Bioconjugation and Fluorescence Labeling of Iron Oxide

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Bioconjugation and Fluorescence Labelling of Iron Oxide Nanoparticles Grafted with Bromomaleimide-terminal Polymers Ruirui Qiao, Lars Esser, Changkui Fu, Cheng Zhang, Jinming Hu, Paulina Ramírez-García, Yuhuan Li, John F. Quinn, Michael R. Whittaker, Andrew K. Whittaker, and Thomas P. Davis Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01282 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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Bioconjugation and Fluorescence Labelling of Iron Oxide Nanoparticles Grafted with Bromomaleimideterminal Polymers Ruirui Qiao,*† Lars Esser,† Changkui Fu,‡§ Cheng Zhang, ‡§ Jinming Hu,∥ Paulina RamírezGarcía, † Yuhuan Li, † John F. Quinn, † Michael R. Whittaker, † Andrew K. Whittaker‡§ and Thomas P. Davis*† ⊥ †

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute

of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville VIC 3052, Australia ‡

Australian Institute for Bioengineering and Nanotechnology and §ARC Centre of Excellence in

Convergent Bio-Nano Science and Technology, The University of Queensland, Brisbane, Old 4072, Australia ∥CAS

Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and

Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China ⊥Department

of Chemistry, University of Warwick, Gibbet Hill, Coventry CV4 7AL, United

Kingdom KEYWORDS. Magnetic nanoparticles, dibromomaleimide, brushed PEG, RAFT, bioconjugation

ACS Paragon Plus Environment

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ABSTRACT. Iron oxide nanoparticles have been widely applied in biomedical applications for their unique physical properties. Despite the relatively mature synthetic approaches for iron oxide nanoparticles, surface modification strategies for obtaining particles with satisfactory biofunctionality are still urgently needed to meet the challenge of nanomedicine. Herein, we report a surface modification and biofunctionalization strategy for iron oxide-based magnetic nanoparticles based on a dibromomaleimide (DBM)-terminated polymer with brushed polyethylene glycol (PEG) chains. PEG acrylate and phosphonate monomers, serving as antibiofouling and surface anchoring compartments for iron oxide nanoparticles, were incorporated utilizing a novel DBM containing reversible addition-fragmentation chain transfer (RAFT) agent. The particles prepared through this new surface architecture possessed high colloidal stability in a physiological buffer and the capacity of covalent conjugation with biomolecules for targeting. Cell tracking of the molecular probes was achieved concomitantly by exploiting DBM conjugation-induced fluorescence of the nanoparticles.


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INTRODUCTION Iron oxide nanoparticles (IONPs) represent a class of inorganic nanoparticles with significant applicability in nanomedicine.1-5

In the past three decades, significant reports have been

published on the preparation of biocompatible IONPs for use in vivo, such as MRI contrast agents, hyperthermia or controlled drug/siRNA delivery.6-10 In addition to the well-established synthetic routes, e.g., thermal decomposition, surface modifications of IONPs have been considered to be a critical strategy to render particles with stability under physiological conditions. In addition, biocompatibility and the capacity for conjugation to biomolecules are also essential for in vivo targeting. Among the synthesis and engineering strategies available, polymer coatings have been employed for enhancing the colloidal stability, biocompatibility and protein avoidance of nanoparticles.11-18 To enable colloidal stability in water and physiological conditions, anchoring groups with strong iron binding affinities such as phosphonic acid or catechol have been employed as terminated groups in polymers.13, 19 Particularly, phosphonic acid have been widely used as anchoring group for IONPs for its high affinity for Fe–OH, forming more stable Fe–O–P bonds than Fe–OH carboxylic acid bond.3 Meanwhile, fluorescence labelling of particles or bioconjugates is desirable for in vitro tracking. Currently, functional polymers that satisfy above design criteria are relatively complex in their syntheses and additional labelling of fluorophores are generally required.20 Reversible-deactivation radical polymerization techniques such as reversible additionfragmentation chain transfer (RAFT) and atom-transfer radical polymerization (ATRP) allow the production of polymers with controlled molecular weight and functional moieties. Recently, dibromomaleimides (DBM) have attracted much attention for their facile and specific conjugation with proteins or peptides containing disulfide bonds.21, 22 Haddleton and O’Reilly et


