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Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells
BBAGEN-28761; No. of pages: 9; 4C: 3 Biochimica et Biophysica Acta xxx (2017) xxx–xxx
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Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells☆ Sara Trabulo a,b,1, Antonio Aires c,d,1, Alexandra Aicher a, Christopher Heeschen a,b,⁎, Aitziber L. Cortajarena c,d,e,⁎⁎ a
Stem Cells & Cancer Group, Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain Centre for Stem Cells in Cancer & Ageing, Barts Cancer Institute, Queen Mary University of London, EC1M 6BQ, UK CIC BiomaGUNE, Parque Tecnológico de San Sebastián, Paseo Miramón 182, Donostia-San Sebastián 20009, Spain d IMDEA Nanociencia and Nanobiotechnology Unit associated to Centro Nacional de Biotecnología (CNB-CSIC), Campus Universitario de Cantoblanco, Madrid 28049, Spain e Ikerbasque, Basque Foundation for Science, Mª Díaz de Haro 3, 48013 Bilbao, Spain b c
a r t i c l e
i n f o
Article history: Received 27 October 2016 Received in revised form 26 December 2016 Accepted 28 January 2017 Available online xxxx Keywords: Pancreatic cancer Magnetic nanoparticles Multifunctionalization Nanocarriers CD47 Gemcitabine Controlled drug release Active targeting Nanomedicine
a b s t r a c t Nanomedicine nowadays offers novel solutions in cancer therapy by introducing multimodal treatments in one single formulation. In addition, nanoparticles act as nanocarriers changing the solubility, biodistribution and efficiency of the therapeutic molecules, thus generating more efficient treatments and reducing their side effects. To apply these novel therapeutic approaches, efforts are focused on the multi-functionalization of the nanoparticles and will open up new avenues to advanced combinational therapies. Pancreatic ductal adenocarcinoma (PDAC) is a cancer with unmet medical needs. Abundant expression of the anti-phagocytosis signal CD47 has also been observed on pancreatic cancer cells, in particular a subset of cancer stem cells (CSCs) responsible for resistance to standard therapy and metastatic potential. CD47 receptor is found on pancreatic cancer and highly expressed on CSCs, but not on normal pancreas. Inhibiting CD47 using monoclonal antibodies has been shown as an effective strategy to treat PDAC in vivo. However, CD47 inhibition effectively slowed tumor growth only in combination with Gemcitabine or Abraxane. In this work, we present the generation of multifunctionalized iron oxide magnetic nanoparticles (MNPs) that include the anti-CD47 antibody and the chemotherapeutic drug Gemcitabine in a single formulation. We demonstrate the in vitro efficacy of the formulation against CD47-positive pancreatic cancer cells. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Magnetic nanoparticles (MNPs) have been explored during the last decades in several scientific fields based on their excellent physical and chemical properties, such as superparamagnetism, good colloidal stability, low toxicity and good biocompatibility [1–8]. In recent years MNPs have been widely investigated for their use in biomedical applications including diagnosis via magnetic biosensing or magnetic resonance imaging (MRI) [9–14], therapy [15–18] and magnetic separation techniques [19–22].
☆ This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader. ⁎ Correspondence to: C. Heeschen, Stem Cells & Cancer Group, Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain. ⁎⁎ Correspondence to: A.L. Cortajarena, CIC BiomaGUNE, Parque Tecnológico de San Sebastián, Paseo Miramón 182, Donostia-San Sebastián 20009, Spain. E-mail addresses: [email protected] (C. Heeschen), [email protected] (A.L. Cortajarena). 1 These authors contributed equally to this work.
Even though therapeutic systems based on magnetic nanoparticles have received a lot of attention in nanomedicine, controlling the targeting of specific cells still remains a challenge. Passively targeted nanoparticles via the enhanced permeability and retention (EPR) effect fail to accomplish tumor specificity in many examples. Therefore, the conjugation of cell-specific targeting ligands on the surface of magnetic nanoparticles that enable receptor-mediated endocytosis can be advantageous, as it can provide specificity, increasing the efficiency of treatments and reducing their side effects [23–25]. A great variety of targeting ligands for cancer diagnosis and therapy have been developed [26]. Generally active targeting is accomplished by the attachment of a targeting ligand on the surface of the MNP, which specifically recognizes a receptor that is over-expressed on the target cells. The most common method for the preparation of actively targeted MNPs is the conjugation of antibodies, antibody fragments, peptides, sugars, or small molecule ligands [27]. Nanoparticles (NPs) are ideal platforms for developing novel combinational therapies, as they act as nanocarriers that can be functionalized with multiple components. NPs developed for biomedical applications require careful physicochemical and targeting design, but also require
http://dx.doi.org/10.1016/j.bbagen.2017.01.035 0304-4165/© 2017 Elsevier B.V. All rights reserved.
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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additional considerations paid to drug loading, transport, and release [25]. By developing advanced multifunctionalization strategies, NPs can be decorated with the desired loads of each active component [28], which include targeting agents, standard chemotherapeutic agents, and novel therapeutic agents (e.g. therapeutic antibodies). The multifunctionalization is performed in a way that first, the NPs are able to carry and protect a significant drug payload; second, multiple therapeutic agents can be properly loaded remaining functionally active; and third, the therapeutic cargo release mechanism and rate are modulated for optimal therapeutic efficacy. In this sense, we have previously shown how to generate multifunctional MNPs with large drug payloads per targeting agent (200:1) [29], which cannot be reached by antibody-based targeted drug delivery approaches [30–32]. Overall, nanomedicine has opened new avenues towards the development of selective, minimally-invasive and efficient therapeutic modalities against several diseases such as cancer. In particular, pancreatic cancer is currently the fifth cause of cancer-related deaths and is characterized by early metastasis, extensive desmoplasia and pronounced resistance to chemotherapy and radiation. Regardless of the extensive research efforts, no major progress has been made regarding improvement in clinical endpoints recently. Since the introduction of the antimetabolite Gemcitabine in the '90s [33], only the use of combination therapies such as Folfirinox [34], and more recently the combination of Gemcitabine with nab-paclitaxel (Abraxane) [35], has been able to moderately extend the median survival, but eventually the vast majority of patients still succumb to the disease. In particular, the studies using Abraxane, which is a colloidal suspension of 130 nm particles prepared by homogenization of paclitaxel with human serum albumin, highlight the importance of drug delivery and targeting [36] on improving the pharmacokinetics [37], the bioavailability [38] and the intra-tumoral accumulation of drugs [39] and therefore the impact that an appropriate delivery can have on the clinical outcome of a given drug. It is therefore of the utmost importance not only to develop new effective anti-PDAC treatments but to develop systems that are able to specifically deliver the combination of the different therapeutic agents to the tumor cells. It has been shown for different types of cancers that distinct populations of cells with stem cell properties, namely cancer stem cells (CSCs), are responsible for resistance to standard therapy and bear metastatic potential. We and others have provided conclusive evidence for such a hierarchical organization in human pancreatic ductal adenocarcinoma (PDAC) [40–42], and the survival of such resistant CSCs during chemotherapy can justify the later relapse of the disease that still happens to most of the patients. We have recently shown that CD47 is expressed on primary PDAC cells and we have demonstrated that inhibiting CD47 function using monoclonal antibodies constitutes an effective strategy to treat PDAC [43]. In summary, we observed that treatment with monoclonal antibodies for CD47 in combination with Gemcitabine or Abraxane significantly reduced primary tumor growth, using PDX models of PDAC. Specifically, we found that anti-CD47 therapy alone had only marginal impact on the size and rate of tumor growth, while treatment in combination with Gemcitabine or Abraxane resulted in efficient growth control of tumors and prevented relapse after discontinuation of treatment [43]. These results suggest that anti-CD47 therapy could be used to treat primary PDAC tumors, but combination with other anti-cancer therapeutic agents, such as Abraxane or Gemcitabine, is needed [43]. Therefore, the development of novel nanotherapies, including actively targeted MNPs with the possibility of combining different therapeutic agents in a single vehicle seems highly attractive to overcome the difficulties of treating pancreatic cancer [44,45]. In this work we introduce a new strategy for pancreatic cancer treatment based on nanotechnology by combining the inhibition of CD47, the chemotherapeutic effect of Gemcitabine, and the efficient vehiculization of those components in a single formulation by using nanoparticles. For this purpose we developed multifunctional MNPs that target a receptor overexpressed in pancreatic cancer cells
allowing at the same time the delivery of therapeutic agents with different mechanisms of action without compromising their biological activity. This approach resulted in an effective delivery of both Gemcitabine and anti-CD47 antibody to pancreatic cancer cells. Thus, MNPs build up a good platform that can be further improved for in vivo applications to administer other drug combinations currently evaluated to increase the efficiency of treatments, while reducing their side effects. 2. Materials and methods 2.1. Materials Purified anti-human CD47 antibodies (B6H12 clone) were purchased from eBioscience (anti-CD47). Gemcitabine (Gem) was purchased from Fluorochem. Ultrapure reagent grade water (18.2 MΩ, Wasserlab) was used in all experiments. Dimercaptosuccinic acid (DMSA) coated MNPs were produced by means of the co-precipitation technique as described before [46] and have been provided by Liquid Research Ltd. (UK). The MNPs (MF66) have a zeta potential: −56 mV, an average core size of 12 ± 3 nm, and a hydrodynamic diameter (Zaverage): 97 nm (PDI: 0.18). These MNPs present good stability in PBS buffer and RPMI cell culture medium with and without 10% of bovine serum, showing a hydrodynamic size of 98 nm (PDI: 0.18) in PBS, 100 nm (PDI: 0.18) in RPMI without serum and 105 nm (PDI: 0.18) in RPMI 10% serum. A Gemcitabine derivative, Gem-S-S-Pyr was prepared according to described procedures [28]. 2.2. Measurements Ultraviolet-visible (UV–Vis) and fluorescence spectra were recorded on a Synergy H4 microplate reader (BioTek) using 96-well plates. Hydrodynamic diameter and zeta potential measurements were determined using a Zetasizer Nano-ZS device (Malvern Instruments). Hydrodynamic diameter and zeta potential were measured from dilute sample suspensions (0.1 mg Fe ml−1) in water at pH 7.4 using a zeta potential cell. High-performance liquid chromatography (HPLC) was performed using a 1260 Infinity HPLC (Agilent Technologies) with a ZORBAX 300SB-C18 column 5 μm, 9.4 × 250 mm. 2.3. Multifunctionalization of MNPs 2.3.1. MNP activation 6 ml of MNPs at 2 mg Fe ml−1 were incubated overnight at 37 °C with 50 μmol of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC)/g Fe, 25 μmol of n-hydroxy succinimide (NHS)/g Fe and 25 μmol of cysteamine hydrochloride/g Fe, previously neutralized by 1 M equivalent of sodium hydroxide (NaOH). After 16 h, the sample was washed by cycles of centrifugation and redispersion in Milli-Q water 3 times. The presence of sulfhydryl groups introduced onto the MNPs was quantitatively measured by reaction with 2,4dinitrothiocyanatebenzene (DNTB) [47]. 2.3.2. Covalent attachment of anti-CD47 antibodies on MNPs A solution of the anti-CD47 (1 mg ml− 1) in 0.01 M 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.15 M sodium chloride (NaCl), pH 8.2, was incubated for 1 h at room temperature with a 5 M equivalent solution of 2-iminothiolane. This strategy, as has been previously described, allows the oriented immobilization of the antibodies on the nanoparticles [29,48]. After that, the modified antibody was purified by gel filtration through a desalting resin (Sephadex G-25) using PBS, 0.002 M (EDTA), pH 7.4. The sulfhydryl groups of MNPs were activated as follows: 2 ml of aqueous suspension of pre-activated MNPs at 2 mg Fe ml− 1 was mixed with 25 μmol/g Fe of 2aldrithiol solution during 2 h at 40 °C. After reaction, 200 μl of brine was added and the sample centrifuged 10 min at 10,000 × g and
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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Fig. 1. General scheme of the multifunctionalization of MNPs.
