONs at different pH values. During 72 h, amount of DOX release was below 10% at pH 7.4 while this value was 44% at pH 4.6. This is a desirable result; because DOX will not be released before reaching tumor region. This pHdependent DOX release is very desirable for targeted cancer therapy. It minimizes the amount of drug release during blood circulation (pH 7.4). Instead, sufficient amount of drug is introduced to cancer cells (pH 4.5 to 6.5).
at very high concentrations at 12 and 24 and 48 h post-injection (Fig. 4a) while non-specific tumor uptake was observed in mice with blocked HER2 receptors (data not shown). Tumor uptake and tumor/ non-tumor ratios (Fig. 4b) show the potential of multifunctional SPIONs for breast tumors bio-probing and imaging. The uptake of the nano carriers in the tumor indicated good tumor-targeting capability. The tumor/muscle ratios for specific and non-specific (data not shown) uptake were 38.40 ± 15.02 and 15.64 ± 7.35 at 48 h post injection respectively which indicated nonspecific uptake due to the enhanced permeation and retention (EPR) effect. A comparison of the biodistribution data of 111In labeled Trastuzumab-conjugated SPIONs in blocked and unblocked tumor bearing mice revealed that uptake of SPIONs was higher in most organs except the tumor, which again indicated the tumor specificity of 111In labeled Trastuzumabconjugated nanocarriers.
4.4. In vitro cell cytotoxicity and specificity analysis 4.6. Tumor imaging In vitro cell cytotoxicity and specificity analysis was performed based on [18] experiment. Fibroblast cell viability of bare, APTES, APTES-PEG, and Trastuzumab conjugated SPIONs is depicted in Fig. 3b at 24 h posttreatment. Results confirmed that APTES and APTES-PEG modified SPIONs are more biocompatible than bare SPIONs. Moreover, SKBr3 cell survival curves for Trastuzumab and Dox conjugated, APTES-PEG Coated SPIONs are shown in Fig. 3c. The relative numbers of SKBr3 cells were reduced to 95.2%, 57.6% and 41.5% for Trastuzumab-free SPIONs, Trastuzumab conjugated SPIONs and Trastuzumab-DOX conjugated SPIONs respectively at 24 h post-treatment.
Tumor region is clearly contrasted in coronal SPECT images of tumor bearing mouse at 4, 24 and 48 h after injection (Fig. 5a). Distinguishable darkening MR images of tumor cells were due to tumor uptake of SPIONs which were specifically delivered to tumor site via decoration with mAb, as shown in Fig. 5b. Imaging after mAb-free SPIONs injection did not lead to darkening in the tumor region as shown in Fig. 5c. Overall, the quantification results obtained from biodistribution studies and SPECT scans matched well, confirming that SPECT scans truly reflected the distribution of 111In labeled Trastuzumab-conjugated Nano-carriers in vivo.
4.5. Biodistribution study
5. Therapeutic efficacy
Biodistribution study revealed that Indium111-labeled TrastuzumabFunctionalized, APTES-PEG Coated SPIONs were accumulated in tumors
Tumor growth in treated mice appeared to be slowing in comparison to controls within one week. After complex administration,
Fig. 3. (A) Dox release profiles of compound at different pH values, (B) cell viability of fibroblast cell for 1. control cells, 2. bare, 3. Aminosilane, 4. APTES-PEG and 5. mAb conjugated SPIONs, (C) relative SKBR3 cell number after treatment with 1. Trastuzumab-free SPIONs, 2. Trastuzumab conjugated SPIONs, and 3. Trastuzumab–Doxorubicin conjugated SPIONs.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx
5
Fig. 4. (A) Biodistribution results at 12, 24 and 48 h post injection. (B) Tumor/Non tumor ratios in tumor bearing mice at 12, 24 and 48 h post injection. Data are presented as mean ± SD (n = 5).
tumor volumes were ∼ 50%, 25 %, 23% lower for treated animals compared with control group at end of each week. This significant therapeutic effect was continued throughout the study (Fig. 6).
Three weeks after injection, tumor volumes for the control mice were fully larger than those in the treated group (0.69 ± 0.06 vs.0.16 ± 0.03 cm 3).