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al., for example, reported the incorporation of DBM into polymers to achieve conjugationinduced fluorescence labeling of proteins and peptides through the formation of dithiomaleimide (DTM) benefiting from the multiple functionality of bromomaleimide (Br for nucleophilic substitution and ene for Michael addition).23,

24

Furthermore, De la Torre et al. reported on

chemoselective reactions with both thiol and amino terminal peptides.25 Herein, biocompatible magnetic nanoparticles based on the thermal decomposition synthesis approach and a surface modification using brushed poly(ethylene) glycol (PEG) polymers containing dibromomaleimide is described, thereby providing a new strategy for the development of selectively targeting nanoprobes. PEG acrylate and phosphonate monomers serving as anti-biofouling and surface anchoring compartments for iron oxide nanoparticles were synthesized using a novel DBM containing RAFT agent. The DBM-containing PEGylated IONPs displayed both excellent colloidal stability and biocompatibility, and the surface functionalization was further exploited for bioconjugation with targeting-biomolecules. EXPERIMENTAL SECTION Materials and apparatus The RAFT agents 4-cyano-4-(((dodecylthio)carbonothioyl)thio) pentatonic acid were purchased from Boron Molecular and used as received. Iron oxide nanocrystals with oleic acid coating were purchased from Ocean NanoTech. NH2-Cy5.5 was purchased from Lumiprobe Corp. All the other chemicals and reagents were purchased from Sigma-Aldrich and used as received. Dulbecco's Modified Eagle Medium (DMEM) culture medium and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). MCF-7 cells from American Type Culture Collection (Manassas, VA) were used were used as received.


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Nuclear Magnetic Resonance (NMR) Spectra. 1H and 13C NMR spectra were recorded on a Bruker AC400F (400 MHz) spectrometer. Chloroform (CDCl3), and DMSO-d6 were used as the solvents, depending on the particular substance being analysed. Gel Permeation Chromatography (GPC). GPC analyses of polymer samples were performed in N,N-dimethylacetamide (DMAc with 0.03% w/v LiBr and 0.05% 2,6-dibutyl-4methylphenol (BHT) using a Shimadzu modular system comprising a DGU-12A degasser, an SIL-10AD automatic injector, and a 5.0 µm bead-size guard column (50 × 7.8 mm) followed by four 300 × 7.8 mm linear Phenogel columns (bead size: a 5.0 µm; pore sizes: 105, 104, 103, and 500 Å) and an RID-10A differential refractive-index detector. The temperature of columns was maintained at 50 °C using a CTO-10A oven, and the flow rate was kept at 1 mL/min using a LC10AT pump. A molecular weight calibration curve was produced using commercial narrow molecular weight distribution polystyrene standards with molecular weights ranging from 500 to 106 g/mol. Polymer solutions at 2−3 mg/mL were prepared in the eluent and filtered through 0.45 µm filters prior to injection. Thermogravimetric analysis (TGA). TGA measurements were performed using a PerkinElmer Pyris 1 TGA and its corresponding Pyris 1 software measuring at a rate of 20 °C min−1 from 30 °C to 600 °C. The weight loss percentage was calculated by the difference between the sample weights at 30 °C and at 600 °C. Transmission Electron Microscope (TEM). TEM images were obtained using a JEOL JEM2011 TEM. Dynamic Light Scattering (DLS). Hydrodynamic size of the particles were analyzed at 298.0 K using Nano ZS (Malvern) equipped with a solid state He−Ne laser (λ = 632.8 nm).