redispersed in 2 ml of 0.01 M sodium phosphate, pH 7.4 and incubated at 4 °C overnight with 200 μl of modified anti-CD47 solution at 700 μg ml− 1 in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.4. After that, the MNP-anti-CD47 was purified by gel filtration through a Sepharose CL-6B column using 0.01 M sodium phosphate buffer, pH 7.4. The remaining sulfhydryl activated groups were blocked at 25 °C for 1 h with 20 μmol/g Fe of 3-mercaptopropionic acid in 0.01 M sodium phosphate, pH 7.4. Finally, MNPs were washed several times with 0.01 M sodium phosphate, pH 7.4 and stored at 4 °C until used. Samples of supernatants before and after the immobilization process were withdrawn and measured using Bradford assay [49]. A reference solution was prepared with exactly the initial antibody concentration and media conditions (pH, ionic strength) and bovine serum albumin (BSA) was used as protein standard. The amount of bound antibodies
was determined from the difference between the not conjugated antibody concentration in the supernatant and the initial antibody (Ab) concentration (μg Ab/mg Fe). 2.3.3. Covalent attachment of Gem and anti-CD47 antibodies on MNPs First, a Gem derivative (2.5 μmol/g Fe) was added to react with sulfhydryl pre-activated MNPs (4 ml at 2 mg Fe ml−1). The covalently immobilized GEM was determined by quantification of the 2pyridinethione released during the reaction (λmax = 343 nm, ε343nm = 8080 M cm− 1). Then the remaining sulfhydryl groups of MNP-Gem were activated as follows: 4 ml of aqueous suspension of sulfhydryl activated MNP-Gem at 2 mg Fe ml− 1 was mixed with 25 μmol/g Fe of 2-aldrithiol for 2 h at 40 °C. After reaction, 200 μl of brine were added and the sample centrifuged 10 min at 10,000 × g
Fig. 2. Cellular uptake of MNPs with or without functionalization with anti-CD47. Prussian blue staining of the pancreatic cell line Panc-1 (a–c) and pancreatic cancer primary cell cultures Panc215 (d–f) and Panc354 (g–i) incubated with non-functionalized MNPs, or MNPs functionalized with anti-CD47 at either 4 or 37 °C. Non-treated cells (NTCs) are shown as control. Main pictures have been acquired with a 10× objective, and insets show 2× zoomed.
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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and redispersed in 4 ml of 0.01 M sodium phosphate, pH 7.4. Finally, the anti-CD47 antibodies were immobilized on MNP-Gem following the same protocol described above for immobilization on MNPs.
After drying the cells, the cover slips were mounted in glass slides using DePeX and finally the cells were observed using light microscopy.
2.4. Drug release studies
2.8. In vitro cytotoxicity assay
The cumulative drug releases from the MNP-Gem and MNP-Gemanti-CD47 were carried out under physiological conditions (pH 7.4 and 37 °C) using two different concentrations of glutathione (GSH) as reducing agent (1 μM and 1 mM of GSH to mimic the extracellular and intracellular conditions, respectively). For each experiment, 4.8 mg of MNP-GEM and MNP-GEM-anti-CD47 were dissolved in 1 ml of 0.01 M phosphate buffer at pH 7.4 containing either 1 μM of GSH or 1 mM GSH and incubated at 37 °C. The amount of GEM released was analyzed at regular time intervals by HPLC using a C18 column, mobile phase water/acetonitrile (80/20), at a flow rate of 0.3 ml min−1, and the absorbance was measured at 270 nm. The percentage of Gem released was calculated from a standard calibration curve of the free-drug solution.
Cell viability was measured using the Resazurin dye (Sigma-Aldrich), which is an indicator of cell viability in proliferation and cytotoxicity assays. To assess cell death, cells were cultured on a 96-well plate at a density of 5 × 104 cells per well in 200 μl of cell culture medium. After 24 h, the growth medium was removed and cells were then incubated overnight at 37 °C in the presence of different concentrations of free Gemcitabine (Gem) (0.1, 0.4 and 1 μM), MNP-Gem and MNPGem-anti-CD47 (0.2 mg Fe ml−1, 4.8 μM Gem, Ab 20 μg/mg Fe). After incubation, cells were washed three times with PBS and then maintained at 37 °C and 5% CO2 incubator. After 7 days, the medium was replaced with cell culture medium containing 10% of Resazurin dye (1 mg ml−1). Cells were maintained at 37 °C and 5% CO2 incubator for approximately 2 h and then a microplate reader was used to determine the amount of Resazurin by measuring the fluorescence of the reaction mixture (λex = 544 nm, λem = 590 nm). 200 μl of 10% of resazurin dye was added to empty wells as a negative control. The viability of the cells was expressed as the percentage of the fluorescence of treated cells (minus the fluorescence of the negative control) in comparison with control, untreated cells (minus the fluorescence of the negative control).
2.5. MNP sterilization We always carried out the MNP sterilization before cell incubation. 1 ml of MNP stock was dispersed by sonication for 5 min and then was filtered through a 0.22 μm Millex-GP filter (Merck-Millipore Darmstadt, Germany). 2.6. Primary human pancreatic cancer cells and cell lines Human PDAC tissues were obtained with written informed consent from all patients and expanded in vivo as patient-derived xenografts (PDXs), as previously described [50]. To generate the primary cell cultures, the PDX tissue fragments were minced, enzymatically digested with collagenase (Stem Cell Technologies, Vancouver, BC) for 90 min at 37 °C and after centrifugation for 5 min at 1200 rpm cell pellets were resuspended and cultured in RPMI supplemented with 10% FBS and 50 units ml− 1 penicillin/streptomycin. These primary cultures were used in vitro only until passage 10. Panc-1 and BxPC3 cell lines were purchased from American Type Culture Collections (Manassas, VA, USA) and were grown as a monolayer in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 50 units ml−1 of penicillin/streptomycin. Primary cancer cells cultures and cell lines were maintained in an incubator at 37 °C in a humidified atmosphere of 95% air and 5% CO2. 2.7. Uptake studies of MNPs and MNP-anti-CD47 To determine the specific targeting of MNP-anti-CD47 for CD47-positive primary cancer cells and cell lines (Panc215, Panc354, and Panc-1) cells were seeded on coverslips and incubated with the different NPs the next day. Cells were incubated for 4 h either at 4 °C or 37 °C, in the presence of MNPs and MNP-anti-CD47 (0.2 mg Fe ml−1, Ab 20 μg/mg Fe), and then washed with PBS and left at 37 °C overnight. After 24 h, cells were washed three times with PBS and then stained for iron content by Prussian blue staining. 2.7.1. Prussian blue staining For Prussian blue staining to evaluate MNP uptake, cells were seeded as described in the section above. Once the incubation time was over, the cells were washed twice with PBS and fixed with ice-cold methanol for 2 min at 4 °C. The cells were then washed twice with PBS, and incubated with a 1:1 mixture of 4% potassium ferrocyanide and 4% hydrochloric acid (Prussian blue staining solution) for 15 min at room temperature. The cells were then washed with distilled water three times and counterstained for cytoplasm with neutral red 0.5% for 2 min at room temperature. The cells were washed with distilled water several times until all the excess neutral red staining came off.
Fig. 3. Cell viability of pancreatic cancer cell lines and primary cell cultures after Gemcitabine treatment. Cells were treated with Gemcitabine, MF66 NPs or MF66-Gem, 24 h after seeding, at the indicated concentrations. Cell viability was measured 6 days after treatment. Mann-Whitney test: **** p b 0.0001, *** p b 0.005, ** p b 0.001 compared to MNPs, ## p b 0.001 compared to equivalent Gemcitabine dose in free form.
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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2.9. Flow cytometry Cells were adjusted to a concentration of 106 cells ml−1 before analysis using a FACS fortessa. DAPI was used for exclusion of dead cells. For the assessment of apoptosis, cells were incubated with DAPI and Annexin V-APC staining kit (BD) according to the manufacturer's instructions. Data were analyzed with FlowJo software. 2.10. Statistical analyses Results are presented as means ± standard deviation unless stated otherwise and significance was determined using the Mann-Whitney test. All analyses were performed using GraphPad Prism. 3. Results and discussion This paper describes the development of a delivery system based on magnetic nanoparticles that allows the targeting of pancreatic cancer cells, resulting in improved uptake by cancer cells and the delivery of two different therapeutic agents with different mechanisms of action. 3.1. Multifunctionalization of MNPs The general strategy followed in order to prepare the functionalized MNP formulations MNP-Gem, MNP-anti-CD47 and MNP-Gem-antiCD47 is shown in Fig. 1. To this end, anti-CD47 antibody functionalized MNPs were produced by the formation of disulfide bonds between the reactive thiol of the modified anti-CD47 antibody with 2-iminothiolane and the activated sulfhydryl groups of the pre-activated MNPs (Fig. 1). The multifunctionalization strategy through disulfide bonds started with the introduction of a controlled amount of Gem followed by
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immobilization of the anti-CD47 antibody on MNP-Gem (Fig. 1). Gem was immobilized onto MNPs though a linker that allows us to perform the release of the Gem selectively in an intracellular environment. The strategy used for the covalent immobilization of the anti-CD47 ensures the correct orientation of the antibody onto the MNPs [29,51]. The synthesis of MNP-Gem started by the reaction of cysteamine with the carboxylic groups of the DMSA coating in the presence of EDC and NHS, leading to a formulation that contains 9 μmol of thiol/g Fe. Then, a Gemcitabine derivative was reacted with the activated MNPs [28]. This conjugation was achieved employing 2.5 μmol of Gem derivative, leading to a formulation that contains 2 μmol Gem/g Fe, with a zeta-potential (ζ) of −50 ± 1 mV and a hydrodynamic diameter (DH) of 97 ± 1 nm (PDI: 0.17). The functionalization of MNPs with antibodies was achieved by the formation of disulfide bonds between the reactive thiol of the modified anti-CD47 antibodies and the activated sulfhydryl groups of the MNPs following the protocol that we previously described [29]. After the reaction, the immobilized antibodies were quantified by the Bradford assay. The standard load obtained of covalently linked anti-CD47 antibodies was 30 mg/g Fe (86%), corresponding to around one antibody molecule per magnetic nanoparticle. The sodium phosphate MNP-anti-CD47 suspension was stable for weeks stored at 4 °C without noticeable precipitation. The physicochemical properties of the formulation were not significantly altered upon the functionalization (ζ: − 47 ± 2 mV and DH: 107 ± 1 nm (PDI: 0.17)). Finally, to obtain MNP-Gem-anti-CD47, firstly the Gem drug derivative was immobilized [28], leading to a formulation that contains 2 μmol Gem/g Fe (ζ: −50 ± 1 mV and DH: 97 ± 1 nm (PDI: 0.17)). Then, the anti-CD47 antibodies were immobilized on MNP-Gem following the protocol that we previously described [29]. The final formulation contains 2 μmol Gem and 30 mg Ab/g Fe, corresponding to 34 molecules
Fig. 4. Cell viability of pancreatic cancer cell lines and primary cell cultures after Gemcitabine treatment. Cells were treated with Gemcitabine, MF66 NPs or MF66-Gemcitabine, 24 h after seeding, at a concentration of 1 μM. Cell viability was measured 6 days after treatment.
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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of Gem and 1 molecule of Ab per MNPs (ζ: −45 ± 1 mV and DH: 109 ± 1 nm (PDI: 0.17)). 3.2. Drug release studies To evaluate the stimuli-response behavior of the MNP-Gem and MNP-Gem-anti-CD47 under a reducing environment, the drug release was monitored at 37 °C in 0.01 M sodium phosphate, pH 7.4 using 1 μM or 1 mM of GSH to mimic the extracellular and intracellular conditions, respectively (data not shown). These formulations showed a similar standard release that we previously described [29] 96–98% when treated with 1 mM GSH (mimicking an intracellular environment) after 6–8 h while only 3–5% of the cargo was released with 1 μM GSH (mimicking the extracellular environment) after 6–8 h. These results show that the release of Gem from these is selective and strongly dependent on the reducing environment, so that it will take place mostly inside the cells and is not affected by the presence of the antibody. 3.3. MNP uptake studies To determine the interaction of MNPs with the cells and the effect of the anti-CD47 antibody in the targeting of MNPs, pancreatic cancer cells expressing the CD47 receptor were incubated for 1 h at 37 °C, with either MNP-anti-CD47 and MNPs (as a negative control), followed by washes with PBS and then incubated for an additional 24 h at 37 °C.