Fig. 5. (A) SPECT, (B) MR images and (C) MRI control images of tumor bearing mice at 12, 24 and 48 h.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
6
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx
6. Discussion We synthesized biocompatible mono-disperse nanoparticles via thermal decomposition method and then modified their surface with APTES-PEG to prevent SPIONs uptake by cells of reticuloendothelial system which lead to increase their presentation time in blood stream. Cytotoxity effect of modified SPIONs in non-tumor cells was relatively low in comparison with bare iron oxide nanoparticles while cell viability of SKBR3 cells were reduced to 95.2%, 57.6% and 41.5% for Trastuzumabfree SPIONs, Trastuzumab conjugated SPIONs and Trastuzumab-DOX conjugated SPIONs respectively. In addition, PEG chains provided suitable platforms for antibody and radiopharmaceutical attachments. In our experiments; all the samples showed a typical superparamagnetic behavior which were indicated in Fig. 2.C. Magnetization property of modified SPIONs which is critical for MR imaging has just been decreased by 10.7% for APTES and 20% for APTES-PEG coated SPIONs in comparison with bare iron oxide nanoparticles. In order to antibody conjugation, Trastuzumab was first thiolated using Traut's reagent and then conjugated to SPIONs via maleimide-thiol coupling reaction. By this method, 63.79 ± 2.60 % of antibody was conjugated onto SPIONs. Previous studies have shown that anti-HER2 conjugation to nanoparticles does not alter their intrinsic specificity and binding affinity to HER2 + cells [30]. SPIONs radiolabeling with 111In radionuclide was performed successfully with yield of 96% using thiolated PCTA bifunctional chelator (BFC). This radiotracer allows highly specific radiolabeling and superior in vivo characteristics with a significantly higher tumor uptake. When receptors were blocked, tumor uptake was decreased from 7.38 ± 1.06, 9.94 ± 10.7 and 12.88 ± 0.76%ID/g to 3.38 ± 0.52 and 5.94 ± 1.02 and 6.12 ± 1.48%ID/g at 12, 24, and 48 h post-injection respectively, indicating the HER2-specific uptake in tumor tissue. In the absence of receptors mediated endocytosis, EPR effect of nanoparticles is a major mechanism of cancer tumor uptake. In comparison with SPIONs-free radiolabeled Trastuzumab (data not shown), tumor uptake was decreased dramatically to 39% of SPIONs conjugated experiments at 48 h post-injection. In addition, clearance from the blood was fast, leading to increasing tumor-to-blood ratios of radioactivity over time (from 1.36 ± 0.20 at 12 h to 8.05 ± 2.0 at 48 h after injection). Nonspecific retention of radioactivity in non-targeted organs and tissues relatively was low. In comparison with previous radiolabeled antibody conjugated SPIONs experiment [18], blood uptake was increased from 1.8 ± 0.15 to 3.1 ± 0.39%ID/g at 24 h after injection due to appropriate nanoparticles surface modifications which lead to SPIONs escape from RES. Results showed high initial blood retention with moderate liver uptake, making them an attractive nano-probe for tumor theranostics. Moreover, pH sensitive DOX release was investigated, and obtained results after 72 h showed that 43% of DOX could be released effectively at tumor pH. This controlled drug release in tumor region helps in effective tumor cells killing and prevents healthy cells homicide. Moreover, enhanced permeability and retention effect of solid tumors for SPIONs and also well decoration of SPIONs with mAb
Fig. 6. Therapeutic evaluation in tumor bearing mice. Data are presented as mean ± SD (n = 3).
leads to effective targeting and drug delivery to tumor site. Simultaneous effects of conjugated radiopharmaceutical, antibody and chemotherapeutic agent to SPIONs lead to impressive tumor suppression as well as effective dual modality MRI/SPECT tumor imaging. SPIONs accumulation in tumor region alters darkening in MR images. During therapeutic assessments, tumor volume was reduced from 0.25 cm 3 to 0.16 cm 3 for treatment group in spite of tumor growth in control group. This means that 36% of tumor volume was decreased during 28 days. Based on our best knowledge this therapeutic efficacy was not achieved in any multifunctional modified SPIONs experiment.
7. Conclusion Specific bio-probes for single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI) have enormous potential for tumor imaging. The bioprobes were developed by Indium111-labeled Trastuzumab-DOX conjugated, APTES-PEG Coated SPIONs. Our experimental results showed that modified SPIONs retain their magnetic properties as well as the ability to specifically localize in HER2 overexpressing tumors. Bio-distribution study revealed that 111In-labeled, TrastuzumabDOX conjugated, APTES treated SPIONs were accumulated at very high concentrations in tumors as a result of EPR effect and HER2 receptor targeting. SPECT and MR imaging indicated that resulting SPIONs could be used for dual-modality imaging. Moreover, promising results of therapeutic evaluation proved theranostic ability of complex. Therapeutic efficacy of SPIONs was increased via circulation time enhancement by appropriate coating and active targeting using Trastuzumab antibody, controlled doxorubicin release in tumor region, and Auger electrons and also gamma rays of 111In radionuclide.
References [1] Loudos G, Kagadis GC, Psimadas D. Current status and future perspectives of in vivo small animal imaging using radiolabeled nanoparticles. Eur J Radiol 2011;78: 287–95. [2] Koo O, Rubinstein I, Onyuksel H. Role of nanotechnology in targeted drug delivery and imaging: a concise review. J Nanomedicine 2005;1(3):193–212. [3] Chen T, Cheng T, Chen C, Hsu SC, et al. Targeted Herceptin–dextran iron oxide nanoparticles for noninvasive imaging of HER2/neu receptors using MRI. J Biol Inorg Chem 2009;14:253–60. [4] Kalambur V, Longmire E, Bischof JC. Cellular level loading and heating of superparamagnetic iron oxide nanoparticles. J Langmuir 2007;23:12329–36. [5] Li Z, Tan B, Allix M, Cooper A, Rosseinsky M. direct coprecipitation route to monodisperse dual-functionalized magnetic iron oxide nanocrystals without size selection. J Small 2008;4:231–9. [6] Schellenberger E, Schnorr J, Reutelingsperger C, et al. Linking proteins with anionic nanoparticles via protamine: ultrasmall protein-coupled probes for magnetic resonance imaging of apoptosis. J Small 2008;4:225–30. [7] Sun C, Veiseh O, Gunn J, Fang C, et al. In vivo MRI detection of gliomas by chlorotoxin-conjugated superparamagnetic nanoprobes. J Small 2008;4:240–9. [8] Brannon-Peppas L, James O. Blanchette. Nanoparticle and targeted systems for cancer therapy. J Adv Drug Deliv Rev 2012;64:206–12. [9] Grobmyer S, Zhou G, Luke G, et al. Nanoparticle delivery for metastatic breast cancer. J Maturitas 2012;73:19–26. [10] Garcia-Bennett A, Nees M, Fadeel B. In search of the holy grail: folate-targeted nanoparticles for cancer therapy. J Biochem Pharmacol 2011;81:976–84. [11] Liu X, Tao H, Yang K, Zhang S, Lee S, Liu Z. Optimization of surface chemistry on single-walled carbon nanotubes for in vivo photothermal ablation of tumors. Biomaterials 2011;32:144–51. [12] Prencipe G, Tabakman S, Welsher K, et al. PEG branched polymer for functionalization of nanomaterials with ultralong blood circulation. J Am Chem Soc 2009;131:4783–7. [13] Liu XW, Tao HQ, Yang K, Zhang S, et al. PEGylated FePt@Fe2O3 core-shell magnetic nanoparticles: potential theranostic applications and in vivo toxicity studies. J Nanomed Nanotechnol Biol Med 2013;9:1077–88. [14] Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. J Oncog 2006;25:4798–811. [15] Rıhova B, Etrych T, Pechar M, et al. Doxorubicin bound to a HPMA copolymer carrier through hydrazone bond is effective also in a cancer cell line with a limited content of lysosomes. J Control Release 2001;74:225–32. [16] Ulbrich K, Etrych T, Chytil P, Pechar M, et al. Polymeric anticancer drugs with pHcontrolled activation. Int J Pharm 2004;277:63–72. [17] Itoa A, Kugaa Y, Hondaa H, Kikkawab H, et al. Magnetite nanoparticle-loaded antiher2 immunoliposomes for combination of antibody therapy with hyperthermia. J Cancer Lett 2004;212(2):167–75.