Functionalization of NaGdF4 nanoparticles with a dibromomaleimide-terminated polymer for MR/optical imaging of thrombosis

Yuhuan Lia, Fangyun Xinab, Jinming Hu*c, Shweta Jagdaled, Thomas P. Davisae, Christoph E. Hagemeyerd and Ruirui Qiao*ae
aARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia. E-mail: ruirui.qiao@monash.edu
bBeijing Key Laboratory of Photo-electronic/Electro-photonic Conversion Materials, Key Laboratory of Cluster Science of Ministry of Education, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, P. R. China
cCAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Science at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, Anhui, China. E-mail: jmhu@ustc.edu.cn
dNanobiotechnology Laboratory, Australian Centre for Blood Diseases, Monash University, Melbourne, VIC 3004, Australia
eARC Centre of Excellence in Convergent Bio-Nano Science and Technology and Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia

Received 16th October 2019 , Accepted 10th December 2019

First published on 10th December 2019


Herein, we report the development of a thrombosis-targeted molecular imaging probe with magnetic resonance (MR) and optical dual-modality capacity using dibromomaleimide (DBM)-bearing polymer-grafted NaGdF4 nanoparticles. The random copolymer of bisphosphonic ester (BPE)-P(OEGA-co-DBM) was first synthesized through reversible addition–fragmentation chain transfer (RAFT) copolymerization of oligo(ethylene glycol)methyl ether acrylate (OEGA) and DBM-based monomers using a BPE-terminated RAFT agent. The resulting polymers were subjected to deprotection with the formation of bisphosphonic acid (BPA) terminals, allowing for the attachment of the as-synthesized BPA-P(OEGA-co-DBM) chains onto the surface of paramagnetic NaGdF4 nanoparticles through the ligand exchange reaction. Azide moieties could be readily incorporated into the hybrid nanoparticles by the coupling reaction between the highly reactive DBM moieties and amine derivatives. Intriguingly, the coupling reaction was characterized by a unique fluorescence turn-on even in aqueous media, which subsequently enabled the fluorescence imaging applications of the resulting hybrid nanoparticle. Furthermore, a single-chain antibody (scFv), which is specifically used for the active conformation of the GPIIb/IIIa integrin, was successfully attached onto the nanoparticles by a strain-promoted copper-free “click” reaction, allowing the targeting of activated platelets in acute thrombosis. The hybrid nanoparticles prepared through this new surface functionalization protocol possessed not only high colloidal stability under physiological conditions but also potential MR/optical imaging capacity. The thrombosis targeting capacity of the hybrid nanoparticle-based probe was then demonstrated by exploiting DBM conjugation-induced fluorescence in living cells.


Introduction

Polymer grafted functional nanomaterials are currently being developed for various biomedical applications ranging from sensing and diagnosis to drug delivery.1–3 These applications often involve the attachment of biomolecules,4,5 such as peptides,6 antibodies,7 folic acid,8 or aptamers,9 for specific targeting of diseases, including tumors and10 atherosclerotic plaques,11 or passing through biological barriers.12 The targeting moieties are required to be linked with the functional cores through either electrostatic interaction or conjugation to the surface of nanoparticles via covalent binding with the functional groups in the polymer coronas.10–15 In comparison with noncovalent adsorption, covalent binding offers better chemical control in terms of providing a robust link with the targeting moieties to avoid the potential desorption of the biomolecules in complex biological environments.16,17 Moreover, strong coordination between the polymer coronas and the inner cores is the first prerequisite for preventing polymer detachment.18 Therefore, well-designed biocompatible polymers that allow both effective bioconjugation and robust surface coordination are commonly required for the surface engineering of inorganic functional nanoparticles.19

Very recently, our group reported a DBM-terminated poly(oligo(ethylene glycol) methyl ether acrylate (POEGA) homopolymer for the surface modification of iron oxide nanoparticles (IONPs).13 Phosphonic acid groups were incorporated into the polymer for their strong coordination with the Fe atoms on the surface of IONPs.20 The polymers, which can be used for cellular tracking of peptide-labelled IONPs, exhibit a peculiar bioconjugation-induced fluorescence emission, arising from the “Michael addition” reaction between DBM and –NH2 or –SH groups.21–23 This emerging fluorescence has been demonstrated to be extremely useful for monitoring the nanoprobes especially for the particles without emission by conventional fluorescence microscopy.