Fig. 6. Cell viability of pancreatic cancer cell lines and primary cell cultures after Gem or anti-CD47 treatment. Cells were treated with Gem, anti-CD47, MNPs, MNP-Gem, MNPanti-CD47, or MNP-Gem-anti-CD47, 24 h after seeding, at the indicated concentrations. Cell viability was measured 6 days after treatment. Mann-Whitney test: * p b 0.05, ns p N 0.05 compared to Gem 1 μM.
Prussian blue staining was used to monitor the presence of iron from the MNPs inside the cells. As shown in Fig. 2, non-functionalized MNPs were barely detected by the Prussian blue staining inside of two primary pancreatic cancer cultures (Panc215 and Panc354), which closely reflect patients'intratumoral heterogeneity and are kept at very low passages as well as the established cell line Panc-1 (Fig. 2, panels b, e, h). On the other hand, MNPs functionalized with anti-CD47 mAb were detected in the cytoplasm of all cell types tested, when incubated at 37 °C (Fig. 2, panels c, f, i). These results demonstrate that functionalizing the MF66 MNPs with anti-CD47 mAb greatly improves their cellular uptake by pancreatic cancer cells. In addition, to demonstrate the selective targeting through CD47 receptors, the cells were incubated with free anti-CD47 prior to the addition of MNP-anti-CD47, which significantly reduced the uptake of MNP-anti-CD47 (data not shown). 3.4. In vitro Gem-MNP cytotoxicity studies
Fig. 5. Apoptosis induction after treatment with anti-CD47 antibody. Cells were treated with anti-CD47, MNPs or MNP-anti-CD47, 24 h after seeding, at the indicated concentrations. The percentage of annexin V-positive cells was determined by flow cytometry 20 h after treatment. Mann-Whitney test: * compared to control, # compared to MNPs.
To assess the efficiency of Gemcitabine delivery to pancreatic cancer cells, we first incubated Panc215 and Panc354 two of the cell types with a range of Gemcitabine concentrations, either in its usual form – free Gem – or conjugated to MNPs (MNP-Gem) (Fig. 3, A and B). From these results it can be observed that, first of all, MNPs by themselves do not cause significant cytotoxicity to the pancreatic cancer cells. The minimal toxicity observed could be due to the fact that the incubation with MNPs can promote changes in both pH and composition of the cell culture media due to their known ability to adsorb components
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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such as proteins (protein corona effect), amino acids, vitamins or ions. When MNPs are attached to the cell surface there might be changes as to how cell-to-cell interactions occur, as some of them might be physically hampered, and while in the cytoplasm, MNPs have been shown to elicit a small increase in cellular oxidative stress through the production of reactive oxygen species (ROS) [52]. Moreover, and as expected, Gemcitabine is slightly less efficient when delivered attached to MNPs than free-Gem, as can be seen more clearly for the lower dose used (0.1 μM). This result is expected since Gemcitabine coupled to the MNPs has another internalization mechanism compared to free Gemcitabine, when administered to cells in vitro. While free-Gem is ready to be taken up by the cells, MNP-Gem internalization is slower and after uptake Gem has to be released from the MNPs to be able to find its way to the nucleus to inhibit the DNA synthesis, causing cell death. Therefore, the lower efficiency of MNP-Gem compared to free-Gem could potentially be explained by a combination of factors including first the slower uptake mechanism of MNP-Gem resulting in an overall lower uptake since not all MNP-Gem are internalized by the cells in the 24 h of incubation time, second the slower mechanism of action once internalized since a release step is required, and third the possibility of the MNP uptake being influenced by the formation of the protein corona [53]. However, it is important to note that the usual concentration at which Gem is used for in vitro studies using primary pancreatic cancer cell cultures is 100 ng ml− 1 (or 0.333 μM) [50]. Our results using 0.4 μM of Gem show that at this or higher concentrations, the effect of free-Gem and MNP-Gem is similar, probably because a saturating concentration is reached. Therefore, the different uptake mechanism and release kinetic coupling of Gemcitabine to the MNPs does not constitute a major hurdle for Gemcitabine administration at its therapeutic concentration. Next we validated these findings on different pancreatic cancer cell types, including the commonly used cell lines Panc-1 and Bx-PC3, by repeating the experiment using only the higher concentration 1 μM (Fig. 4). The results show that, for this concentration, the effect of free-Gem or MNP-Gem is comparable in all cases. The proposed system is of great interest as it shows that Gemcitabine can be linked to the MNPs without losing its cytotoxic activity and can therefore be combined with other drugs in the same NPs to create a multifunctionalized platform that delivers a combination of treatments. Moreover, if it could be demonstrated that the multifunctionalized MNPs accumulate specifically in the tumors, lower doses of the active compounds would be necessary as the biodistribution would be more favorable.
3.5. Apoptosis induction by MNP-anti-CD47 We have shown before that CD47 is expressed on pancreatic cancer (stem) cells and that the anti-CD47 antibody not only enables phagocytosis of these cells by macrophages, but also directly induced apoptosis of the cancer cells, while exerting no effect on non-malignant cells [43]. Here we have shown already that coupling anti-CD47 on the surface of MNPs greatly improves their uptake by pancreatic cancer cells, but we also wanted to confirm whether the anti-CD47 retains its ability to induce apoptosis in pancreatic cancer cells, after being immobilized on the surface of MNPs. As can be seen in Fig. 5, there is a dose dependent increase on the percentage of Annexin V-positive cells upon treatment with free anti-CD47. When administered at the same dose of 4 μM but in the form of MNP-anti-CD47, the anti-CD47 antibody promotes similar levels of apoptosis induction in the pancreatic cancer cells tested Panc215 and Panc354. Therefore, the functionalization on MNPs with anti-CD47 not only increases their uptake by pancreatic cancer cells (Fig. 2) but also has a therapeutic effect of promoting the apoptosis of these cells (Fig. 5). It can be concluded at this point that both Gem and the anti-CD47 antibody retain their biological function after being conjugated to MNPs.
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3.6. In vitro cytotoxicity studies of multifunctionalized MNPs We next wanted to determine if we could achieve even higher levels of cytotoxicity on pancreatic cancer cells by using multifunctionalized NPs combining Gemcitabine and anti-CD47 conjugated to MNPs. The results shown in Fig. 6 show that the effect of anti-CD47 on apoptosis that we observed 20 h after treatment (Fig. 5) is not observed anymore when the cell viability was measured 6 days after treatment. This could be due to the fact that only a small population of cells is affected by this treatment, potentially the cancer stem cells, and the effect is masked by the proliferation of the more differentiated cells. These findings are in line with our previous findings that showed that CD47 targeting as a single treatment strategy was not effective in vivo, while the combination with chemotherapy was able to promote long lasting tumor regression [43]. Since no effect on cytotoxicity was observed for anti-CD47 antibody alone or conjugated to MNPs, it was not surprising that there was no additional in vitro cytotoxicity when we combined Gemcitabine and anti-CD47 in the same NPs. However, this result does not mean that there would not be a measurable effect in vivo as in this context multiple administrations of the therapeutic combination are usually performed, and a layer by layer elimination of the tumoral tissue is expected to happen. Nevertheless, the fact that there was no decrease on the cytotoxic activity that must attributed to Gem, given the lack of effect of anti-CD47 antibody alone, demonstrates the potential of producing MNPs combining different types of therapeutic agents without them losing their biological activities. It would be interesting to further modify this delivery platform to deliver more or different drugs, such as paclitaxel, and/or to conjugate them with antibodies that bear an even greater level of tumor specificity, as well as with molecules that prolong circulation time and avoid the recognition of these delivery systems by the reticuloendothelial system (RES) and their uptake by liver cells, such as poly(ethylene glycol) (PEG), in order to make them suitable for intravenous administration. 4. Conclusions We have developed a multifunctional nanoformulation of MNPs for selective multimodal treatment of pancreatic cancer, including Gemcitabine as a chemotherapeutic drug and anti-CD47 antibody as adjuvant of the treatment. The designed immobilization strategy preserves both the stability and activity of the conjugated molecules. The multifunctionalization does not affect the selective drug release, which only occurs under reducing intracellular conditions, thus keeping the intracellular selectivity of the treatment. In addition, the cytotoxic activity of the drug delivered by the nanoparticles is comparable to the effect of the free drug at therapeutic drug concentrations. The immobilized antiCD47 antibody retains its targeting activity, thus increasing the delivery of the nanoformulation to the cancer cells during short incubation times. In addition, the tagged anti-CD47 antibody shows efficient induction of apoptosis in pancreatic cancer cells as its effect is larger compared to the free antibody. These data suggest that the apoptotic activity of the antibody is not only preserved but can be enhanced by its delivery by MNPs. Finally the cytotoxic activity of the multifunctional nanoformulation is not increased in the in vitro studies. This result is not unexpected since the specific apoptotic effect of the anti-CD47 antibody might preferentially affect the small subpopulation of CSCs and these effects are not easily detectable among bulk cancer cells in an in vitro set up. Therefore, since the effects of targeting CSCs responsible for the tumor relapse by using the anti-CD47 antibody have been demonstrated in preclinical in vivo studies, the developed platform bears great potential for future therapeutic development. Certainly, before using these nanoparticles in vivo, the platform needs to be further optimized in order to be stable in circulation and invisible to the reticuloendothelial system (RES) to avoid premature clearance, which is typically done by covering the nanoparticles with proteins or polymers, such as BSA or polyethyleneglycol (PEG) [54]. Also, as the use of antibodies can evoke
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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an immune response, an antibody fragment or synthetic peptide with the bioactive region could be used instead of the whole antibody. Transparency Document The Transparency document associated with this article can be found, in the online version. Acknowledgements This work was supported by EU-FP7 MULTIFUN project (no. 262943), and by the Comunidad de Madrid NANOFRONTMAG-CM project (S2013/MIT-2850). References [1] U. Jeong, X. Teng, Y. Wang, H. Yang, Y. Xia, Superparamagnetic colloids: controlled synthesis and niche applications, Adv. Mater. 19 (2007) 33–60. [2] C. Cheng, F. Xu, H. Gu, Facile synthesis and morphology evolution of magnetic iron oxide nanoparticles in different polyol processes, New J. Chem. 35 (2011) 1072–1079. [3] M. Mahmoudi, S. Sant, B. Wang, S. Laurent, T. Sen, Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy, Adv. Drug Deliv. Rev. 63 (2011) 24–46. [4] A. Ito, M. Shinkai, H. Honda, T. Kobayashi, Medical application of functionalized magnetic nanoparticles, J. Biosci. Bioeng. 100 (2005) 1–11. [5] J. Xie, S. Jon, Magnetic nanoparticle-based theranostics, Theranostics 2 (2012) 122–124. [6] A.P. Khandhar, R.M. Ferguson, H. Arami, K.M. Krishnan, Monodisperse magnetite nanoparticle tracers for in vivo magnetic particle imaging, Biomaterials 34 (2013) 3837–3845. [7] S.C. Tang, I.M. Lo, Magnetic nanoparticles: essential factors for sustainable environmental applications, Water Res. 47 (2013) 2613–2632. [8] R. Hudson, Y. Feng, R.S. Varma, A. Moores, Bare magnetic nanoparticles: sustainable synthesis and applications in catalytic organic transformations, Green Chem. 16 (2014) 4493–4505. [9] J.M. Perez, F.J. Simeone, Y. Saeki, L. Josephson, R. Weissleder, Viral-induced self-assembly of magnetic nanoparticles allows the detection of viral particles in biological media, J. Am. Chem. Soc. 125 (2003) 10192–10193. [10] W. Li, S. Tutton, A.T. Vu, L. Pierchala, B.S. Li, J.M. Lewis, P.V. Prasad, R.R. Edelman, First-pass contrast-enhanced magnetic resonance angiography in humans using ferumoxytol, a novel ultrasmall superparamagnetic iron oxide (USPIO)-based blood pool agent, J. Magn. Reson. Imaging 21 (2005) 46–52. [11] M.A. McAteer, N.R. Sibson, C. von Zur Muhlen, J.E. Schneider, A.S. Lowe, N. Warrick, K.M. Channon, D.C. Anthony, R.P. Choudhury, In vivo magnetic resonance imaging of acute brain inflammation using microparticles of iron oxide, Nat. Med. 13 (2007) 1253–1258. [12] C.C. You, O.R. Miranda, B. Gider, P.S. Ghosh, I.B. Kim, B. Erdogan, S.A. Krovi, U.H. Bunz, V.M. Rotello, Detection and identification of proteins using nanoparticle-fluorescent polymer ‘chemical nose’ sensors, Nat. Nanotechnol. 2 (2007) 318–323. [13] V. Hsieh, A. Jasanoff, Bioengineered probes for molecular magnetic resonance imaging in the nervous system, ACS Chem. Neurosci. 3 (2012) 593–602. [14] S. Gandhi, H. Arami, K.M. Krishnan, Detection of cancer-specific proteases using magnetic relaxation of peptide-conjugated nanoparticles in biological environment, Nano Lett. 16 (2016) 3668–3674. [15] J. Dobson, Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery, Gene Ther. 13 (2006) 283–287. [16] B. Thiesen, A. Jordan, Clinical applications of magnetic nanoparticles for hyperthermia, Int. J. Hyperth. 24 (2008) 467–474. [17] J.D. Hood, M. Bednarski, R. Frausto, S. Guccione, R.A. Reisfeld, R. Xiang, D.A. Cheresh, Tumor regression by targeted gene delivery to the neovasculature, Science 296 (2002) 2404–2407. [18] S. Laurent, S. Dutz, U.O. Hafeli, M. Mahmoudi, Magnetic fluid hyperthermia: focus on superparamagnetic iron oxide nanoparticles, Adv. Colloid Interf. Sci. 166 (2011) 8–23. [19] J.S. Beveridge, J.R. Stephens, M.E. Williams, The use of magnetic nanoparticles in analytical chemistry, Annu Rev Anal Chem (Palo Alto, Calif) 4 (2011) 251–273. [20] M.A. Ahmed, S.M. Ali, S.I. El-Dek, A. Galal, Magnetite–hematite nanoparticles prepared by green methods for heavy metal ions removal from water, Mater. Sci. Eng. B 178 (2013) 744–751. [21] L. Borlido, A.M. Azevedo, A.C. Roque, M.R. Aires-Barros, Magnetic separations in biotechnology, Biotechnol. Adv. 31 (2013) 1374–1385. [22] J. He, M. Huang, D. Wang, Z. Zhang, G. Li, Magnetic separation techniques in sample preparation for biological analysis: a review, J. Pharm. Biomed. Anal. 101 (2014) 84–101. [23] B.J. Boyd, Past and future evolution in colloidal drug delivery systems, Expert Opin. Drug Deliv. 5 (2008) 69–85. [24] V.P. Torchilin, Multifunctional nanocarriers, Adv. Drug Deliv. Rev. 58 (2006) 1532–1555. [25] O. Veiseh, J.W. Gunn, M. Zhang, Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging, Adv. Drug Deliv. Rev. 62 (2010) 284–304.