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UV−Visible spectrophotometry. All UV−vis spectra were acquired on a Shimadzu UV-3600 UV−vis-NIR spectrophotometer in quartz cuvettes of 10 mm path length. Fluorescence spectrometry. The fluorescent emission spectra were obtained using a Shimadzu RF-5301 pc fluorescence spectrophotometer in quartz cuvettes of 10 mm path length. Fourier Transform Infrared (FT-IR) Spectra. Fourier transform infrared (FT-IR) spectra were recorded on a Shimadzu IRTracer-100 FT-IR spectrometer under attenuated total reflectance (ATR). The spectra were collected over 64 scans with a spectral resolution of 4 cm−1. Relaxivity. Relaxivity of IOPNs were performed at 3 Tesla on a Siemens MAGNETOM Trio MR scanner. The T2 relaxation times were determined using an inversion recovery (IR)-prepared turbo spin echo (TSE) imaging pulse sequence using a variety of inversion times (TIs) ranging from 25 ms to 3970 ms. The T2 relaxation times were obtained by conducting TSE images with a TR of 4,000 ms and increasing echo times (TEs) of 10, 20, 40, 80, 140, 300, 600, 1000 ms. Repetition time (TR) was set to be 4 s and slice thickness was 6 mm for all experiments. Cell binding assays for IONPs-based probes. Fluorescence microscopy studies were carried out to evaluate the binding affinity of the IONPs-based nanoprobes to cancer cells. In detail, breast cancer cell line MCF-7 were cultured using DMEM supplemented with 15% FBS and maintained in a humidified environment containing 5% CO2 and air at 37 °C. 2×105 MCF-7 cells were seeded in the wells of 8-well chamber slides and incubated overnight at 37 °C under 5% CO2 to allow firm adherence. After being rinsed with PBS, the cells were incubated with the nanoprobes at 37 °C under 5% CO2 for 2 h. After that, the cells were rinsed three times with PBS and stained with DRAQ5. The fluorescence micrographs were captured with a Leica TCS SP8 confocal microscope.


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Cell viability assays. Alarma Blue assay was used for the cell viability evaluation. HEK293 (human embryonic kidney cell 293, ATCC) were grown in Dulbecco’s Modified Eagle Media (DMEM) culture media with 10% Fetal Bovine Serum (FBS). HEK293 cells (1.5 ×104 cells/well) were exposed to materials (10, 50, 100, 200 and 400 µg/mL) for 24 h in 96-well plates, with the final volume of 100 µL. Cell culture medium was used as a control. After exposure, the suspensions were removed and the cells were incubated with 10% Alamar Blue (Invitrogen) for 4 h at 37 °C. A microplate reader (CLARIOstar, BMG LABTECH) was used to read the fluorescence at 560 nm excitation and 590 nm emission. Background values (10% Alamar Blue in cell culture medium) were subtracted from each well and the average fluorescent intensity of the triplicates was calculated to indicate cell viability. The membrane integrity and cell morphology were evaluated by the coincubation of Calcein AM and propidium iodide (PI) staining: 2 µM Calcein AM and 4 µM PI for 15 min at 37 °C. 1 mM TBHP was used as positive control and incubated with the cells for 20 min before the staining. Cells were examined under a Leica TCS SP8 confocal microscope. RESULTS AND DISCUSSION The DBM-functional chain transfer agent (CTA) was prepared according to Scheme 1. First, N(2-hydroxyethyl)-3,4-DBM (3) was synthesized in a two-step reaction: (i) dibromomaleimide (1) was activated using methyl chloroformate and N-methylmorpholine and (ii) the N-methoxycarbonyl activated product (2) was then further reacted with ethanolamine at room temperature to obtain N-(2-hydroxyethyl)-3,4-DBM (3).26 Finally, the DBM-terminated CTA (4) as obtained by a Steglich esterification of aforementioned compound with the carboxylic acid-terminated Rgroup of 4-cyano-4-(((dodecylthio)carbonothioyl)thio) pentanoic acid. The NMR spectrum as displayed in Figure S1 clearly demonstrated the successful synthesis of the desired product.