Currently, the reliable and rapid diagnosis of thrombosis in cardiovascular disease (CVD) is widely required in clinical treatment, because conventional clinical/diagnostic techniques rely heavily on indirect detection methodologies such as reduction of flow or vessel narrowing in the respective vessels. Therefore, it is important to come up with a more direct and targeted visualization of thrombi through an imaging platform in order to achieve the accurate diagnosis of thrombotic occlusion as well as improve the clinical outcome of thrombotic disease. With regard to the most crucial contrast agents in T1-weighted magnetic resonance imaging (MRI), gadolinium (Gd)-based nanoparticles have received increasing attention over the last couple of years.10,11 Previously we have reported the development of molecular imaging probes by using NaGdF4, rare-earth element-doped nanoparticles (NaGdF4:Yb,Er) or core–shell structured particles (NaGdF4:Yb,Er@NaGdF4) for the in vivo imaging of tumors, lymph node metastasis, and atherosclerotic plaques in cardiovascular disease models.15 Due to the intrinsic paramagnetic properties of these probes, as well as the combination of upconverting properties generated from doping of rare earth elements, these probes have shown great promise as alternatives for Gd chelate-based MRI contrast agents.

Herein, by building on a multifunctional polymer platform with the incorporation of DBM-based monomers, followed by a coupling reaction with azide-terminated PEG, we demonstrate the surface engineering of NaGdF4 with activated platelet-specific scFv24,25 through a copper-free “click” reaction, which can be potentially used as a MRI contrast agent for thrombosis imaging.26 Moreover, owing to the fluorescence emission induced by the conjugation between DBM and amine groups, the probe was then evaluated using a cellular model of activated integrin expression as seen in thrombosis by confocal microscopy.

Experimental

Synthetic procedures

Chemicals and apparatus. The chemicals and solvents used for the synthesis of NaGdF4 nanoparticles, DBM-based monomers and polymers were purchased from Sigma-Aldrich and used as received. A5 cells expressing non-activated GPIIb/IIIa and Clone3 (C3) cells expressing activated GPIIb/IIIa receptors were used as reported previously.27 FITC labelled PAC-1 antibodies were purchased from BD Biosciences. All culture reagents were supplied by Life Technologies, Gibco-BRL.
Synthesis of the chain transfer agent (CTA). Under the protection of nitrogen, tetramethyl(((2-hydroxyethyl)azanediyl) bis(methylene))bis(phosphonate) (0.305 g, 1 mmol) was dissolved in 3 mL anhydrous dichloromethane (DCM) with 150 μL triethylamine in an ice-water bath. Then 2-bromopropanoyl bromide (0.214 g, 1 mmol) was added dropwise, and the mixture was stirred overnight. After completion of the reaction, the solution was filtered and was sequentially washed with saturated NaHCO3 and deionized H2O three times and dried over anhydrous MgSO4. After evaporation of the solvent, 2-(bis((dimethoxyphosphoryl)methyl)amino)ethyl 2-bromopropanoate was obtained.

Next, n-butylthiol (1.79 mL, 0.02 mol) was added in 19.2 mL anhydrous DCM. Then 4.98 mL trimethylamine and CS2 (1.01 mL, 0.01 mol) were added, and the mixture was stirred at room temperature for 2 h. Next, 2-(bis((dimethoxyphosphoryl)methyl)amino)ethyl 2-bromopropanoate (4.39 g, 0.01 mol) in 6.4 mL DCM was added and the mixture was stirred overnight at room temperature. The reaction mixture was washed with deionized water three times, dried with MgSO4 and evaporated to obtain a crude RAFT agent. The crude product was further purified by flash column chromatography.