[26] J.D. Byrne, T. Betancourt, L. Brannon-Peppas, Active targeting schemes for nanoparticle systems in cancer therapeutics, Adv. Drug Deliv. Rev. 60 (2008) 1615–1626. [27] M. Talelli, A. Aires, M. Marciello, Protein-modified magnetic nanoparticles for biomedical applications, Curr. Org. Chem. 20 (2016) 1252–1261. [28] A. Latorre, P. Couleaud, A. Aires, A.L. Cortajarena, Á. Somoza, Multifunctionalization of magnetic nanoparticles for controlled drug release: a general approach, Eur. J. Med. Chem. 82 (2014) 355–362. [29] A. Aires, S. Ocampo, B. Simões, J. Rodríguez, J. Cadenas, P. Couleaud, K. Spence, A. Latorre, R. Miranda, A. Somoza, R. Clarke, J. Carrascosa, A. Cortajarena, Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44positive cancer cells, Nanotechnology 27 (2016) 065103. [30] M.A. Firer, G. Gellerman, Targeted drug delivery for cancer therapy: the other side of antibodies, J. Hematol. Oncol. 5 (2012) 70. [31] Y. Sun, F. Yu, B.W. Sun, Antibody-drug conjugates as targeted cancer therapeutics, Yao Xue Xue Bao 44 (2009) 943–952. [32] C. Peters, S. Brown, Antibody-drug conjugates as novel anti-cancer chemotherapeutics, Biosci. Rep. 35 (2015). [33] H.A. Burris III, M.J. Moore, J. Andersen, M.R. Green, M.L. Rothenberg, M.R. Modiano, M.C. Cripps, R.K. Portenoy, A.M. Storniolo, P. Tarassoff, R. Nelson, F.A. Dorr, C.D. Stephens, D.D. Von Hoff, Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial, J. Clin. Oncol. 15 (1997) 2403–2413. [34] T. Conroy, F. Desseigne, M. Ychou, O. Bouche, R. Guimbaud, Y. Becouarn, A. Adenis, J.L. Raoul, S. Gourgou-Bourgade, C. de la Fouchardiere, J. Bennouna, J.B. Bachet, F. Khemissa-Akouz, D. Pere-Verge, C. Delbaldo, E. Assenat, B. Chauffert, P. Michel, C. Montoto-Grillot, M. Ducreux, U. Groupe Tumeurs Digestives of, P. Intergroup, FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer, N. Engl. J. Med. 364 (2011) 1817–1825. [35] D.D. Von Hoff, T. Ervin, F.P. Arena, E.G. Chiorean, J. Infante, M. Moore, T. Seay, S.A. Tjulandin, W.W. Ma, M.N. Saleh, M. Harris, M. Reni, S. Dowden, D. Laheru, N. Bahary, R.K. Ramanathan, J. Tabernero, M. Hidalgo, D. Goldstein, E. Van Cutsem, X. Wei, J. Iglesias, M.F. Renschler, Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine, N. Engl. J. Med. 369 (2013) 1691–1703. [36] N. Desai, V. Trieu, B. Damascelli, P. Soon-Shiong, SPARC expression correlates with tumor response to albumin-bound paclitaxel in head and neck cancer patients, Transl. Oncol. 2 (2009) 59–64. [37] A. Sparreboom, C.D. Scripture, V. Trieu, P.J. Williams, T. De, A. Yang, B. Beals, W.D. Figg, M. Hawkins, N. Desai, Comparative preclinical and clinical pharmacokinetics of a cremophor-free, nanoparticle albumin-bound paclitaxel (ABI-007) and paclitaxel formulated in Cremophor (Taxol), Clin. Cancer Res. 11 (2005) 4136–4143. [38] E.R. Gardner, W.L. Dahut, C.D. Scripture, J. Jones, J.B. Aragon-Ching, N. Desai, M.J. Hawkins, A. Sparreboom, W.D. Figg, Randomized crossover pharmacokinetic study of solvent-based paclitaxel and nab-paclitaxel, Clin. Cancer Res. 14 (2008) 4200–4205. [39] N.P. Desai, V. Trieu, L.Y. Hwang, R. Wu, P. Soon-Shiong, W.J. Gradishar, Improved effectiveness of nanoparticle albumin-bound (nab) paclitaxel versus polysorbatebased docetaxel in multiple xenografts as a function of HER2 and SPARC status, Anti-Cancer Drugs 19 (2008) 899–909. [40] P.C. Hermann, S.L. Huber, T. Herrler, A. Aicher, J.W. Ellwart, M. Guba, C.J. Bruns, C. Heeschen, Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer, Cell Stem Cell 1 (2007) 313–323. [41] I. Miranda-Lorenzo, J. Dorado, E. Lonardo, S. Alcala, A.G. Serrano, J. Clausell-Tormos, M. Cioffi, D. Megias, S. Zagorac, A. Balic, M. Hidalgo, M. Erkan, J. Kleeff, A. Scarpa, B. Sainz Jr., C. Heeschen, Intracellular autofluorescence: a biomarker for epithelial cancer stem cells, Nat. Methods 11 (2014) 1161–1169. [42] C. Li, D.G. Heidt, P. Dalerba, C.F. Burant, L. Zhang, V. Adsay, M. Wicha, M.F. Clarke, D.M. Simeone, Identification of pancreatic cancer stem cells, Cancer Res. 67 (2007) 1030–1037. [43] M. Cioffi, S. Trabulo, M. Hidalgo, E. Costello, W. Greenhalf, M. Erkan, J. Kleeff, B. Sainz Jr., C. Heeschen, Inhibition of CD47 effectively targets pancreatic cancer stem cells via dual mechanisms, Clin. Cancer Res. 21 (2015) 2325–2337. [44] M. Au, T.I. Emeto, J. Power, V.N. Vangaveti, H.C. Lai, Emerging therapeutic potential of nanoparticles in pancreatic cancer: a systematic review of clinical trials, Biomedicine 4 (2016) 20. [45] E. Fernandes, J.A. Ferreira, P. Andreia, L. Luis, S. Barroso, B. Sarmento, L.L. Santos, New trends in guided nanotherapies for digestive cancers: a systematic review, J. Control. Release 209 (2015) 288–307. [46] S. Kossatz, R. Ludwig, H. Dähring, V. Ettelt, G. Rimkus, M. Marciello, G. Salas, V. Patel, F.J. Teran, I. Hilger, High therapeutic efficiency of magnetic hyperthermia in xenograft models achieved with moderate temperature increases in the tumor area, Pharm. Res. 31 (12) (2014) 3274–3288. [47] C.T.E, in: C.T.E (Ed.), Disulfide Bonds Between Cysteine Residues Protein Structure: A Practical Approach, IRL Press at Oxford University Press, Oxford 1989, pp. 155–167. [48] S. Puertas, M. Moros, R. Fernández-Pacheco, M.R. Ibarra, V. Grazú, J.M. de la Fuente, Designing novel nano-immunoassays: antibody orientation versus sensitivity, J. Phys. D. Appl. Phys. 43 (2010) 474012–474019. [49] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [50] M.T. Mueller, P.C. Hermann, J. Witthauer, B. Rubio-Viqueira, S.F. Leicht, S. Huber, J.W. Ellwart, M. Mustafa, P. Bartenstein, J.G. D'Haese, M.H. Schoenberg, F. Berger, K.W. Jauch, M. Hidalgo, C. Heeschen, Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer, Gastroenterology 137 (2009) 1102–1113.
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
S. Trabulo et al. / Biochimica et Biophysica Acta xxx (2017) xxx–xxx [51] C. Grüttner, K. Müller, J. Teller, F. Westphal, A. Foreman, R. Ivkov, Synthesis and antibody conjugation of magnetic nanoparticles with improved specific power absorption rates for alternating magnetic field cancer therapy, J. Magn. Magn. Mater. 311 (2007) 181–186. [52] H. Markides, M. Rotherham, A.J. El Haj, Biocompatibility and toxicity of magnetic nanoparticles in regenerative medicine, J. Nanomater. 2012 (2012) 1–11.
9
[53] M. Hadjidemetriou, Z. Al-Ahmady, M. Mazza, R.F. Collins, K. Dawson, K. Kostarelos, In vivo biomolecule corona around blood-circulating, clinically used and antibodytargeted lipid bilayer nanoscale vesicles, ACS Nano 9 (2015) 8142–8156. [54] A. Aires, S.M. Ocampo, D. Cabrera, L. de la Cueva, G. Salas, F.J. Teran, A.L. Cortajarena, BSA-coated magnetic nanoparticles for improved therapeutic properties, J. Mater. Chem. B 3 (2015) 6239–6247.