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Scheme 1. Synthesis of dibromomaleimide (DBM) functional polymers with multi-phosphonate groups Next a DBM-terminated poly(oligoethylene glycol) methyl ether acrylate (poly(OEGA)) homopolymer (5, Scheme 1) was synthesized by RAFT polymerization using OEGA (Mn = 480) as monomer, 4 as CTA, AIBN as initiator and 1,4-dioxane as solvent. The polymerization was carried out for 24 h before being stopped by exposure to air. After purification, the polymer POEGA-DBM was characterized using NMR and GPC. The NMR spectrum as shown in Figure 1 confirmed the successful synthesis of POEGA-DBM with a number-average molecular weight of 9160 g/mol and a relatively low polydispersity (Table 1 and Figure S3). Subsequently, the POEGA-DBM polymer was used as a macro-RAFT agent for chain extension. In this second polymerization step, phosphonate acrylate (PA) monomers prepared by esterification of dimethyl (2-hydroxyethyl)phosphonate with acryloyl chloride were added for to imbue the polymer with multiple phosphonic acid groups for the strong binding affinity to the surface of the iron oxide nanoparticles.27 In this case the addition of phosphonate monomers can be well-controlled via the monomer feed concentration. POEGA-b-PA-DBM block copolymers were subsequently isolated by precipitation into mixture of diethyl ether and petroleum ether and characterized by 1H and


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31

P NMR (Figure 1). GPC analysis (Figure S3) of polymer 1 and 2 demonstrated a narrow

molecular weight distribution (PDI = 1.20 and 1.22, respectively), indicating well-controlled polymerizations by RAFT (Table 1).

Table 1. A summary of experimental conditions and the molecular weights measured for both homopolymer and copolymer, determined by 1H NMR and GPC.

RAFT

Monomers

AIBN

Reaction

agent

(mmol)

(mmol)

time (h)

Conver.

Mn

Mn

(NMR)

(GPC)

(%)

(mmol)

PDI (Mw/Mn)

(g/mol)

(g/mol)

POEGADBM

0.135

3.4

0.013

24

35

9160

7400

1.20

0.05

0.5

0.005

24

44

10100

7800

1.22

POEGAb-PADBM

Prior to surface grafting of the IONPs, the phosphonic ester groups of POEGA-b-PA-DBM end groups were dealkylated using trimethylsilyl bromide (TMSBr) to expose the phophonic acid groups, thereby creating POEGA-b-PPA-DBM (Figure 2a). This product was confirmed by 31P NMR exemplified by an upfield shift of the 31 phosphorus NMR signal from 29.70 ppm to 28.23 ppm and 1H NMR by the appearance of a broad peak at 5.09 ppm originating from the hydroxyl group of phosphonic acid, confirming the successful deprotection of the phosphonic ester groups (Figure S4a and b).


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Figure 1. 1H NMR and 31P NMR of POEGA-b-PA-DBM polymers.

The POEGA-b-PPA-DBM polymers were then mixed with oleic acid coated IONPs in THF under 40 °C for a ligand exchange process based on the strong binding affinity of the phosphonic acid group for iron (Figure 2b). The PEGylated IONPs were redissolved in water and characterized to have a uniform size (13.5 ± 0.99 nm, average of 300 particles) as revealed by transmission electron microscopy (TEM) (Figure 2c and d), consistent with the original particles before ligand exchange (13.5 ± 1.2 nm, Figure S5a and b). The grafting of the polymers on the IONPs was validated using Fourier transform infrared spectroscopy (FTIR) (Figure 2e). The spectra of the PEGylated IONPs proved consistent with the spectra taken from POEGA-b-PPA-DBM polymers displaying ester and ether bonds signals at 1728 and 1095 cm−1, respectively. Meanwhile, the shift of the ester signal from 1708 to 1732 cm-1 further demonstrated the surface exchange from oleic acid to polymers. The amount of polymer grafting


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on the surface was investigated by thermogravimetric analysis (TGA) and the weight loss was determined to be 31% for PEGylated IONPs (Figure S6), much higher than the TGA result obtained from single phosphonic acid-terminated brushed PEG.13

Figure 2. (a) Deprotection of the phosphonate groups and (b) surface ligand exchange of the oleic acid coated iron oxide nanoparticles; (c) TEM and (d) size histogram of the nanoparticles; (e) FTIR spectra of oleic acid coated IONPs, POEGA-b-PPA-DBM polymer and POEGA-bPPA-DBM polymer-grafted IONPs.