Synthesis of the DBM-based monomer. The DBM-based monomer was synthesized according to the previous report.23 To a solution of 2-hydroxyethyl acrylate (13 g, 0.1 mol) and triethylamine (TEA, 15 mL, 0.107 mol) in chloroform (CHCl3) (300 mL), 2-bromoacetyl chloride (8.3 mL, 0.1 mol) was added dropwise in an ice-water bath. After stirring for two days, the excess 2-bromoacetyle chloride was quenched by the addition of methanol (5 mL). After methanol addition, the solution was stirred for another 30 min and then poured into saturated aqueous NaHCO3 (100 mL) before being washed twice with water (100 mL). The organic layer was collected and dried over anhydrous MgSO4, filtered and concentrated via rotary evaporation to obtain a brown oil. The product was further purified by column chromatography to obtain a colourless oil.

Subsequently, 2,3-dibromomaleimide (5.39 g, 21.1 mmol) and K2CO3 (2.92 g, 21.1 mmol) in acetone (200 mL) were added dropwise into 2-(2-bromoacetoxy)ethyl acrylate (4.56 g, 19.2 mmol). The solution was stirred at room temperature for 16 h before the solvent was removed by evaporation. The residue was dissolved in diethyl ether (300 mL) and washed with deionized water (200 mL × 6) and brine (200 mL) and dried over anhydrous MgSO4. The crude product was further purified by column chromatography.

Synthesis of BPA-P(OEGA-co-DBM) and BPA-P(OEGA-co-DBM-N3) copolymers. Typically, 28.4 mg (0.06 mmol) BPE-based CTA, 1 mg (0.006 mmol) azobisisobutyronitrile (AIBN), 0.5 g (45 mmol) OEGA monomer, and 0.5 g (5 mmol) DBM-based monomer were mixed in 2 mL of DMF, followed by degassing with nitrogen for 30 min. The polymerization was conducted at 70 °C for 24 h. The BPE-P(OEGA-co-DBM) copolymer was purified by precipitation into diethyl ether three times and dried under vacuum.

The deprotection of BPE was carried out as follows: 0.5 g (0.05 mmol) of the obtained copolymer was dissolved in 3 mL of anhydrous DCM in a glass vial equipped with a magnetic stirrer bar and sealed with a septum. The solution was stirred at 0 °C under a nitrogen flow, and a solution of trimethylsilyl bromide (TMSBr, 0.1725 g, 147.5 μL, 1 mmol) in 2 mL of DCM was added dropwise. The reaction mixture was stirred for 24 h at room temperature. At the end of the reaction, excess TMSBr and DCM were removed by evaporation under vacuum. After removal of TMSBr, methanol (5 mL) was added, and the mixture was stirred for another 24 h at room temperature. Afterwards, the resulting BPA-P(OEGA-co-DBM) copolymer was precipitated into an excess amount of petroleum ether (B.R. 40–60 °C) and washed with a mixture (1/1; v/v) of petroleum ether and diethyl ether. The remaining solvent was removed in a vacuum oven overnight at 35 °C, and the final product was analysed by 1H NMR.

The conjugation of the BPA-P(O EGA-co-DBM) copolymer (1 eq.) with N3-OEG8-NH2 (5.5 eq.) was further processed in tetrahydrofuran (THF, 20 mL) with sodium carbonate (12.5 eq.) at room temperature for 30 min.28 The obtained polymers were purified by precipitation with a mixture (1/1; v/v) of petroleum ether and diethyl ether.