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx [18] Zolata HR, Afarideh H, Abbasi-Davani F. Radio immunoconjugated, Dox-loaded, surface-modified superparamagnetic iron oxide nanoparticles (SPIONs) as a bioprobe for breast cancer tumor theranostics. J Radioanal Nucl Chem 2014;301: 451–60. [19] Misri R, Meier D, Yung A, Kozlowski P, et al. Development and evaluation of a dualmodality (MRI/SPECT) molecular imaging bioprobe. J Nanomed Nanotechnol Biol Med 2012;8:1007–16. [20] Heidari Majd M, Asgari D, Barar J, et al. Tamoxifen loaded folic acid armed PEGylated magnetic nanoparticles for targeted imaging and therapy of cancer. J Colloids Surf B Biointerfaces 2013;106:117–25. [21] Yang X, Hong H, Grailer JJ, et al. RGD-functionalized, DOX-conjugated, and 64Cu-labeled superparamagnetic iron oxide nanoparticles for targeted anticancer drug delivery and SPECT/MR imaging. Biomaterials 2011;32: 4151–60. [22] Xu Z, Shen C, Hou Y, et al. Oleylamine as both reducing agent and stabilizer in a facile synthesis of magnetite nanoparticles. J Chem Mater 2009;21:1778–80. [23] Xie J, Xu C, Xu Z, Hou Y, et al. Linking hydrophilic macromolecules to monodisperse magnetite (Fe3O4) nanoparticles via trichloro- s-triazine. J Chem Mater 2006;18:5401–3.
7
[24] Feng B, Hong RY, Wang LS, Guo L, et al. Synthesis of Fe3O4/APTES/PEG diacid functionalized magnetic nanoparticles for MR imaging. J Colloids Surf A Physicochim Eng Aspects 2008;328(1–3):52–9. [25] Xiao Y, Hong H, Matson VZ, et al. Gold nanorods conjugated with doxorubicin and cRGD for combined anti-cancer drug delivery and PET imaging. J Theranostics 2012;2(8):757–68. [26] Yousefpour P, Atyabi F, Vasheghani-Farahani E, et al. Targeted delivery of doxorubicin-utilizing chitosan nanoparticles surface-functionalized with anti-Her2 trastuzumab. Int J Nanomedicine 2011;6:1977–90. [27] Steinhauser I, Spänkuch B, Strebhardt K, Langer K. Trastuzumab-modified nanoparticles: optimisation of preparation and uptake in cancer cells. J Biomater 2006; 27(28):4975–83. [28] Weber C, Reiss S, Langer K. Preparation of surface modified protein nanoparticles by introduction of sulfhydryl groups. Int J Pharm 2000;211(1–2):67–78. [29] Germershaus O, Merdan T, Bakowsky U. Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting Her2-expressing tumors. Bioconjug Chem 2006;17:1190–9. [30] Debotton N, Zer H, Parnes M. A quantitative evaluation of the molecular binding affinity between a monoclonal antibody conjugated to a nanoparticle and an antigen by surface plasmon resonance. Eur J Pharm Biopharm 2010;74(2):148–56.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
Contents lists available at ScienceDirect
Nuclear Medicine and Biology journal homepage: www.elsevier.com/locate/nucmedbio
Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles Hamidreza Zolata a, Fereydoun Abbasi Davani b,⁎, Hossein Afarideh a a b
AmirKabir University of Technology, Energy Engineering and Physics Faculty Shahid Beheshti university of Tehran, Radiation Application Department
a r t i c l e
i n f o
Article history: Received 23 July 2014 Received in revised form 19 September 2014 Accepted 24 September 2014 Available online xxxx Keywords: Superparamagnetic iron oxide nanoparticles Doxorubicin Trastuzumab Indium-111 SPECT/MR imaging
a b s t r a c t Indium-111 labeled, Trastuzumab-Doxorubicin Conjugated, and APTES-PEG coated magnetic nanoparticles were designed for tumor targeting, drug delivery, controlled drug release, and dual-modal tumor imaging. Superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by thermal decomposition method to obtain narrow size particles. To increase SPIONs circulation time in blood and decrease its cytotoxicity in healthy tissues, SPIONs surface was modified with 3-Aminopropyltriethoxy Silane (APTES) and then were functionalized with N-Hydroxysuccinimide (NHS) ester of Polyethylene Glycol Maleimide (NHS-PEG-Mal) to conjugate with thiolated 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid (PCTA) bifunctional chelator (BFC) and Trastuzumab antibody. In order to tumor SPECT/MR imaging, SPIONs were labeled with Indium-111 (T1/2 = 2.80d). NHS ester of monoethyl malonate (MEM-NHS) was used for conjugation of Doxorubicin (DOX) chemotherapeutic agent onto SPIONs surface. Mono-Ethyl Malonate allows DOX molecules to be attached to SPIONs via pH-sensitive hydrazone bonds which lead to controlled drug release in tumor region. Active and passive tumor targeting were achieved through incorporated anti-HER2 (Trastuzumab) antibody and EPR effect of solid tumors for nanoparticles respectively. In addition to in vitro assessments of modified SPIONs in SKBR3 cell lines, their theranostic effects were evaluated in HER2 + breast tumor bearing BALB/c mice via biodistribution study, dual-modal molecular imaging and tumor diameter measurements. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Nanotechnology takes advantage of the special properties of various materials when they are in the scale of a few nanometers. Since most of potential therapeutics has poor pharmacokinetics and biopharmaceutical properties, there is a need to develop suitable drug delivery systems that distribute the therapeutically active drug molecule only to the site of action, without affecting healthy organs and tissues [1,2]. Superparamagnetic iron oxide nanoparticles (SPIONs) with appropriate surface modification have been widely used for numerous in vivo applications, such as magnetic resonance imaging (MRI) contrast enhancement, immunoassay, and drug delivery [3–7]. These areas include delivery by avoiding the reticuloendothelial system (RES), utilizing the enhanced permeability and retention effect (EPR) and tumor targeting. Nanoparticles will usually be taken up by the liver, spleen and other parts of the RES depending on their surface characteristics. Particles with more hydrophobic surfaces will preferentially be taken up by the liver, followed by the spleen and lungs. Particles with longer circulation times, and hence greater ability to target to the site of interest, should be 100 nm or less in diameter and have a hydrophilic surface in order to reduce clearance
⁎ Corresponding author. E-mail addresses: [email protected] (H. Zolata), [email protected] (F. Abbasi Davani).