Dynamic light scattering (DLS) analysis was conducted to characterize the aqueous dispersion of the resultant nanoparticles and the hydrodynamic size of the particles in water and PBS buffer was characterized to be 41.25 nm and 40.78 nm, respectively (Figure 3a). The strong binding affinity of the multi-phosphonic acid groups enabled very high colloidal stability in both water


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and PBS buffer in 15 days (Figure 3a). No aggregation was observed in either water or PBS solution after a 2-month observation and the nanoparticle sizes (identified using TEM) remained unchanged, indicative of long-term stability. Next, the superparamagnetic properties of the PEGylated IONPs were investigated using a 3T MRI. The r2 relaxivity (efficiency to increase MRI contrast) was extracted from the linear regression fits of the experimental data, as shown in Figure 3b, and was determined to be 142 mM-1s-1. In comparison to the commercialized products, such as Feridex® (93 mM-1S-1 under 3T MRI) and Resovist® (143 mM mM-1S-1 under 3T MRI), the as-prepared IONP provided satisfied performance.28

Figure 3. (a) Hydrodynamic size of the PEGylated IONPs in H2O and PBS; (b) T2 relaxation rate (R2) of PEGylated IONPs in water against the concentration of Fe ion determined by 3T MRI;


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(c) conjugation of POEGA-b-PPA-DBM polymer with peptide; (d) fluorescence spectra of POEGA-b-PPA-DBM and POEGA-b-PPA-PBM. In recent literature DBM has been described as a reagent for facile conjugation with thiolated biomolecules under mild conditions.22, 29-31 DBM conjugates have also been demonstrated to be inherently fluorescent in the visible range obviating the requirement for an additional fluorescent tag.22, 23 In the present study, DBM-terminated IONPs were conjugated with biomolecules via amino substitution. In order to investigate the conjugation-induced fluorescence properties of the hybrid nanoparticles, tumor specific GRP78-binding peptide (CRLLDTNRPFLPY) was conjugated to the DBM-terminated polymers in aqueous media with 2.5 eq. of sodium carbonate (see the reaction scheme in Figure 3c). Fluorescence spectra collected from the peptidefunctionalized bromomaleimide (PBM) polymer showed a significant emission at ~510 nm in contrast to the unreacted polymers (Figure 3d), indicating the successful conjugation reaction between POEGA-b-PPA-DBM and GRP78-binding peptides.30 We further coupled a nearinfrared (NIR) fluorescent dye Cy5.5 to the POEGA-b-PPA-DBM coated IONPs through DBM and amine-functionalized Cy5.5 (Figure S7a). The fluorescence spectra of IONPs-Cy5.5 showed two different emissions (Figure S7c, d) at ~700 nm (λex = 684 nm) and ~530 nm (λex = 400 nm) whereas the DBM coated IONPs yielded no significant emission (Figure S7b) when irradiated at the two different wavelengths. As expected, the fluorescence intensity at ~530 nm was much weaker – however it is still powerful enough to allow for identification.20 Therefore, we envisage that the straightforward POEGA-b-PPA-DBM modification strategy resulting in stable and trackable IONPs can be readily scaled up and exploited for biomedical applications. We then carried out in vitro experiments to explore the possibility of specific cellular recognition by designing a tumor specific IONP-anti-GRP78 probe.32 The 530 nm emission was


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not observed using spectrophotofluorometry because of quenching induced by the IONP core. In comparison to the control group (Figure 4a), an emission from the conjugates was obtained under confocal microscopy after 2 h incubation with the MCF-7 breast cancer cell line (Figure 4b), showing that the IONP-anti-GRP78 probes can be tracked using the fluorescence of PBM elicited by the IONP-anti-GRP78 probe. Co-localization images show that the probes were successfully taken up by the tumor cells and were mainly located in the cell cytoplasm. A major advantage of this fluorescence tracking through PBM is that the emission is generated solely from the conjugates and not the nanoparticle or the biomolecule.

Figure 4. (a) Confocal images of IONPs and (b) IONP-anti-GRP78 probe incubated with MCF-7 cell line. Left to right: images under different channels including the nuclei stained by DRAQ5, PBM fluorescence and the merged images. Scale bars = 10 µm.