Synthesis of oleic acid (OA)-coated NaGdF4 nanoparticles. Oleic acid-coated NaGdF4 nanoparticles were synthesized according to the previous report.29 GdCl3·6H2O (0.371 g, 1 mmol) was dissolved in a mixture of OA (14 mL) and 1-octadecene (16 mL). The solution was heated to 150 °C under nitrogen protection to form a homogeneous solution and then slowly added into 10 mL of methanol solution containing NaOH (0.100 g, 2.5 mmol) and NH4F (0.148 g, 4 mmol) after it was cooled down to room temperature. Next, the reaction mixture was stirred at 50 °C for 30 min and then the residual methanol was removed under vacuum for 10 min at 100 °C. After that, the reaction mixture was heated to 300 °C under atmospheric pressure with an electromantle, and maintained at 300 °C for 1 h under nitrogen protection. The reaction was terminated by cooling the reaction mixture down to room temperature. The resultant NaGdF4 nanoparticles were precipitated by ethanol, collected by centrifugation, washed with ethanol several times, and finally redispersed in THF or cyclohexane for further experiments.
Grafting polymers on NaGdF4 nanoparticles. 10 mg of the purified NaGdF4 particles capped with oleic acid and 100 mg of BPA-P(OEGA-co-DBM) or BPA-P(OEGA-co-DBM-N3) copolymers were dissolved in 5 mL of THF. The ligand exchange reaction was allowed to proceed overnight at 40 °C. Then, the resulting particles were precipitated into cyclohexane, followed by washing with cyclohexane three times, and finally dried under vacuum at room temperature. The resultant particles were dissolved in deionized water and purified by ultrafiltration with an Amicon Ultra centrifugal filter (molecular weight cut-off (MWCO): 100 kDa). TGA analysis was performed using freeze-dried powders.
Bioconjugation of NaGdF4@BPA-P(OEGA-co-DBM-N3) nanoparticles with the scFv antibody. For strain-promoted azide–alkyne cycloaddition, anti-GPIIb/IIIa-scFv-BCN was conjugated to the “click” N3 fusion partner as described previously25,30,31 with a ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]10 and 1[thin space (1/6-em)]:[thin space (1/6-em)]50 at pH 7 in PBS buffer for 3 h at room temperature under oxygen-free conditions. After the reaction, the samples were washed twice with PBS using spin columns (Millipore, MWCO: 10 kDa).

Characterization

Nuclear magnetic resonance (NMR) spectroscopy. 1H NMR spectra were recorded on a Bruker AC400F (400 MHz) spectrometer. Chloroform was used as the solvent, 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-4-methylphenol (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: 5.0 μm; pore sizes: 105, 104, 103, and 500 Å) and an RID-10A differential refractive-index detector. The temperature of the columns was maintained at 50 °C using a CTO-10A oven, and the flow rate was maintained at 1 mL min−1 using an LC-10AT 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−1. 2–3 mg mL−1 polymer solutions 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 at a rate of 20 °C min−1 from 50 to 800 °C. The weight loss percentage was calculated by the difference between the sample weights at 50 °C and 800 °C.
Transmission electron microscopy (TEM). TEM images were obtained using a JEOL JEM-2011 TEM. Energy-dispersive X-ray spectroscopy (EDS) maps were measured using a JEOL JEM-ARM200f scanning transmission electron microscope (STEM).
Dynamic light scattering (DLS). The hydrodynamic size of the particles was analysed at 298.0 K using a Nano ZS (Malvern) equipped with a solid state He–Ne laser (λ = 632.8 nm).
Fluorescence spectrometry. The fluorescence emission spectra were obtained using a Shimadzu RF-5301 pc fluorescence spectrophotometer in quartz cuvettes of 10 mm path length.
Fourier transform infrared (FTIR) spectrometry. FTIR spectra were recorded on a Shimadzu IRTracer-100 FTIR spectrometer under attenuated total reflectance (ATR). The spectra were collected over 64 scans with a spectral resolution of 4 cm−1.
Relaxivity. The T1 relaxation times of the nanoparticle solution with a series of concentrations loaded in 5 mm glass tubes were determined on a Bruker BioSpec 94/30 USR 9.4 T small animal MRI scanner using an RAREVTR sequence (TE = 30.87 ms, TR = 69.14–1000 ms, FOV = 40 × 40 mm, SI = 1 mm, matrix = 256 × 256, measurement time = 4 minutes 49 seconds).
Cell viability assays. Alamar Blue assay was used for cell viability evaluation. HEK293 cells (human embryonic kidney cell 293, ATCC) were grown in Dulbecco's Modified Eagle's Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% pen–strep. A5 and C3 were grown in DMEM with 10% FBS, 1% pen–strep, 1% L-glutamine, 1% non-essential amino acid (NEAA) and 700 μg geneticin for selection. HEK293 cells (1.5 × 104 cells per well) were exposed to NaGdF4@BPA-P(OEGA-co-DBM-N3) (5, 10, 25, 50 and 100 μg mL−1) for 24 h in 96-well black bottom plates. 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 2 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 fluorescence intensity of the triplicate was calculated to indicate cell viability.