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Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells☆ Sara Trabulo a,b,1, Antonio Aires c,d,1, Alexandra Aicher a, Christopher Heeschen a,b,⁎, Aitziber L. Cortajarena c,d,e,⁎⁎ a
Stem Cells & Cancer Group, Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain Centre for Stem Cells in Cancer & Ageing, Barts Cancer Institute, Queen Mary University of London, EC1M 6BQ, UK CIC BiomaGUNE, Parque Tecnológico de San Sebastián, Paseo Miramón 182, Donostia-San Sebastián 20009, Spain d IMDEA Nanociencia and Nanobiotechnology Unit associated to Centro Nacional de Biotecnología (CNB-CSIC), Campus Universitario de Cantoblanco, Madrid 28049, Spain e Ikerbasque, Basque Foundation for Science, Mª Díaz de Haro 3, 48013 Bilbao, Spain b c
a r t i c l e
i n f o
Article history: Received 27 October 2016 Received in revised form 26 December 2016 Accepted 28 January 2017 Available online xxxx Keywords: Pancreatic cancer Magnetic nanoparticles Multifunctionalization Nanocarriers CD47 Gemcitabine Controlled drug release Active targeting Nanomedicine
a b s t r a c t Nanomedicine nowadays offers novel solutions in cancer therapy by introducing multimodal treatments in one single formulation. In addition, nanoparticles act as nanocarriers changing the solubility, biodistribution and efficiency of the therapeutic molecules, thus generating more efficient treatments and reducing their side effects. To apply these novel therapeutic approaches, efforts are focused on the multi-functionalization of the nanoparticles and will open up new avenues to advanced combinational therapies. Pancreatic ductal adenocarcinoma (PDAC) is a cancer with unmet medical needs. Abundant expression of the anti-phagocytosis signal CD47 has also been observed on pancreatic cancer cells, in particular a subset of cancer stem cells (CSCs) responsible for resistance to standard therapy and metastatic potential. CD47 receptor is found on pancreatic cancer and highly expressed on CSCs, but not on normal pancreas. Inhibiting CD47 using monoclonal antibodies has been shown as an effective strategy to treat PDAC in vivo. However, CD47 inhibition effectively slowed tumor growth only in combination with Gemcitabine or Abraxane. In this work, we present the generation of multifunctionalized iron oxide magnetic nanoparticles (MNPs) that include the anti-CD47 antibody and the chemotherapeutic drug Gemcitabine in a single formulation. We demonstrate the in vitro efficacy of the formulation against CD47-positive pancreatic cancer cells. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Magnetic nanoparticles (MNPs) have been explored during the last decades in several scientific fields based on their excellent physical and chemical properties, such as superparamagnetism, good colloidal stability, low toxicity and good biocompatibility [1–8]. In recent years MNPs have been widely investigated for their use in biomedical applications including diagnosis via magnetic biosensing or magnetic resonance imaging (MRI) [9–14], therapy [15–18] and magnetic separation techniques [19–22].
☆ This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader. ⁎ Correspondence to: C. Heeschen, Stem Cells & Cancer Group, Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain. ⁎⁎ Correspondence to: A.L. Cortajarena, CIC BiomaGUNE, Parque Tecnológico de San Sebastián, Paseo Miramón 182, Donostia-San Sebastián 20009, Spain. E-mail addresses: [email protected] (C. Heeschen), [email protected] (A.L. Cortajarena). 1 These authors contributed equally to this work.
Even though therapeutic systems based on magnetic nanoparticles have received a lot of attention in nanomedicine, controlling the targeting of specific cells still remains a challenge. Passively targeted nanoparticles via the enhanced permeability and retention (EPR) effect fail to accomplish tumor specificity in many examples. Therefore, the conjugation of cell-specific targeting ligands on the surface of magnetic nanoparticles that enable receptor-mediated endocytosis can be advantageous, as it can provide specificity, increasing the efficiency of treatments and reducing their side effects [23–25]. A great variety of targeting ligands for cancer diagnosis and therapy have been developed [26]. Generally active targeting is accomplished by the attachment of a targeting ligand on the surface of the MNP, which specifically recognizes a receptor that is over-expressed on the target cells. The most common method for the preparation of actively targeted MNPs is the conjugation of antibodies, antibody fragments, peptides, sugars, or small molecule ligands [27]. Nanoparticles (NPs) are ideal platforms for developing novel combinational therapies, as they act as nanocarriers that can be functionalized with multiple components. NPs developed for biomedical applications require careful physicochemical and targeting design, but also require
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Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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additional considerations paid to drug loading, transport, and release [25]. By developing advanced multifunctionalization strategies, NPs can be decorated with the desired loads of each active component [28], which include targeting agents, standard chemotherapeutic agents, and novel therapeutic agents (e.g. therapeutic antibodies). The multifunctionalization is performed in a way that first, the NPs are able to carry and protect a significant drug payload; second, multiple therapeutic agents can be properly loaded remaining functionally active; and third, the therapeutic cargo release mechanism and rate are modulated for optimal therapeutic efficacy. In this sense, we have previously shown how to generate multifunctional MNPs with large drug payloads per targeting agent (200:1) [29], which cannot be reached by antibody-based targeted drug delivery approaches [30–32]. Overall, nanomedicine has opened new avenues towards the development of selective, minimally-invasive and efficient therapeutic modalities against several diseases such as cancer. In particular, pancreatic cancer is currently the fifth cause of cancer-related deaths and is characterized by early metastasis, extensive desmoplasia and pronounced resistance to chemotherapy and radiation. Regardless of the extensive research efforts, no major progress has been made regarding improvement in clinical endpoints recently. Since the introduction of the antimetabolite Gemcitabine in the '90s [33], only the use of combination therapies such as Folfirinox [34], and more recently the combination of Gemcitabine with nab-paclitaxel (Abraxane) [35], has been able to moderately extend the median survival, but eventually the vast majority of patients still succumb to the disease. In particular, the studies using Abraxane, which is a colloidal suspension of 130 nm particles prepared by homogenization of paclitaxel with human serum albumin, highlight the importance of drug delivery and targeting [36] on improving the pharmacokinetics [37], the bioavailability [38] and the intra-tumoral accumulation of drugs [39] and therefore the impact that an appropriate delivery can have on the clinical outcome of a given drug. It is therefore of the utmost importance not only to develop new effective anti-PDAC treatments but to develop systems that are able to specifically deliver the combination of the different therapeutic agents to the tumor cells. It has been shown for different types of cancers that distinct populations of cells with stem cell properties, namely cancer stem cells (CSCs), are responsible for resistance to standard therapy and bear metastatic potential. We and others have provided conclusive evidence for such a hierarchical organization in human pancreatic ductal adenocarcinoma (PDAC) [40–42], and the survival of such resistant CSCs during chemotherapy can justify the later relapse of the disease that still happens to most of the patients. We have recently shown that CD47 is expressed on primary PDAC cells and we have demonstrated that inhibiting CD47 function using monoclonal antibodies constitutes an effective strategy to treat PDAC [43]. In summary, we observed that treatment with monoclonal antibodies for CD47 in combination with Gemcitabine or Abraxane significantly reduced primary tumor growth, using PDX models of PDAC. Specifically, we found that anti-CD47 therapy alone had only marginal impact on the size and rate of tumor growth, while treatment in combination with Gemcitabine or Abraxane resulted in efficient growth control of tumors and prevented relapse after discontinuation of treatment [43]. These results suggest that anti-CD47 therapy could be used to treat primary PDAC tumors, but combination with other anti-cancer therapeutic agents, such as Abraxane or Gemcitabine, is needed [43]. Therefore, the development of novel nanotherapies, including actively targeted MNPs with the possibility of combining different therapeutic agents in a single vehicle seems highly attractive to overcome the difficulties of treating pancreatic cancer [44,45]. In this work we introduce a new strategy for pancreatic cancer treatment based on nanotechnology by combining the inhibition of CD47, the chemotherapeutic effect of Gemcitabine, and the efficient vehiculization of those components in a single formulation by using nanoparticles. For this purpose we developed multifunctional MNPs that target a receptor overexpressed in pancreatic cancer cells
allowing at the same time the delivery of therapeutic agents with different mechanisms of action without compromising their biological activity. This approach resulted in an effective delivery of both Gemcitabine and anti-CD47 antibody to pancreatic cancer cells. Thus, MNPs build up a good platform that can be further improved for in vivo applications to administer other drug combinations currently evaluated to increase the efficiency of treatments, while reducing their side effects. 2. Materials and methods 2.1. Materials Purified anti-human CD47 antibodies (B6H12 clone) were purchased from eBioscience (anti-CD47). Gemcitabine (Gem) was purchased from Fluorochem. Ultrapure reagent grade water (18.2 MΩ, Wasserlab) was used in all experiments. Dimercaptosuccinic acid (DMSA) coated MNPs were produced by means of the co-precipitation technique as described before [46] and have been provided by Liquid Research Ltd. (UK). The MNPs (MF66) have a zeta potential: −56 mV, an average core size of 12 ± 3 nm, and a hydrodynamic diameter (Zaverage): 97 nm (PDI: 0.18). These MNPs present good stability in PBS buffer and RPMI cell culture medium with and without 10% of bovine serum, showing a hydrodynamic size of 98 nm (PDI: 0.18) in PBS, 100 nm (PDI: 0.18) in RPMI without serum and 105 nm (PDI: 0.18) in RPMI 10% serum. A Gemcitabine derivative, Gem-S-S-Pyr was prepared according to described procedures [28]. 2.2. Measurements Ultraviolet-visible (UV–Vis) and fluorescence spectra were recorded on a Synergy H4 microplate reader (BioTek) using 96-well plates. Hydrodynamic diameter and zeta potential measurements were determined using a Zetasizer Nano-ZS device (Malvern Instruments). Hydrodynamic diameter and zeta potential were measured from dilute sample suspensions (0.1 mg Fe ml−1) in water at pH 7.4 using a zeta potential cell. High-performance liquid chromatography (HPLC) was performed using a 1260 Infinity HPLC (Agilent Technologies) with a ZORBAX 300SB-C18 column 5 μm, 9.4 × 250 mm. 2.3. Multifunctionalization of MNPs 2.3.1. MNP activation 6 ml of MNPs at 2 mg Fe ml−1 were incubated overnight at 37 °C with 50 μmol of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC)/g Fe, 25 μmol of n-hydroxy succinimide (NHS)/g Fe and 25 μmol of cysteamine hydrochloride/g Fe, previously neutralized by 1 M equivalent of sodium hydroxide (NaOH). After 16 h, the sample was washed by cycles of centrifugation and redispersion in Milli-Q water 3 times. The presence of sulfhydryl groups introduced onto the MNPs was quantitatively measured by reaction with 2,4dinitrothiocyanatebenzene (DNTB) [47]. 2.3.2. Covalent attachment of anti-CD47 antibodies on MNPs A solution of the anti-CD47 (1 mg ml− 1) in 0.01 M 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.15 M sodium chloride (NaCl), pH 8.2, was incubated for 1 h at room temperature with a 5 M equivalent solution of 2-iminothiolane. This strategy, as has been previously described, allows the oriented immobilization of the antibodies on the nanoparticles [29,48]. After that, the modified antibody was purified by gel filtration through a desalting resin (Sephadex G-25) using PBS, 0.002 M (EDTA), pH 7.4. The sulfhydryl groups of MNPs were activated as follows: 2 ml of aqueous suspension of pre-activated MNPs at 2 mg Fe ml− 1 was mixed with 25 μmol/g Fe of 2aldrithiol solution during 2 h at 40 °C. After reaction, 200 μl of brine was added and the sample centrifuged 10 min at 10,000 × g and
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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Fig. 1. General scheme of the multifunctionalization of MNPs.