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Figure 5. (a) Alarma Blue and (b) Calcein AM assays for evaluating the cytotoxicity of the IONPs.

The biosafety profile of the POEGA-PPA-DBM polymer (Figure S8) and the as-prepared IONPs (Figure 5) was further assessed on human embryonic kidney cells 293 (HEK293) by using Alarma Blue assay and Calcein AM staining. As shown in Figure 5a, no significant cytotoxicity was observed after 24 h incubation with IONPs in a concentration range of 10-400


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µg Fe/mL. Cell membrane integrity and cellular morphology were further evaluated by Calcein AM/propidium iodide (PI) staining after 24 h exposure to 400 µg Fe/mL IONPs (Figure 5b). The green fluorescence from Calcein AM staining HEK293 cells was uniformly diffused in cell cytoplasm, indicating maintained membrane integrity. Moreover, no PI staining (red fluorescence) was observed in the cells in comparison to the 1 mM tert-Butyl hydroperoxide solution (TBHP) positive control group. The high viability and membrane integrity support a satisfactory safety profile of POEGA-b-PPA-DBM grafted IONPs. CONCLUSIONS In summary, we have established a surface grafting strategy for magnetic nanoparticles using a dibromomaleimide RAFT diblock copolymer containing brushed POEGA and phosphonic acid monomers. The strong binding affinity of the multiple phosphonic acids beget iron oxide particles with a high grafting density and an excellent colloidal stability in aqueous systems under physiological conditions. The simple method described herein can be easily adapted to the surface grafting of other metal-based inorganic nanoparticles, e.g. Gd-based nanoparticles, thanks to the ease of controlling the sequence length of phosphonic acid units. The dibromomaleimide-terminated

surface

ligand

enables

conjugation

with

amino-ended

biomolecules under basic conditions following a simple reaction procedure. Most importantly, the conjugation-induced fluorescence facilitates tracking of the magnetic nanoparticle-based conjugates through an emission at ~510-530 nm. The in vitro experiments demonstrated successful recognition of breast cancer cells through the labelling of GRP78-binding peptides. Therefore, we propose that a polymer design strategy based on DBM functionality will offer a powerful tool for the bioconjugation of nanoparticles.


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Supporting Information. Experimental details for polymer synthesis; 1HNMR spectra of the dibromomaleimide-CTA,

2-(dimethoxyphosphoryl)ethyl

acrylate,

POEGA-b-PPA-DBM

polymers, GPC analysis of the polymers; TGA analysis of the polymer grafted iron oxide particles, UV-Vis absorption spectra of IONPs and fluorescence spectra of IONPs-Cy5.5. Corresponding Author *Email: [email protected], [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes Any additional relevant notes should be placed here. ACKNOWLEDGMENT This work was supported by the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (project number CE140100036) and National Natural Science Foundation of China (81571746). RQ thanks Dr. Shiyang Tang from UoW for the TEM characterization of the iron oxide particles. REFERENCES (1) Qiao, R. R.; Yang, C. H.; Gao, M. Y., Superparamagnetic iron oxide nanoparticles: from preparations to in vivo MRI applications. J Mater Chem 2009, 19, 6274-6293. (2) Turcheniuk, K.; Tarasevych, A. V.; Kukhar, V. P.; Boukherroub, R.; Szunerits, S., Recent advances in surface chemistry strategies for the fabrication of functional iron oxide based magnetic nanoparticles. Nanoscale 2013, 5, 10729-10752.


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Bioconjugation and Fluorescence Labelling of Iron Oxide Nanoparticles Grafted with Bromomaleimideterminal Polymers Ruirui Qiao,*† Lars Esser,† Changkui Fu,‡§ Cheng Zhang, ‡§ Jinming Hu,∥ Paulina RamírezGarcía, † Yuhuan Li, † John F. Quinn, † Michael R. Whittaker, † Andrew K. Whittaker‡§ and Thomas P. Davis*† ⊥


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Bioconjugation and Fluorescence Labeling of Iron Oxide

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