Membrane integrity and cell morphology were evaluated by co-incubation with calcein AM and propidium iodide (PI) stains: 2 μM calcein AM and 4 μM PI for 15 min at 37 °C. H2O2 (1 mM) was used as the positive control and incubated with the cells for 20 min before staining. The cells were examined under a Leica TCS SP8 confocal microscope.

Cellular uptake study. A5 and C3 cells were used for the cellular binding study. The samples were diluted in 1× PBS and added to the existing media. After 24 h of incubation, the cells were washed with PBS three times and observed via confocal microscopy (40×).

After 24 h of incubation, 5 × 105 cells per sample were digested overnight using concentrated hydrochloric acid and diluted with ultrapure water before analysis. The amount of Gd3+ ions was quantitatively analyzed through ICP-OES (PerkinElmer Optima) measurement.

Results and discussion

Synthesis of the bisphosphonic ester (BPE)-terminated CTA

The phosphate group has been reported to be a versatile functional group for the surface anchoring of inorganic nanoparticles, including iron oxide nanoparticles, gold nanoparticles, quantum dots, and upconversion nanoparticles.18 In our previous work, a bisphosphonic acid-terminated polymer was used for the surface modification of Gd-based nanoparticles due to its strong multidentate Gd3+ binding ability, endowing the particles with superior colloidal stability in both water and physiological media.29 Herein, we firstly synthesized the BPE-functional CTA according to the process shown in Fig. S1. The 1H NMR spectrum (Fig. S2) clearly demonstrated the successful synthesis of the desired product.32