redispersed in 2 ml of 0.01 M sodium phosphate, pH 7.4 and incubated at 4 °C overnight with 200 μl of modified anti-CD47 solution at 700 μg ml− 1 in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.4. After that, the MNP-anti-CD47 was purified by gel filtration through a Sepharose CL-6B column using 0.01 M sodium phosphate buffer, pH 7.4. The remaining sulfhydryl activated groups were blocked at 25 °C for 1 h with 20 μmol/g Fe of 3-mercaptopropionic acid in 0.01 M sodium phosphate, pH 7.4. Finally, MNPs were washed several times with 0.01 M sodium phosphate, pH 7.4 and stored at 4 °C until used. Samples of supernatants before and after the immobilization process were withdrawn and measured using Bradford assay [49]. A reference solution was prepared with exactly the initial antibody concentration and media conditions (pH, ionic strength) and bovine serum albumin (BSA) was used as protein standard. The amount of bound antibodies
was determined from the difference between the not conjugated antibody concentration in the supernatant and the initial antibody (Ab) concentration (μg Ab/mg Fe). 2.3.3. Covalent attachment of Gem and anti-CD47 antibodies on MNPs First, a Gem derivative (2.5 μmol/g Fe) was added to react with sulfhydryl pre-activated MNPs (4 ml at 2 mg Fe ml−1). The covalently immobilized GEM was determined by quantification of the 2pyridinethione released during the reaction (λmax = 343 nm, ε343nm = 8080 M cm− 1). Then the remaining sulfhydryl groups of MNP-Gem were activated as follows: 4 ml of aqueous suspension of sulfhydryl activated MNP-Gem at 2 mg Fe ml− 1 was mixed with 25 μmol/g Fe of 2-aldrithiol for 2 h at 40 °C. After reaction, 200 μl of brine were added and the sample centrifuged 10 min at 10,000 × g
Fig. 2. Cellular uptake of MNPs with or without functionalization with anti-CD47. Prussian blue staining of the pancreatic cell line Panc-1 (a–c) and pancreatic cancer primary cell cultures Panc215 (d–f) and Panc354 (g–i) incubated with non-functionalized MNPs, or MNPs functionalized with anti-CD47 at either 4 or 37 °C. Non-treated cells (NTCs) are shown as control. Main pictures have been acquired with a 10× objective, and insets show 2× zoomed.
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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and redispersed in 4 ml of 0.01 M sodium phosphate, pH 7.4. Finally, the anti-CD47 antibodies were immobilized on MNP-Gem following the same protocol described above for immobilization on MNPs.
After drying the cells, the cover slips were mounted in glass slides using DePeX and finally the cells were observed using light microscopy.
2.4. Drug release studies
2.8. In vitro cytotoxicity assay
The cumulative drug releases from the MNP-Gem and MNP-Gemanti-CD47 were carried out under physiological conditions (pH 7.4 and 37 °C) using two different concentrations of glutathione (GSH) as reducing agent (1 μM and 1 mM of GSH to mimic the extracellular and intracellular conditions, respectively). For each experiment, 4.8 mg of MNP-GEM and MNP-GEM-anti-CD47 were dissolved in 1 ml of 0.01 M phosphate buffer at pH 7.4 containing either 1 μM of GSH or 1 mM GSH and incubated at 37 °C. The amount of GEM released was analyzed at regular time intervals by HPLC using a C18 column, mobile phase water/acetonitrile (80/20), at a flow rate of 0.3 ml min−1, and the absorbance was measured at 270 nm. The percentage of Gem released was calculated from a standard calibration curve of the free-drug solution.
Cell viability was measured using the Resazurin dye (Sigma-Aldrich), which is an indicator of cell viability in proliferation and cytotoxicity assays. To assess cell death, cells were cultured on a 96-well plate at a density of 5 × 104 cells per well in 200 μl of cell culture medium. After 24 h, the growth medium was removed and cells were then incubated overnight at 37 °C in the presence of different concentrations of free Gemcitabine (Gem) (0.1, 0.4 and 1 μM), MNP-Gem and MNPGem-anti-CD47 (0.2 mg Fe ml−1, 4.8 μM Gem, Ab 20 μg/mg Fe). After incubation, cells were washed three times with PBS and then maintained at 37 °C and 5% CO2 incubator. After 7 days, the medium was replaced with cell culture medium containing 10% of Resazurin dye (1 mg ml−1). Cells were maintained at 37 °C and 5% CO2 incubator for approximately 2 h and then a microplate reader was used to determine the amount of Resazurin by measuring the fluorescence of the reaction mixture (λex = 544 nm, λem = 590 nm). 200 μl of 10% of resazurin dye was added to empty wells as a negative control. The viability of the cells was expressed as the percentage of the fluorescence of treated cells (minus the fluorescence of the negative control) in comparison with control, untreated cells (minus the fluorescence of the negative control).
2.5. MNP sterilization We always carried out the MNP sterilization before cell incubation. 1 ml of MNP stock was dispersed by sonication for 5 min and then was filtered through a 0.22 μm Millex-GP filter (Merck-Millipore Darmstadt, Germany). 2.6. Primary human pancreatic cancer cells and cell lines Human PDAC tissues were obtained with written informed consent from all patients and expanded in vivo as patient-derived xenografts (PDXs), as previously described [50]. To generate the primary cell cultures, the PDX tissue fragments were minced, enzymatically digested with collagenase (Stem Cell Technologies, Vancouver, BC) for 90 min at 37 °C and after centrifugation for 5 min at 1200 rpm cell pellets were resuspended and cultured in RPMI supplemented with 10% FBS and 50 units ml− 1 penicillin/streptomycin. These primary cultures were used in vitro only until passage 10. Panc-1 and BxPC3 cell lines were purchased from American Type Culture Collections (Manassas, VA, USA) and were grown as a monolayer in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 50 units ml−1 of penicillin/streptomycin. Primary cancer cells cultures and cell lines were maintained in an incubator at 37 °C in a humidified atmosphere of 95% air and 5% CO2. 2.7. Uptake studies of MNPs and MNP-anti-CD47 To determine the specific targeting of MNP-anti-CD47 for CD47-positive primary cancer cells and cell lines (Panc215, Panc354, and Panc-1) cells were seeded on coverslips and incubated with the different NPs the next day. Cells were incubated for 4 h either at 4 °C or 37 °C, in the presence of MNPs and MNP-anti-CD47 (0.2 mg Fe ml−1, Ab 20 μg/mg Fe), and then washed with PBS and left at 37 °C overnight. After 24 h, cells were washed three times with PBS and then stained for iron content by Prussian blue staining. 2.7.1. Prussian blue staining For Prussian blue staining to evaluate MNP uptake, cells were seeded as described in the section above. Once the incubation time was over, the cells were washed twice with PBS and fixed with ice-cold methanol for 2 min at 4 °C. The cells were then washed twice with PBS, and incubated with a 1:1 mixture of 4% potassium ferrocyanide and 4% hydrochloric acid (Prussian blue staining solution) for 15 min at room temperature. The cells were then washed with distilled water three times and counterstained for cytoplasm with neutral red 0.5% for 2 min at room temperature. The cells were washed with distilled water several times until all the excess neutral red staining came off.
Fig. 3. Cell viability of pancreatic cancer cell lines and primary cell cultures after Gemcitabine treatment. Cells were treated with Gemcitabine, MF66 NPs or MF66-Gem, 24 h after seeding, at the indicated concentrations. Cell viability was measured 6 days after treatment. Mann-Whitney test: **** p b 0.0001, *** p b 0.005, ** p b 0.001 compared to MNPs, ## p b 0.001 compared to equivalent Gemcitabine dose in free form.
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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2.9. Flow cytometry Cells were adjusted to a concentration of 106 cells ml−1 before analysis using a FACS fortessa. DAPI was used for exclusion of dead cells. For the assessment of apoptosis, cells were incubated with DAPI and Annexin V-APC staining kit (BD) according to the manufacturer's instructions. Data were analyzed with FlowJo software. 2.10. Statistical analyses Results are presented as means ± standard deviation unless stated otherwise and significance was determined using the Mann-Whitney test. All analyses were performed using GraphPad Prism. 3. Results and discussion This paper describes the development of a delivery system based on magnetic nanoparticles that allows the targeting of pancreatic cancer cells, resulting in improved uptake by cancer cells and the delivery of two different therapeutic agents with different mechanisms of action. 3.1. Multifunctionalization of MNPs The general strategy followed in order to prepare the functionalized MNP formulations MNP-Gem, MNP-anti-CD47 and MNP-Gem-antiCD47 is shown in Fig. 1. To this end, anti-CD47 antibody functionalized MNPs were produced by the formation of disulfide bonds between the reactive thiol of the modified anti-CD47 antibody with 2-iminothiolane and the activated sulfhydryl groups of the pre-activated MNPs (Fig. 1). The multifunctionalization strategy through disulfide bonds started with the introduction of a controlled amount of Gem followed by
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immobilization of the anti-CD47 antibody on MNP-Gem (Fig. 1). Gem was immobilized onto MNPs though a linker that allows us to perform the release of the Gem selectively in an intracellular environment. The strategy used for the covalent immobilization of the anti-CD47 ensures the correct orientation of the antibody onto the MNPs [29,51]. The synthesis of MNP-Gem started by the reaction of cysteamine with the carboxylic groups of the DMSA coating in the presence of EDC and NHS, leading to a formulation that contains 9 μmol of thiol/g Fe. Then, a Gemcitabine derivative was reacted with the activated MNPs [28]. This conjugation was achieved employing 2.5 μmol of Gem derivative, leading to a formulation that contains 2 μmol Gem/g Fe, with a zeta-potential (ζ) of −50 ± 1 mV and a hydrodynamic diameter (DH) of 97 ± 1 nm (PDI: 0.17). The functionalization of MNPs with antibodies was achieved by the formation of disulfide bonds between the reactive thiol of the modified anti-CD47 antibodies and the activated sulfhydryl groups of the MNPs following the protocol that we previously described [29]. After the reaction, the immobilized antibodies were quantified by the Bradford assay. The standard load obtained of covalently linked anti-CD47 antibodies was 30 mg/g Fe (86%), corresponding to around one antibody molecule per magnetic nanoparticle. The sodium phosphate MNP-anti-CD47 suspension was stable for weeks stored at 4 °C without noticeable precipitation. The physicochemical properties of the formulation were not significantly altered upon the functionalization (ζ: − 47 ± 2 mV and DH: 107 ± 1 nm (PDI: 0.17)). Finally, to obtain MNP-Gem-anti-CD47, firstly the Gem drug derivative was immobilized [28], leading to a formulation that contains 2 μmol Gem/g Fe (ζ: −50 ± 1 mV and DH: 97 ± 1 nm (PDI: 0.17)). Then, the anti-CD47 antibodies were immobilized on MNP-Gem following the protocol that we previously described [29]. The final formulation contains 2 μmol Gem and 30 mg Ab/g Fe, corresponding to 34 molecules
Fig. 4. Cell viability of pancreatic cancer cell lines and primary cell cultures after Gemcitabine treatment. Cells were treated with Gemcitabine, MF66 NPs or MF66-Gemcitabine, 24 h after seeding, at a concentration of 1 μM. Cell viability was measured 6 days after treatment.
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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of Gem and 1 molecule of Ab per MNPs (ζ: −45 ± 1 mV and DH: 109 ± 1 nm (PDI: 0.17)). 3.2. Drug release studies To evaluate the stimuli-response behavior of the MNP-Gem and MNP-Gem-anti-CD47 under a reducing environment, the drug release was monitored at 37 °C in 0.01 M sodium phosphate, pH 7.4 using 1 μM or 1 mM of GSH to mimic the extracellular and intracellular conditions, respectively (data not shown). These formulations showed a similar standard release that we previously described [29] 96–98% when treated with 1 mM GSH (mimicking an intracellular environment) after 6–8 h while only 3–5% of the cargo was released with 1 μM GSH (mimicking the extracellular environment) after 6–8 h. These results show that the release of Gem from these is selective and strongly dependent on the reducing environment, so that it will take place mostly inside the cells and is not affected by the presence of the antibody. 3.3. MNP uptake studies To determine the interaction of MNPs with the cells and the effect of the anti-CD47 antibody in the targeting of MNPs, pancreatic cancer cells expressing the CD47 receptor were incubated for 1 h at 37 °C, with either MNP-anti-CD47 and MNPs (as a negative control), followed by washes with PBS and then incubated for an additional 24 h at 37 °C.