by macro-phages [8]. Tumor vessels are known to have large fenestrations which result in preferential accumulation of nanomaterials within tumors [9]. One well-known strategy to reduce proteins binding to nanoparticles is attaching PEG chains; this is also thought to prevent NP uptake by cells of the RES [10–13]. Most conventional chemotherapeutic agents, for example, doxorubicin, cisplatin or paclitaxel, elicit mitochondrial permeabilization in an indirect fashion by triggering perturbations of intermediate metabolism or by increasing the concentration of proapoptotic second messengers [14]. Design and synthesis of non-targeted or antibody targeted biodegradable PEG coupled with doxorubicin attached through acidsensitive hydrazone bond was reported [15,16]. Targeted drug therapy, potentially combined with simultaneous imaging modalities to monitor the delivery of drugs to specific tissues. The potential to deliver active chemotherapeutic drugs in the vicinity or directly within specific tumors via receptor mediated pathways, and to image tumors through the use of nanoparticles has been conceptually and experimentally shown for several classes of nanoparticles [10]. Anti-HER2 antibody can induce antitumor responses, and can be used in delivering drugs to HER2-overexressing cancer [17,18]. Furthermore, dual-modality SPECT/MR imaging bioprobes would allow for simultaneous dynamic imaging of structure and function, facilitate noninvasive monitoring of treatment, and directly provide information on pharmacokinetics and metabolism of drugs [19].
http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007 0969-8051/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
2
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx
2. Experiment 2.1. Materials Iron (III) acetylacetonate, benzyl ether (99%), oleylamine (N 70%), 3-aminopropyltriethoxysilane (APTES), mono-ethyl malonate (MEM), and anhydrous hydrazine were provided by Sigma Aldrich, USA. Trastuzumab (Herceptin) from Roche (Germany) and p-SCN-Bn-PCTA from Macrocyclics (USA). All other chemicals were of analytical reagent grade. 2.2. Preparation of bare SPIONs Hydrophobic SPIONPs were prepared by thermal decomposition method to obtain uniform and narrow-sized SPIONs [20–23]. Briefly, 1.4 gr Fe (acac) 3 was dissolved in a mixture of 20 mL benzyl ether and 20 mL oleylamine that was mingled by a magnetic stirrer. The solution was dehydrated at 120 °C for 1 hr and then quickly heated up to 280 °C for 2 h. After cooling, 65 mL ethanol was added to the mixture and precipitated by centrifuge at 6000 rpm. Attained mixture was eluted with ethanol for three times. The product was re-dispersed in 20 ml hexane and stored at 4 °C. 2.3. SPIONs treatment with amino-silane 5 ml of (3-aminopropyl) triethoxysilane (APTES) is placed into an aqueous solution of an acid (pH = 4) that acts as a catalyst [24]. APTES was hydrolyzed, and then a condensation reaction takes place to form a silane polymer. In the hydrolysis reaction, alkoxide groups are replaced by hydroxyl groups to form reactive silanol groups, which condense with other silanol groups to produce siloxane bonds. The solution of silane polymer was added to 30 ml of the SPIONs in a flask. The mixture was stirred for 4 h at 65 °C under argon protection. After cooling, the product was eluted with DI water and ethanol, pursued by drying in vacuum at room temperature [24].
Afterward, Trastuzumab was dissolved at a concentration of 1 mg/mL in phosphate buffer (pH 8.0). Trastuzumab solution (1 mL) was incubated with 40.2 μL (50-fold molar excess) of 2-iminothiolane (Traut's reagent) solution (5.7 mg in 5.0 mL phosphate buffer, pH 8.0) [26,27]. The thiolation reaction was stirred gently for 5 hrs at room temperature. Unreacted Traut's reagent was excluded by centrifugation three times with 30 kDa cutoff centrifugal ultrafilters (Millipore Corporation) at 4000 g and 10 °C for 15 minutes. The amount of thiol groups within the antibody were quantified through disulfide building with 5, 50dithio-bis-2 (nitrobenzoic acid) (Ellman's reagent) which 2-nitro-5thiobenzoic acid (TNB) was released stoichiometrically and could be quantified photometrically at 412 nm [27,28]. 2.7. Thiolated Trastuzumab and PCTA-SH conjugation onto the SPIONs Thiolated Trastuzumab and PCTA conjugation onto the SPIONs was performed by a reaction between the maleimide groups of the PEG conjugated on the surface of SPIONs and the attached SH group of the Trastuzumab and PCTA. The molar ratio for thiolated Trastuzumab: PCTA was set at 2:1, and total molar values of Trastuzumab and PCTA were equal to molar value (0.0042 mM) of PEG-Mal. Thiolated trastuzumab (0.0028 mM) and PCTA-SH solution (0.0014 mM) were added into the 10 mL water solution containing 10 mg modified SPIONs at room temperature for 6 h under a N2 atmosphere. 2.8.