Synthesis of the DBM-based monomer and BPA-P(OEGA-co-DBM) copolymer

The DBM-based monomer and BPA-P(OEGA-co-DBM) copolymer were synthesized according to Fig. 1a and b. Briefly, the DBM-based monomer was prepared in a two-step reaction: (i) bromoacetyl acrylate was synthesized by the reaction of 2-hydroxyethyl acrylate (1) and bromoacetyl chloride (2). As shown in Fig. S3, the formation of the product 2-(2-bromoacetoxy)ethyl acrylate (3) was confirmed by 1H NMR. (ii) Compound 3 was further reacted with 2,3-dibromomaleimide (4) to obtain the DBM-based monomer (5). The 1H and 13C NMR spectra, as displayed in Fig. S4 and S5, clearly demonstrated the successful preparation of the DBM-based monomer with a molecular weight of 433.7 g mol−1 (corresponding to [M + Na]+) as determined by liquid chromatography mass spectrometry (LC-MS). Next, a copolymer of BPE-P(OEGA-co-DBM), which consists of the DBM-based monomer and OEGA (number average molecular weight (Mn) of 480 g mol−1), was synthesized via RAFT polymerization (Fig. 1b (1)) for 4 hours. The 1H and 31P NMR spectra after purification (Fig. S6) suggested the successful incorporation of both DBM and OEGA in the BPE-P(OEGA-co-DBM) copolymer with an Mn of 18[thin space (1/6-em)]600 g mol−1 and a polydispersity of 1.39 by GPC analysis (Fig. S7). Afterwards, the phosphonic ester groups of BPE-P(OEGA-co-DBM) were hydrolysed in the presence of trimethylsilyl bromide (TMSBr) to expose the phosphonic acid groups, thereby creating BPA-P(OEGA-co-DBM) (Fig. 1b (2)). The complete conversion from BPE-P(OEGA-co-DBM) into BPA-P(OEGA-co-DBM) was confirmed by 31P NMR, revealing an upfield shift of the 31P signal from 25.8 to 9.9 ppm (Fig. 2). After the successful synthesis of BPA-P(OEGA-co-DBM), the fluorescent copolymer of BPA-P(OEGA-co-DBM-N3) was achieved by treating the BPA-P(OEGA-co-DBM) precursor with N3-OEG8-NH2 for 30 min (Fig. 3 (1)). The successful introduction of the azide moiety was indicated by FTIR, showing the appearance of the characteristic signal of azide residues at ∼2100 cm−1 compared to the BPA-P(OEGA-co-DBM) precursor (Fig. 4a). The reaction between DBM and amine groups corresponds to a chemico-fluorescent response, leading to a green emission at ∼510 nm. To investigate the conjugation-induced fluorescence property, fluorescence spectra (Fig. 4b and c) under UV 400 nm excitation were collected from the corresponding copolymers. A macroscopic image (Fig. S8) was obtained under 365 nm UV irradiation. The azide-functionalized BPA-P(OEGA-co-DBM-N3) polymer displayed a fluorescence switch on performance, which was exemplified by a significantly higher intensity at ∼510 nm emission in contrast to the BPA-P(OEGA-co-DBM) precursor in both THF (Fig. 4b) and water (Fig. 4c), respectively. Hence, a successful coupling reaction of BPA-P(OEGA-co-DBM) with azide groups is identified.
image file: c9py01568j-f1.tif
Fig. 1 (a) Synthesis of the DBM-based monomer and (b) synthesis of the BPA-P(OEGA-co-DBM) copolymer with BPE terminals via RAFT polymerization and subsequent deprotection of the BPE groups.

image file: c9py01568j-f2.tif
Fig. 2 1H and 31P NMR (inset) spectra (CDCl3) of the BPA-P(OEGA-co-DBM) polymer.

image file: c9py01568j-f3.tif
Fig. 3 Reaction between DBM in the BPA-P(OEGA-co-DBM) copolymer and azide groups (1); surface ligand exchange of the oleic acid coated NaGdF4 nanoparticles (2, 3).

image file: c9py01568j-f4.tif
Fig. 4 FTIR (a) and fluorescence spectra (b, in THF; c, in H2O) of BPA-P(OEGA-co-DBM) and BPA-P(OEGA-co-DBM-N3) polymers.