Fig. 6. Cell viability of pancreatic cancer cell lines and primary cell cultures after Gem or anti-CD47 treatment. Cells were treated with Gem, anti-CD47, MNPs, MNP-Gem, MNPanti-CD47, or MNP-Gem-anti-CD47, 24 h after seeding, at the indicated concentrations. Cell viability was measured 6 days after treatment. Mann-Whitney test: * p b 0.05, ns p N 0.05 compared to Gem 1 μM.
Prussian blue staining was used to monitor the presence of iron from the MNPs inside the cells. As shown in Fig. 2, non-functionalized MNPs were barely detected by the Prussian blue staining inside of two primary pancreatic cancer cultures (Panc215 and Panc354), which closely reflect patients'intratumoral heterogeneity and are kept at very low passages as well as the established cell line Panc-1 (Fig. 2, panels b, e, h). On the other hand, MNPs functionalized with anti-CD47 mAb were detected in the cytoplasm of all cell types tested, when incubated at 37 °C (Fig. 2, panels c, f, i). These results demonstrate that functionalizing the MF66 MNPs with anti-CD47 mAb greatly improves their cellular uptake by pancreatic cancer cells. In addition, to demonstrate the selective targeting through CD47 receptors, the cells were incubated with free anti-CD47 prior to the addition of MNP-anti-CD47, which significantly reduced the uptake of MNP-anti-CD47 (data not shown). 3.4. In vitro Gem-MNP cytotoxicity studies
Fig. 5. Apoptosis induction after treatment with anti-CD47 antibody. Cells were treated with anti-CD47, MNPs or MNP-anti-CD47, 24 h after seeding, at the indicated concentrations. The percentage of annexin V-positive cells was determined by flow cytometry 20 h after treatment. Mann-Whitney test: * compared to control, # compared to MNPs.
To assess the efficiency of Gemcitabine delivery to pancreatic cancer cells, we first incubated Panc215 and Panc354 two of the cell types with a range of Gemcitabine concentrations, either in its usual form – free Gem – or conjugated to MNPs (MNP-Gem) (Fig. 3, A and B). From these results it can be observed that, first of all, MNPs by themselves do not cause significant cytotoxicity to the pancreatic cancer cells. The minimal toxicity observed could be due to the fact that the incubation with MNPs can promote changes in both pH and composition of the cell culture media due to their known ability to adsorb components
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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such as proteins (protein corona effect), amino acids, vitamins or ions. When MNPs are attached to the cell surface there might be changes as to how cell-to-cell interactions occur, as some of them might be physically hampered, and while in the cytoplasm, MNPs have been shown to elicit a small increase in cellular oxidative stress through the production of reactive oxygen species (ROS) [52]. Moreover, and as expected, Gemcitabine is slightly less efficient when delivered attached to MNPs than free-Gem, as can be seen more clearly for the lower dose used (0.1 μM). This result is expected since Gemcitabine coupled to the MNPs has another internalization mechanism compared to free Gemcitabine, when administered to cells in vitro. While free-Gem is ready to be taken up by the cells, MNP-Gem internalization is slower and after uptake Gem has to be released from the MNPs to be able to find its way to the nucleus to inhibit the DNA synthesis, causing cell death. Therefore, the lower efficiency of MNP-Gem compared to free-Gem could potentially be explained by a combination of factors including first the slower uptake mechanism of MNP-Gem resulting in an overall lower uptake since not all MNP-Gem are internalized by the cells in the 24 h of incubation time, second the slower mechanism of action once internalized since a release step is required, and third the possibility of the MNP uptake being influenced by the formation of the protein corona [53]. However, it is important to note that the usual concentration at which Gem is used for in vitro studies using primary pancreatic cancer cell cultures is 100 ng ml− 1 (or 0.333 μM) [50]. Our results using 0.4 μM of Gem show that at this or higher concentrations, the effect of free-Gem and MNP-Gem is similar, probably because a saturating concentration is reached. Therefore, the different uptake mechanism and release kinetic coupling of Gemcitabine to the MNPs does not constitute a major hurdle for Gemcitabine administration at its therapeutic concentration. Next we validated these findings on different pancreatic cancer cell types, including the commonly used cell lines Panc-1 and Bx-PC3, by repeating the experiment using only the higher concentration 1 μM (Fig. 4). The results show that, for this concentration, the effect of free-Gem or MNP-Gem is comparable in all cases. The proposed system is of great interest as it shows that Gemcitabine can be linked to the MNPs without losing its cytotoxic activity and can therefore be combined with other drugs in the same NPs to create a multifunctionalized platform that delivers a combination of treatments. Moreover, if it could be demonstrated that the multifunctionalized MNPs accumulate specifically in the tumors, lower doses of the active compounds would be necessary as the biodistribution would be more favorable.
3.5. Apoptosis induction by MNP-anti-CD47 We have shown before that CD47 is expressed on pancreatic cancer (stem) cells and that the anti-CD47 antibody not only enables phagocytosis of these cells by macrophages, but also directly induced apoptosis of the cancer cells, while exerting no effect on non-malignant cells [43]. Here we have shown already that coupling anti-CD47 on the surface of MNPs greatly improves their uptake by pancreatic cancer cells, but we also wanted to confirm whether the anti-CD47 retains its ability to induce apoptosis in pancreatic cancer cells, after being immobilized on the surface of MNPs. As can be seen in Fig. 5, there is a dose dependent increase on the percentage of Annexin V-positive cells upon treatment with free anti-CD47. When administered at the same dose of 4 μM but in the form of MNP-anti-CD47, the anti-CD47 antibody promotes similar levels of apoptosis induction in the pancreatic cancer cells tested Panc215 and Panc354. Therefore, the functionalization on MNPs with anti-CD47 not only increases their uptake by pancreatic cancer cells (Fig. 2) but also has a therapeutic effect of promoting the apoptosis of these cells (Fig. 5). It can be concluded at this point that both Gem and the anti-CD47 antibody retain their biological function after being conjugated to MNPs.
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3.6. In vitro cytotoxicity studies of multifunctionalized MNPs We next wanted to determine if we could achieve even higher levels of cytotoxicity on pancreatic cancer cells by using multifunctionalized NPs combining Gemcitabine and anti-CD47 conjugated to MNPs. The results shown in Fig. 6 show that the effect of anti-CD47 on apoptosis that we observed 20 h after treatment (Fig. 5) is not observed anymore when the cell viability was measured 6 days after treatment. This could be due to the fact that only a small population of cells is affected by this treatment, potentially the cancer stem cells, and the effect is masked by the proliferation of the more differentiated cells. These findings are in line with our previous findings that showed that CD47 targeting as a single treatment strategy was not effective in vivo, while the combination with chemotherapy was able to promote long lasting tumor regression [43]. Since no effect on cytotoxicity was observed for anti-CD47 antibody alone or conjugated to MNPs, it was not surprising that there was no additional in vitro cytotoxicity when we combined Gemcitabine and anti-CD47 in the same NPs. However, this result does not mean that there would not be a measurable effect in vivo as in this context multiple administrations of the therapeutic combination are usually performed, and a layer by layer elimination of the tumoral tissue is expected to happen. Nevertheless, the fact that there was no decrease on the cytotoxic activity that must attributed to Gem, given the lack of effect of anti-CD47 antibody alone, demonstrates the potential of producing MNPs combining different types of therapeutic agents without them losing their biological activities. It would be interesting to further modify this delivery platform to deliver more or different drugs, such as paclitaxel, and/or to conjugate them with antibodies that bear an even greater level of tumor specificity, as well as with molecules that prolong circulation time and avoid the recognition of these delivery systems by the reticuloendothelial system (RES) and their uptake by liver cells, such as poly(ethylene glycol) (PEG), in order to make them suitable for intravenous administration. 4. Conclusions We have developed a multifunctional nanoformulation of MNPs for selective multimodal treatment of pancreatic cancer, including Gemcitabine as a chemotherapeutic drug and anti-CD47 antibody as adjuvant of the treatment. The designed immobilization strategy preserves both the stability and activity of the conjugated molecules. The multifunctionalization does not affect the selective drug release, which only occurs under reducing intracellular conditions, thus keeping the intracellular selectivity of the treatment. In addition, the cytotoxic activity of the drug delivered by the nanoparticles is comparable to the effect of the free drug at therapeutic drug concentrations. The immobilized antiCD47 antibody retains its targeting activity, thus increasing the delivery of the nanoformulation to the cancer cells during short incubation times. In addition, the tagged anti-CD47 antibody shows efficient induction of apoptosis in pancreatic cancer cells as its effect is larger compared to the free antibody. These data suggest that the apoptotic activity of the antibody is not only preserved but can be enhanced by its delivery by MNPs. Finally the cytotoxic activity of the multifunctional nanoformulation is not increased in the in vitro studies. This result is not unexpected since the specific apoptotic effect of the anti-CD47 antibody might preferentially affect the small subpopulation of CSCs and these effects are not easily detectable among bulk cancer cells in an in vitro set up. Therefore, since the effects of targeting CSCs responsible for the tumor relapse by using the anti-CD47 antibody have been demonstrated in preclinical in vivo studies, the developed platform bears great potential for future therapeutic development. Certainly, before using these nanoparticles in vivo, the platform needs to be further optimized in order to be stable in circulation and invisible to the reticuloendothelial system (RES) to avoid premature clearance, which is typically done by covering the nanoparticles with proteins or polymers, such as BSA or polyethyleneglycol (PEG) [54]. Also, as the use of antibodies can evoke
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
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an immune response, an antibody fragment or synthetic peptide with the bioactive region could be used instead of the whole antibody. Transparency Document The Transparency document associated with this article can be found, in the online version. Acknowledgements This work was supported by EU-FP7 MULTIFUN project (no. 262943), and by the Comunidad de Madrid NANOFRONTMAG-CM project (S2013/MIT-2850). References [1] U. Jeong, X. Teng, Y. Wang, H. Yang, Y. Xia, Superparamagnetic colloids: controlled synthesis and niche applications, Adv. Mater. 19 (2007) 33–60. [2] C. Cheng, F. Xu, H. Gu, Facile synthesis and morphology evolution of magnetic iron oxide nanoparticles in different polyol processes, New J. Chem. 35 (2011) 1072–1079. [3] M. Mahmoudi, S. Sant, B. Wang, S. Laurent, T. Sen, Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy, Adv. Drug Deliv. Rev. 63 (2011) 24–46. [4] A. Ito, M. Shinkai, H. Honda, T. Kobayashi, Medical application of functionalized magnetic nanoparticles, J. Biosci. Bioeng. 100 (2005) 1–11. [5] J. Xie, S. Jon, Magnetic nanoparticle-based theranostics, Theranostics 2 (2012) 122–124. [6] A.P. Khandhar, R.M. Ferguson, H. Arami, K.M. Krishnan, Monodisperse magnetite nanoparticle tracers for in vivo magnetic particle imaging, Biomaterials 34 (2013) 3837–3845. [7] S.C. Tang, I.M. Lo, Magnetic nanoparticles: essential factors for sustainable environmental applications, Water Res. 47 (2013) 2613–2632. [8] R. Hudson, Y. Feng, R.S. Varma, A. Moores, Bare magnetic nanoparticles: sustainable synthesis and applications in catalytic organic transformations, Green Chem. 16 (2014) 4493–4505. [9] J.M. Perez, F.J. Simeone, Y. Saeki, L. Josephson, R. Weissleder, Viral-induced self-assembly of magnetic nanoparticles allows the detection of viral particles in biological media, J. Am. Chem. Soc. 125 (2003) 10192–10193. [10] W. Li, S. Tutton, A.T. Vu, L. Pierchala, B.S. Li, J.M. Lewis, P.V. Prasad, R.R. Edelman, First-pass contrast-enhanced magnetic resonance angiography in humans using ferumoxytol, a novel ultrasmall superparamagnetic iron oxide (USPIO)-based blood pool agent, J. Magn. Reson. Imaging 21 (2005) 46–52. [11] M.A. McAteer, N.R. Sibson, C. von Zur Muhlen, J.E. Schneider, A.S. Lowe, N. Warrick, K.M. Channon, D.C. Anthony, R.P. Choudhury, In vivo magnetic resonance imaging of acute brain inflammation using microparticles of iron oxide, Nat. Med. 13 (2007) 1253–1258. [12] C.C. You, O.R. Miranda, B. Gider, P.S. Ghosh, I.B. Kim, B. Erdogan, S.A. Krovi, U.H. Bunz, V.M. Rotello, Detection and identification of proteins using nanoparticle-fluorescent polymer ‘chemical nose’ sensors, Nat. Nanotechnol. 2 (2007) 318–323. [13] V. Hsieh, A. Jasanoff, Bioengineered probes for molecular magnetic resonance imaging in the nervous system, ACS Chem. Neurosci. 3 (2012) 593–602. [14] S. Gandhi, H. Arami, K.M. Krishnan, Detection of cancer-specific proteases using magnetic relaxation of peptide-conjugated nanoparticles in biological environment, Nano Lett. 16 (2016) 3668–3674. [15] J. Dobson, Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery, Gene Ther. 13 (2006) 283–287. [16] B. Thiesen, A. Jordan, Clinical applications of magnetic nanoparticles for hyperthermia, Int. J. Hyperth. 24 (2008) 467–474. [17] J.D. Hood, M. Bednarski, R. Frausto, S. Guccione, R.A. Reisfeld, R. Xiang, D.A. Cheresh, Tumor regression by targeted gene delivery to the neovasculature, Science 296 (2002) 2404–2407. [18] S. Laurent, S. Dutz, U.O. Hafeli, M. Mahmoudi, Magnetic fluid hyperthermia: focus on superparamagnetic iron oxide nanoparticles, Adv. Colloid Interf. Sci. 166 (2011) 8–23. [19] J.S. Beveridge, J.R. Stephens, M.E. Williams, The use of magnetic nanoparticles in analytical chemistry, Annu Rev Anal Chem (Palo Alto, Calif) 4 (2011) 251–273. [20] M.A. Ahmed, S.M. Ali, S.I. El-Dek, A. Galal, Magnetite–hematite nanoparticles prepared by green methods for heavy metal ions removal from water, Mater. Sci. Eng. B 178 (2013) 744–751. [21] L. Borlido, A.M. Azevedo, A.C. Roque, M.R. Aires-Barros, Magnetic separations in biotechnology, Biotechnol. Adv. 31 (2013) 1374–1385. [22] J. He, M. Huang, D. Wang, Z. Zhang, G. Li, Magnetic separation techniques in sample preparation for biological analysis: a review, J. Pharm. Biomed. Anal. 101 (2014) 84–101. [23] B.J. Boyd, Past and future evolution in colloidal drug delivery systems, Expert Opin. Drug Deliv. 5 (2008) 69–85. [24] V.P. Torchilin, Multifunctional nanocarriers, Adv. Drug Deliv. Rev. 58 (2006) 1532–1555. [25] O. Veiseh, J.W. Gunn, M. Zhang, Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging, Adv. Drug Deliv. Rev. 62 (2010) 284–304.