111
In-labeling of SPIONs and radiolabeling assessment
111 InCl3 was obtained from Radiation Application School cyclotron (Cyclone-30, IBA). 111InCl3 (74 MBq) was diluted in 400 μL of sodium acetate buffer (pH 6.5, 0.1 M) and added to the PCTA-conjugated SPIONs solution. The mixture was incubated at 40 °C for 90 min followed by testing the radiochemical purity by RTLC (Whatman No. 2) using a RTLC scanner (Bioscan Inc.). For to animal studies, 111In-labeled SPIONs were purified using PD10 column with PBS. Exerted procedures to prepare multifunctional SPIONs are depicted in Fig. 1. Obtained specific activity was 7.18 MBq/mg of SPIONs.
2.4. SPIONs conjugation with Mal-PEG-NHS and MEM-NHS 3. In vitro and in vivo assay Synthesis of MEM-NHS and its conjugation with Mal-PEG-NHS altogether onto SPIONs surface were performed according to previous experiments [21,25]. Briefly, 1.70 mg MEM-NHS and 20 mg Mal-PEGNHS (Mw: 5000) were added to 10 mL DI water containing 10 mg SPIONs under a N2 atmosphere. After 30 min of N2 bubbling, the mixture was allowed to react for 5 h at room temperature. After the reaction was complete, the solution was purified by dialysis against DI water for 2 days. The solution was then filtered through a 0.22 μm membrane followed by freeze-drying. 2.5. Conjugation of the DOX molecules onto the SPIONPs DOX molecules were conjugated onto SPIONs through a two-phase reaction [21]. Briefly, MEM methoxy group was substituted with hydrazide in anhydrous DMSO at 40 °C for 24 h. Obtained SPIONs were purified by dialysis using an ammonia solution (0.25%) for 48 h before freeze-drying. Then DOX (0.006 mM) was bound to hydrazide group of SPIONs (10 mL) through an acid-sensitive hydrazone linkage. The reaction was performed in DMSO solution at room temperature for 24 h with an excess amount of DOX which was removed by dialysis using DI water and then purified by Sephadex gel column. 2.6. Thiolation of Trastuzumab and p-SCN-Bn-PCTA The PCTA-SH was prepared by a reaction between the NCS group of p-SCN-Bn-PCTA and the amino group of 2-aminoethanethiol hydrochloride in the presence of triethanol-amine. The reaction was allowed to stay in DI water at room temperature for 3 h under a N2 atmosphere.
The SKBr3 cell line (1.0 × 10 6cells/well) and mouse fibroblasts (L929, adhesive) (1.0 × 104cells/well) were obtained from the National Cell Bank of Iran (NCBI), Pasteur Institute (Tehran, Iran). Mouse fibroblasts were seeded on glass cover slips in 96 well plates and incubated for 24 h. After the 24 h incubation period, medium containing control cells, bare-SPIONs, APTES and APTES-PEG Coated SPIONs (0.2 mM iron) were added to the wells, and cells were incubated at 37 °C for 1 h. The medium was then extracted, and the cells were allowed to rest. At 24 h post-treatment, the cell viability was assessed by the MTT assay using an ELISA reader (microreader, Hyperion) at 540 nm. In order to in vivo assay of complex, female BALB/c mice (20–25 g, 6–8 weeks old) were subcutaneously injected with 3 × 105 SKBR3 cells (expressing high levels of HER2) in the flank. The organ distribution experiments were performed when the tumor volumes reached 0.5 cm in diameter. For tumor imaging, mice (n = 5) were injected intravenously with Trastuzumab-Functionalized, DOX-Conjugated, Indium111labeled, APTES-PEG Coated SPIONs (100 μL, 3.6 MBq, 20 μgm Ab, and 0.5 mg Fe) via the tail vein. At 12, 24 and 48 h after injection, the animals were anesthetized with ether. Images were recorded using a clinical dual-head coincidence gamma camera (GE, USA). Moreover, a clinical 1.5 T MRI scanner (SIEMENS) was used to measure T2-weighted signal intensities (SI) for in vivo MRI studies. For biodistribution study, animals were sacrificed by ether; their organs were removed and weighed. Amount of incorporated 111In was measured with a HPGe gamma detector (CANBERRA). Specificity of Trastuzumab modified SPIONs was determined through HER2 receptors blocking by Trastuzumab (20 μg) injection. For therapeutic evaluation, solid tumors were allowed to
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx
3
Fig. 1. Exerted procedures to prepare multifunctional SPIONs.
develop over a period of 3 weeks, and animals were randomized into control and treatment groups (n = 3) having no significant difference in tumor volumes or body weights. On day 8 animals in the treatment group received injections of radiolabeled modified SPIONs (3.6 MBq). Tumor volumes were determined each week until on day 28. All animal experiments were performed in compliance with the regulations of our institution.
shown in Fig. 2a–b. Moreover, the magnetization of the SPIONs in a variable magnetic field was measured using a vibrating sample magnetometer (VSM) with a sensitivity of 10 − 3 emu and magnetic field up to 8 kOe. Bare and surface modified SPIONs show magnetic behavior. Obtained magnetization values for bare SPIONs, SPIONs treated by APTES and PEG coated SPIONs were 65 emu/g, 58 emu/g and 52 emu/g respectively as shown in Fig. 2c which indicates their potential as a contrast agent for MR imaging.