Preparation of copolymer-coated NaGdF4 nanoparticles

We further grafted the BPA-terminated copolymers onto the NaGdF4 nanoparticles via a ligand exchange process in THF at 40 °C by taking advantage of the BPA terminals, which provide strong binding affinity to the surface of inorganic nanoparticles (Fig. 3). The azide-functionalized polymer coated NaGdF4 nanoparticles (i.e., NaGdF4@BPA-P(OEGA-co-DBM-N3)) were re-dispersed in water and characterized by TEM and DLS analyses. As shown in Fig. 5a and b, the achieved NaGdF4@BPA-P(OEGA-co-DBM-N3) nanoparticles have a uniform size (14.2 ± 2.5 nm) as revealed by TEM. The hydrodynamic sizes of the particles in water and PBS buffer were determined to be 64.5 and 64.3 nm, respectively (Fig. 5c). The hydrophilic PEG together with the strong binding affinity of BPA groups enabled high colloidal stability and water solubility of the nanoparticles in both water and PBS buffer. Next, the amount of polymer grafting on the surface of NaGdF4 nanoparticles was investigated by TGA and the weight loss was 49.9% for NaGdF4@BPA-P(OEGA-co-DBM-N3), which is identical to that for azide free NaGdF4@BPA-P(OEGA-co-DBM) with a 50.9% polymer coating amount (Fig. 5d). This result revealed that the functionalization of DBM moieties with azide residues did not adversely affect the covalent binding between the NaGdF4 nanoparticles and copolymer chains. Meanwhile, a comparison of the fluorescence spectra showed that NaGdF4@BPA-P(OEGA-co-DBM-N3) has a significantly higher fluorescence intensity than NaGdF4@BPA-P(OEGA-co-DBM), indicating the successful glowing effect of the resultant nanoparticles after grafting with BPA-P(OEGA-co-DBM-N3) (Fig. 5e). Furthermore, the paramagnetic property of NaGdF4@BPA-P(OEGA-co-DBM-N3) was also evaluated in water against the concentration of Gd3+ ions by ultra-high-field (9.4 T) MRI. The r1 relaxivity (efficiency to increase the MRI contrast) was extracted from the linear regression fits of the experimental data, as shown in Fig. 5f, and was determined to be 0.74 mM−1 s−1 under 9.4 T MRI. In comparison with previously reported NaGdF4 NPs under a high magnetic field (7 T MRI), NaGdF4@BPA-P(OEGA-co-DBM-N3) showed comparable r1 relaxivity (0.746 mM−1 s−1 vs. 0.74 mM−1 s−1).33 Therefore, the resulting hybrid nanoparticles can be used not only for fluorescence imaging by taking advantage of the emerging fluorescence of DBM moieties after modification with amine residues but also for MR imaging by means of the inorganic cores of NaGdF4.
image file: c9py01568j-f5.tif
Fig. 5 (a) TEM, (b) size histogram and (c) hydrodynamic size distributions of the BPA-P(OEGA-co-DBM-N3) polymer-grafted NaGdF4 nanoparticles in water and PBS; (d) TGA and (e) fluorescence spectra of BPA-P(OEGA-co-DBM) and BPA-P(OEGA-co-DBM-N3) polymer-grafted NaGdF4 nanoparticles; (f) T1 relaxation rate (r1) of BPA-P(OEGA-co-DBM-N3) polymer-grafted NaGdF4 nanoparticles in water against the concentration of Gd3+ ions determined by 9.4 T MRI.

To explore the potential biomedical applications of these hybrid nanoparticles, the biosafety property of NaGdF4@BPA-P(OEGA-co-DBM-N3) was first assessed on HEK293 using Alamar Blue assay and calcein AM/PI staining. As shown in Fig. 6a, no significant cytotoxicity was observed after 24 h of incubation with the nanoparticles in a concentration range of 5–100 μg Gd3+ per mL. Cell membrane integrity and cellular morphology were further evaluated by calcein AM/PI staining after 24 h of exposure to 100 μg Gd3+ per mL (Fig. 6b). The green fluorescence observed on staining live cells with calcein AM was uniformly diffused in the cytoplasm, indicating that membrane integrity and cell viability were maintained. Moreover, no PI staining (red fluorescence) was observed in the cells in comparison with the positive control group (H2O2, 1 mM, 20 min of incubation). The high viability and membrane integrity support a satisfactory safety property of BPA-P(OEGA-co-DBM-N3) grafted NaGdF4 nanoparticles.


image file: c9py01568j-f6.tif
Fig. 6 (a) Alamar Blue assay and (b) calcein AM/PI staining for evaluating the cytotoxicity of NaGdF4@BPA-P(OEGA-co-DBM-N3): (i) control; (ii) NaGdF4@BPA-P(OEGA-co-DBM-N3), 100 μg mL−1; (iii) H2O2, 1 mM. Scale bar = 50 μm.