[26] J.D. Byrne, T. Betancourt, L. Brannon-Peppas, Active targeting schemes for nanoparticle systems in cancer therapeutics, Adv. Drug Deliv. Rev. 60 (2008) 1615–1626. [27] M. Talelli, A. Aires, M. Marciello, Protein-modified magnetic nanoparticles for biomedical applications, Curr. Org. Chem. 20 (2016) 1252–1261. [28] A. Latorre, P. Couleaud, A. Aires, A.L. Cortajarena, Á. Somoza, Multifunctionalization of magnetic nanoparticles for controlled drug release: a general approach, Eur. J. Med. Chem. 82 (2014) 355–362. [29] A. Aires, S. Ocampo, B. Simões, J. Rodríguez, J. Cadenas, P. Couleaud, K. Spence, A. Latorre, R. Miranda, A. Somoza, R. Clarke, J. Carrascosa, A. Cortajarena, Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44positive cancer cells, Nanotechnology 27 (2016) 065103. [30] M.A. Firer, G. Gellerman, Targeted drug delivery for cancer therapy: the other side of antibodies, J. Hematol. Oncol. 5 (2012) 70. [31] Y. Sun, F. Yu, B.W. Sun, Antibody-drug conjugates as targeted cancer therapeutics, Yao Xue Xue Bao 44 (2009) 943–952. [32] C. Peters, S. Brown, Antibody-drug conjugates as novel anti-cancer chemotherapeutics, Biosci. Rep. 35 (2015). [33] H.A. Burris III, M.J. Moore, J. Andersen, M.R. Green, M.L. Rothenberg, M.R. Modiano, M.C. Cripps, R.K. Portenoy, A.M. Storniolo, P. Tarassoff, R. Nelson, F.A. Dorr, C.D. Stephens, D.D. Von Hoff, Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial, J. Clin. Oncol. 15 (1997) 2403–2413. [34] T. Conroy, F. Desseigne, M. Ychou, O. Bouche, R. Guimbaud, Y. Becouarn, A. Adenis, J.L. Raoul, S. Gourgou-Bourgade, C. de la Fouchardiere, J. Bennouna, J.B. Bachet, F. Khemissa-Akouz, D. Pere-Verge, C. Delbaldo, E. Assenat, B. Chauffert, P. Michel, C. Montoto-Grillot, M. Ducreux, U. Groupe Tumeurs Digestives of, P. Intergroup, FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer, N. Engl. J. Med. 364 (2011) 1817–1825. [35] D.D. Von Hoff, T. Ervin, F.P. Arena, E.G. Chiorean, J. Infante, M. Moore, T. Seay, S.A. Tjulandin, W.W. Ma, M.N. Saleh, M. Harris, M. Reni, S. Dowden, D. Laheru, N. Bahary, R.K. Ramanathan, J. Tabernero, M. Hidalgo, D. Goldstein, E. Van Cutsem, X. Wei, J. Iglesias, M.F. Renschler, Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine, N. Engl. J. Med. 369 (2013) 1691–1703. [36] N. Desai, V. Trieu, B. Damascelli, P. Soon-Shiong, SPARC expression correlates with tumor response to albumin-bound paclitaxel in head and neck cancer patients, Transl. Oncol. 2 (2009) 59–64. [37] A. Sparreboom, C.D. Scripture, V. Trieu, P.J. Williams, T. De, A. Yang, B. Beals, W.D. Figg, M. Hawkins, N. Desai, Comparative preclinical and clinical pharmacokinetics of a cremophor-free, nanoparticle albumin-bound paclitaxel (ABI-007) and paclitaxel formulated in Cremophor (Taxol), Clin. Cancer Res. 11 (2005) 4136–4143. [38] E.R. Gardner, W.L. Dahut, C.D. Scripture, J. Jones, J.B. Aragon-Ching, N. Desai, M.J. Hawkins, A. Sparreboom, W.D. Figg, Randomized crossover pharmacokinetic study of solvent-based paclitaxel and nab-paclitaxel, Clin. Cancer Res. 14 (2008) 4200–4205. [39] N.P. Desai, V. Trieu, L.Y. Hwang, R. Wu, P. Soon-Shiong, W.J. Gradishar, Improved effectiveness of nanoparticle albumin-bound (nab) paclitaxel versus polysorbatebased docetaxel in multiple xenografts as a function of HER2 and SPARC status, Anti-Cancer Drugs 19 (2008) 899–909. [40] P.C. Hermann, S.L. Huber, T. Herrler, A. Aicher, J.W. Ellwart, M. Guba, C.J. Bruns, C. Heeschen, Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer, Cell Stem Cell 1 (2007) 313–323. [41] I. Miranda-Lorenzo, J. Dorado, E. Lonardo, S. Alcala, A.G. Serrano, J. Clausell-Tormos, M. Cioffi, D. Megias, S. Zagorac, A. Balic, M. Hidalgo, M. Erkan, J. Kleeff, A. Scarpa, B. Sainz Jr., C. Heeschen, Intracellular autofluorescence: a biomarker for epithelial cancer stem cells, Nat. Methods 11 (2014) 1161–1169. [42] C. Li, D.G. Heidt, P. Dalerba, C.F. Burant, L. Zhang, V. Adsay, M. Wicha, M.F. Clarke, D.M. Simeone, Identification of pancreatic cancer stem cells, Cancer Res. 67 (2007) 1030–1037. [43] M. Cioffi, S. Trabulo, M. Hidalgo, E. Costello, W. Greenhalf, M. Erkan, J. Kleeff, B. Sainz Jr., C. Heeschen, Inhibition of CD47 effectively targets pancreatic cancer stem cells via dual mechanisms, Clin. Cancer Res. 21 (2015) 2325–2337. [44] M. Au, T.I. Emeto, J. Power, V.N. Vangaveti, H.C. Lai, Emerging therapeutic potential of nanoparticles in pancreatic cancer: a systematic review of clinical trials, Biomedicine 4 (2016) 20. [45] E. Fernandes, J.A. Ferreira, P. Andreia, L. Luis, S. Barroso, B. Sarmento, L.L. Santos, New trends in guided nanotherapies for digestive cancers: a systematic review, J. Control. Release 209 (2015) 288–307. [46] S. Kossatz, R. Ludwig, H. Dähring, V. Ettelt, G. Rimkus, M. Marciello, G. Salas, V. Patel, F.J. Teran, I. Hilger, High therapeutic efficiency of magnetic hyperthermia in xenograft models achieved with moderate temperature increases in the tumor area, Pharm. Res. 31 (12) (2014) 3274–3288. [47] C.T.E, in: C.T.E (Ed.), Disulfide Bonds Between Cysteine Residues Protein Structure: A Practical Approach, IRL Press at Oxford University Press, Oxford 1989, pp. 155–167. [48] S. Puertas, M. Moros, R. Fernández-Pacheco, M.R. Ibarra, V. Grazú, J.M. de la Fuente, Designing novel nano-immunoassays: antibody orientation versus sensitivity, J. Phys. D. Appl. Phys. 43 (2010) 474012–474019. [49] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [50] M.T. Mueller, P.C. Hermann, J. Witthauer, B. Rubio-Viqueira, S.F. Leicht, S. Huber, J.W. Ellwart, M. Mustafa, P. Bartenstein, J.G. D'Haese, M.H. Schoenberg, F. Berger, K.W. Jauch, M. Hidalgo, C. Heeschen, Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer, Gastroenterology 137 (2009) 1102–1113.
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035
S. Trabulo et al. / Biochimica et Biophysica Acta xxx (2017) xxx–xxx [51] C. Grüttner, K. Müller, J. Teller, F. Westphal, A. Foreman, R. Ivkov, Synthesis and antibody conjugation of magnetic nanoparticles with improved specific power absorption rates for alternating magnetic field cancer therapy, J. Magn. Magn. Mater. 311 (2007) 181–186. [52] H. Markides, M. Rotherham, A.J. El Haj, Biocompatibility and toxicity of magnetic nanoparticles in regenerative medicine, J. Nanomater. 2012 (2012) 1–11.
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[53] M. Hadjidemetriou, Z. Al-Ahmady, M. Mazza, R.F. Collins, K. Dawson, K. Kostarelos, In vivo biomolecule corona around blood-circulating, clinically used and antibodytargeted lipid bilayer nanoscale vesicles, ACS Nano 9 (2015) 8142–8156. [54] A. Aires, S.M. Ocampo, D. Cabrera, L. de la Cueva, G. Salas, F.J. Teran, A.L. Cortajarena, BSA-coated magnetic nanoparticles for improved therapeutic properties, J. Mater. Chem. B 3 (2015) 6239–6247.
Please cite this article as: S. Trabulo, et al., Multifunctionalized iron oxide nanoparticles for selective targeting of pancreatic cancer cells, Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/j.bbagen.2017.01.035