4. Results 4.1. SPIONs characterization
4.2. SPIONs radiolabeling and antibody conjugation assay
The particle size and morphology of core and amino-silane treated and PEG coated SPIONs were investigated by TEM (ZEISS Model EM10C) and SEM (FEG LECO). APTES-PEG coated SPIONs with spherical shape and an average diameter of 16 ± 0.27 nm were observed, as
Using RTLC, SPIONs radiolabeling efficiency was measured about 97.6% (± 1.2). The highest number of 0.74 ± 0.52 sulfhydryl groups per trastuzumab was introduced. The amount of antibody was determined by UV spectrometry using Warburg's formula [3,29]. We
Fig. 2. TEM (A) and SEM (B) micrographs of APTES-PEG coated SPIONs, (C) magnetization curves obtained by VSM at room temperature of (a) Fe3O4 MNPs; (b) Fe3O4 MNPs treated by APTES; (c) PEG coated Fe3O4 MNPs.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
4
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx
determined that 63.79 ± 2.60 % of thiolated trastuzumab was conjugated to SPIONs surface via maleimide-thiol coupling reaction. 4.3. Drug release study Amount of released DOX was analyzed photometrically at 485 nm [18]. Fig. 3a shows DOX release profiles of modified SPIONs at different pH values. During 72 h, amount of DOX release was below 10% at pH 7.4 while this value was 44% at pH 4.6. This is a desirable result; because DOX will not be released before reaching tumor region. This pHdependent DOX release is very desirable for targeted cancer therapy. It minimizes the amount of drug release during blood circulation (pH 7.4). Instead, sufficient amount of drug is introduced to cancer cells (pH 4.5 to 6.5).
at very high concentrations at 12 and 24 and 48 h post-injection (Fig. 4a) while non-specific tumor uptake was observed in mice with blocked HER2 receptors (data not shown). Tumor uptake and tumor/ non-tumor ratios (Fig. 4b) show the potential of multifunctional SPIONs for breast tumors bio-probing and imaging. The uptake of the nano carriers in the tumor indicated good tumor-targeting capability. The tumor/muscle ratios for specific and non-specific (data not shown) uptake were 38.40 ± 15.02 and 15.64 ± 7.35 at 48 h post injection respectively which indicated nonspecific uptake due to the enhanced permeation and retention (EPR) effect. A comparison of the biodistribution data of 111In labeled Trastuzumab-conjugated SPIONs in blocked and unblocked tumor bearing mice revealed that uptake of SPIONs was higher in most organs except the tumor, which again indicated the tumor specificity of 111In labeled Trastuzumabconjugated nanocarriers.
4.4. In vitro cell cytotoxicity and specificity analysis 4.6. Tumor imaging In vitro cell cytotoxicity and specificity analysis was performed based on [18] experiment. Fibroblast cell viability of bare, APTES, APTES-PEG, and Trastuzumab conjugated SPIONs is depicted in Fig. 3b at 24 h posttreatment. Results confirmed that APTES and APTES-PEG modified SPIONs are more biocompatible than bare SPIONs. Moreover, SKBr3 cell survival curves for Trastuzumab and Dox conjugated, APTES-PEG Coated SPIONs are shown in Fig. 3c. The relative numbers of SKBr3 cells were reduced to 95.2%, 57.6% and 41.5% for Trastuzumab-free SPIONs, Trastuzumab conjugated SPIONs and Trastuzumab-DOX conjugated SPIONs respectively at 24 h post-treatment.
Tumor region is clearly contrasted in coronal SPECT images of tumor bearing mouse at 4, 24 and 48 h after injection (Fig. 5a). Distinguishable darkening MR images of tumor cells were due to tumor uptake of SPIONs which were specifically delivered to tumor site via decoration with mAb, as shown in Fig. 5b. Imaging after mAb-free SPIONs injection did not lead to darkening in the tumor region as shown in Fig. 5c. Overall, the quantification results obtained from biodistribution studies and SPECT scans matched well, confirming that SPECT scans truly reflected the distribution of 111In labeled Trastuzumab-conjugated Nano-carriers in vivo.
4.5. Biodistribution study
5. Therapeutic efficacy
Biodistribution study revealed that Indium111-labeled TrastuzumabFunctionalized, APTES-PEG Coated SPIONs were accumulated in tumors
Tumor growth in treated mice appeared to be slowing in comparison to controls within one week. After complex administration,
Fig. 3. (A) Dox release profiles of compound at different pH values, (B) cell viability of fibroblast cell for 1. control cells, 2. bare, 3. Aminosilane, 4. APTES-PEG and 5. mAb conjugated SPIONs, (C) relative SKBR3 cell number after treatment with 1. Trastuzumab-free SPIONs, 2. Trastuzumab conjugated SPIONs, and 3. Trastuzumab–Doxorubicin conjugated SPIONs.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx
5
Fig. 4. (A) Biodistribution results at 12, 24 and 48 h post injection. (B) Tumor/Non tumor ratios in tumor bearing mice at 12, 24 and 48 h post injection. Data are presented as mean ± SD (n = 5).
tumor volumes were ∼ 50%, 25 %, 23% lower for treated animals compared with control group at end of each week. This significant therapeutic effect was continued throughout the study (Fig. 6).
Three weeks after injection, tumor volumes for the control mice were fully larger than those in the treated group (0.69 ± 0.06 vs.0.16 ± 0.03 cm 3).
Fig. 5. (A) SPECT, (B) MR images and (C) MRI control images of tumor bearing mice at 12, 24 and 48 h.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
6
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx
6. Discussion We synthesized biocompatible mono-disperse nanoparticles via thermal decomposition method and then modified their surface with APTES-PEG to prevent SPIONs uptake by cells of reticuloendothelial system which lead to increase their presentation time in blood stream. Cytotoxity effect of modified SPIONs in non-tumor cells was relatively low in comparison with bare iron oxide nanoparticles while cell viability of SKBR3 cells were reduced to 95.2%, 57.6% and 41.5% for Trastuzumabfree SPIONs, Trastuzumab conjugated SPIONs and Trastuzumab-DOX conjugated SPIONs respectively. In addition, PEG chains provided suitable platforms for antibody and radiopharmaceutical attachments. In our experiments; all the samples showed a typical superparamagnetic behavior which were indicated in Fig. 2.C. Magnetization property of modified SPIONs which is critical for MR imaging has just been decreased by 10.7% for APTES and 20% for APTES-PEG coated SPIONs in comparison with bare iron oxide nanoparticles. In order to antibody conjugation, Trastuzumab was first thiolated using Traut's reagent and then conjugated to SPIONs via maleimide-thiol coupling reaction. By this method, 63.79 ± 2.60 % of antibody was conjugated onto SPIONs. Previous studies have shown that anti-HER2 conjugation to nanoparticles does not alter their intrinsic specificity and binding affinity to HER2 + cells [30]. SPIONs radiolabeling with 111In radionuclide was performed successfully with yield of 96% using thiolated PCTA bifunctional chelator (BFC). This radiotracer allows highly specific radiolabeling and superior in vivo characteristics with a significantly higher tumor uptake. When receptors were blocked, tumor uptake was decreased from 7.38 ± 1.06, 9.94 ± 10.7 and 12.88 ± 0.76%ID/g to 3.38 ± 0.52 and 5.94 ± 1.02 and 6.12 ± 1.48%ID/g at 12, 24, and 48 h post-injection respectively, indicating the HER2-specific uptake in tumor tissue. In the absence of receptors mediated endocytosis, EPR effect of nanoparticles is a major mechanism of cancer tumor uptake. In comparison with SPIONs-free radiolabeled Trastuzumab (data not shown), tumor uptake was decreased dramatically to 39% of SPIONs conjugated experiments at 48 h post-injection. In addition, clearance from the blood was fast, leading to increasing tumor-to-blood ratios of radioactivity over time (from 1.36 ± 0.20 at 12 h to 8.05 ± 2.0 at 48 h after injection). Nonspecific retention of radioactivity in non-targeted organs and tissues relatively was low. In comparison with previous radiolabeled antibody conjugated SPIONs experiment [18], blood uptake was increased from 1.8 ± 0.15 to 3.1 ± 0.39%ID/g at 24 h after injection due to appropriate nanoparticles surface modifications which lead to SPIONs escape from RES. Results showed high initial blood retention with moderate liver uptake, making them an attractive nano-probe for tumor theranostics. Moreover, pH sensitive DOX release was investigated, and obtained results after 72 h showed that 43% of DOX could be released effectively at tumor pH. This controlled drug release in tumor region helps in effective tumor cells killing and prevents healthy cells homicide. Moreover, enhanced permeability and retention effect of solid tumors for SPIONs and also well decoration of SPIONs with mAb
Fig. 6. Therapeutic evaluation in tumor bearing mice. Data are presented as mean ± SD (n = 3).
leads to effective targeting and drug delivery to tumor site. Simultaneous effects of conjugated radiopharmaceutical, antibody and chemotherapeutic agent to SPIONs lead to impressive tumor suppression as well as effective dual modality MRI/SPECT tumor imaging. SPIONs accumulation in tumor region alters darkening in MR images. During therapeutic assessments, tumor volume was reduced from 0.25 cm 3 to 0.16 cm 3 for treatment group in spite of tumor growth in control group. This means that 36% of tumor volume was decreased during 28 days. Based on our best knowledge this therapeutic efficacy was not achieved in any multifunctional modified SPIONs experiment.
7. Conclusion Specific bio-probes for single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI) have enormous potential for tumor imaging. The bioprobes were developed by Indium111-labeled Trastuzumab-DOX conjugated, APTES-PEG Coated SPIONs. Our experimental results showed that modified SPIONs retain their magnetic properties as well as the ability to specifically localize in HER2 overexpressing tumors. Bio-distribution study revealed that 111In-labeled, TrastuzumabDOX conjugated, APTES treated SPIONs were accumulated at very high concentrations in tumors as a result of EPR effect and HER2 receptor targeting. SPECT and MR imaging indicated that resulting SPIONs could be used for dual-modality imaging. Moreover, promising results of therapeutic evaluation proved theranostic ability of complex. Therapeutic efficacy of SPIONs was increased via circulation time enhancement by appropriate coating and active targeting using Trastuzumab antibody, controlled doxorubicin release in tumor region, and Auger electrons and also gamma rays of 111In radionuclide.
References [1] Loudos G, Kagadis GC, Psimadas D. Current status and future perspectives of in vivo small animal imaging using radiolabeled nanoparticles. Eur J Radiol 2011;78: 287–95. [2] Koo O, Rubinstein I, Onyuksel H. Role of nanotechnology in targeted drug delivery and imaging: a concise review. J Nanomedicine 2005;1(3):193–212. [3] Chen T, Cheng T, Chen C, Hsu SC, et al. Targeted Herceptin–dextran iron oxide nanoparticles for noninvasive imaging of HER2/neu receptors using MRI. J Biol Inorg Chem 2009;14:253–60. [4] Kalambur V, Longmire E, Bischof JC. Cellular level loading and heating of superparamagnetic iron oxide nanoparticles. J Langmuir 2007;23:12329–36. [5] Li Z, Tan B, Allix M, Cooper A, Rosseinsky M. direct coprecipitation route to monodisperse dual-functionalized magnetic iron oxide nanocrystals without size selection. J Small 2008;4:231–9. [6] Schellenberger E, Schnorr J, Reutelingsperger C, et al. Linking proteins with anionic nanoparticles via protamine: ultrasmall protein-coupled probes for magnetic resonance imaging of apoptosis. J Small 2008;4:225–30. [7] Sun C, Veiseh O, Gunn J, Fang C, et al. In vivo MRI detection of gliomas by chlorotoxin-conjugated superparamagnetic nanoprobes. J Small 2008;4:240–9. [8] Brannon-Peppas L, James O. Blanchette. Nanoparticle and targeted systems for cancer therapy. J Adv Drug Deliv Rev 2012;64:206–12. [9] Grobmyer S, Zhou G, Luke G, et al. Nanoparticle delivery for metastatic breast cancer. J Maturitas 2012;73:19–26. [10] Garcia-Bennett A, Nees M, Fadeel B. In search of the holy grail: folate-targeted nanoparticles for cancer therapy. J Biochem Pharmacol 2011;81:976–84. [11] Liu X, Tao H, Yang K, Zhang S, Lee S, Liu Z. Optimization of surface chemistry on single-walled carbon nanotubes for in vivo photothermal ablation of tumors. Biomaterials 2011;32:144–51. [12] Prencipe G, Tabakman S, Welsher K, et al. PEG branched polymer for functionalization of nanomaterials with ultralong blood circulation. J Am Chem Soc 2009;131:4783–7. [13] Liu XW, Tao HQ, Yang K, Zhang S, et al. PEGylated FePt@Fe2O3 core-shell magnetic nanoparticles: potential theranostic applications and in vivo toxicity studies. J Nanomed Nanotechnol Biol Med 2013;9:1077–88. [14] Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. J Oncog 2006;25:4798–811. [15] Rıhova B, Etrych T, Pechar M, et al. Doxorubicin bound to a HPMA copolymer carrier through hydrazone bond is effective also in a cancer cell line with a limited content of lysosomes. J Control Release 2001;74:225–32. [16] Ulbrich K, Etrych T, Chytil P, Pechar M, et al. Polymeric anticancer drugs with pHcontrolled activation. Int J Pharm 2004;277:63–72. [17] Itoa A, Kugaa Y, Hondaa H, Kikkawab H, et al. Magnetite nanoparticle-loaded antiher2 immunoliposomes for combination of antibody therapy with hyperthermia. J Cancer Lett 2004;212(2):167–75.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
H. Zolata et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx [18] Zolata HR, Afarideh H, Abbasi-Davani F. Radio immunoconjugated, Dox-loaded, surface-modified superparamagnetic iron oxide nanoparticles (SPIONs) as a bioprobe for breast cancer tumor theranostics. J Radioanal Nucl Chem 2014;301: 451–60. [19] Misri R, Meier D, Yung A, Kozlowski P, et al. Development and evaluation of a dualmodality (MRI/SPECT) molecular imaging bioprobe. J Nanomed Nanotechnol Biol Med 2012;8:1007–16. [20] Heidari Majd M, Asgari D, Barar J, et al. Tamoxifen loaded folic acid armed PEGylated magnetic nanoparticles for targeted imaging and therapy of cancer. J Colloids Surf B Biointerfaces 2013;106:117–25. [21] Yang X, Hong H, Grailer JJ, et al. RGD-functionalized, DOX-conjugated, and 64Cu-labeled superparamagnetic iron oxide nanoparticles for targeted anticancer drug delivery and SPECT/MR imaging. Biomaterials 2011;32: 4151–60. [22] Xu Z, Shen C, Hou Y, et al. Oleylamine as both reducing agent and stabilizer in a facile synthesis of magnetite nanoparticles. J Chem Mater 2009;21:1778–80. [23] Xie J, Xu C, Xu Z, Hou Y, et al. Linking hydrophilic macromolecules to monodisperse magnetite (Fe3O4) nanoparticles via trichloro- s-triazine. J Chem Mater 2006;18:5401–3.
7
[24] Feng B, Hong RY, Wang LS, Guo L, et al. Synthesis of Fe3O4/APTES/PEG diacid functionalized magnetic nanoparticles for MR imaging. J Colloids Surf A Physicochim Eng Aspects 2008;328(1–3):52–9. [25] Xiao Y, Hong H, Matson VZ, et al. Gold nanorods conjugated with doxorubicin and cRGD for combined anti-cancer drug delivery and PET imaging. J Theranostics 2012;2(8):757–68. [26] Yousefpour P, Atyabi F, Vasheghani-Farahani E, et al. Targeted delivery of doxorubicin-utilizing chitosan nanoparticles surface-functionalized with anti-Her2 trastuzumab. Int J Nanomedicine 2011;6:1977–90. [27] Steinhauser I, Spänkuch B, Strebhardt K, Langer K. Trastuzumab-modified nanoparticles: optimisation of preparation and uptake in cancer cells. J Biomater 2006; 27(28):4975–83. [28] Weber C, Reiss S, Langer K. Preparation of surface modified protein nanoparticles by introduction of sulfhydryl groups. Int J Pharm 2000;211(1–2):67–78. [29] Germershaus O, Merdan T, Bakowsky U. Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting Her2-expressing tumors. Bioconjug Chem 2006;17:1190–9. [30] Debotton N, Zer H, Parnes M. A quantitative evaluation of the molecular binding affinity between a monoclonal antibody conjugated to a nanoparticle and an antigen by surface plasmon resonance. Eur J Pharm Biopharm 2010;74(2):148–56.
Please cite this article as: Zolata H., et al, Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles, Nucl Med Biol (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.09.007