Bioconjugation with a single-chain antibody by the “click” reaction

CVD is one of the leading causes of death and disability responsible for substantial medical expense worldwide, so there is an ongoing need for the development of novel cardiovascular diagnosis and therapy approaches.34 Followed by plaque rupture,35 acute thrombosis is recognized as an excellent pathophysiological risk marker for CVD due to the presence of numerous specific epitopes,36 such as activated coagulation factors,37 produced by the coagulation cascade,38 as well as several adhesion receptors on activated platelets.39,40 Hence, the introduction of the targeting strategy will greatly improve the efficiency of diagnostic and therapeutic agents. GPIIb/IIIa receptors mediate platelet aggregation via fibrinogen binding and promote thrombus propagation. As they are only expressed on the platelets, they are an ideal target for molecular imaging and/or drug delivery.41 Thus, the direct observation of thrombosis based on the GPIIb/IIIa receptor is considered to be beneficial for the diagnosis and guidance of CVD therapy in clinical application. Based on our previous studies, scFv that specifically binds to activated GPIIb/IIIa receptors has shown excellent specificity towards platelets.42

Therefore, in this study, the scFv antibody terminated by a bicyclo-[6.1.0]-nonyne (BCN) group that specifically binds to activated GPIIb/IIIa receptors expressed on platelets was covalently conjugated to NaGdF4@BPA-P(OEGA-co-DBM-N3) (NP) via the azide linker by a “click” reaction at room temperature in water (Fig. 7a). The targeting functionalized NP-scFv was formulated and optimized at different molar ratios of NP and scFv (1[thin space (1/6-em)]:[thin space (1/6-em)]10 and 1[thin space (1/6-em)]:[thin space (1/6-em)]50) based on the hydrodynamic sizes and zeta potentials (Fig. 7b and c). After the conjugation of the antibody, the hydrodynamic size of the nanoparticle increased, and the size distribution became broader, especially at a ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]50. Therefore, the conjugates with a 1[thin space (1/6-em)]:[thin space (1/6-em)]10 ratio of NP and scFv were used for further in vitro experiments. In the meantime, the surface charge of NP-scFv reverses to a negative value due to the electronegativity of the introduced antibody, illustrating the successful covalent conjugation between NP and scFv. Meanwhile, according to the reaction yield reported in our previous work,25 the amount of scFv in the NP-scFv (1[thin space (1/6-em)]:[thin space (1/6-em)]10) conjugate can be estimated to be ∼10 per particle.


image file: c9py01568j-f7.tif
Fig. 7 (a) Conjugation of NaGdF4@BPA-P(OEGA-co-DBM-N3) with the scFv-BCN antibody; (b) hydrodynamic size and (c) zeta potential of the NP and NP-scFv at ratios of 1[thin space (1/6-em)]:[thin space (1/6-em)]10 and 1[thin space (1/6-em)]:[thin space (1/6-em)]50.

We then carried out in vitro experiments to explore the possibility of specific cellular recognition by the resultant NP-scFv (1[thin space (1/6-em)]:[thin space (1/6-em)]10) and NP in stably transfected CHO cells, including A5 (non-activated GPIIb/IIIa receptor) and C3 (activated GPIIb/IIIa receptor). Prior to the cellular recognition, the receptor expression on the cells was confirmed (Fig. S9) using a PAC-1 antibody. In comparison with the control and NP group, the significant binding of the conjugate NP-scFv to C3 cells was clearly observed by confocal microscopy after 24 h of incubation (Fig. 8a). Furthermore, the quantitative analysis of cellular internalization by ICP-OES (Fig. 8b) and using ImageJ (Fig. 8c) confirmed a higher binding affinity compared with the control and NP group, demonstrating the excellent targeting ability of NP-scFv.


image file: c9py01568j-f8.tif
Fig. 8 The cellular internalization of NP and NP-scFv (0.1 mg mL−1) in A5 and C3 cells was determined by confocal microscopy (a) and quantitatively measured by ICP-OES (b) after 24 h of incubation. The corresponding fluorescence intensities of the confocal images were quantitatively analyzed using ImageJ (c). Scale bars = 10 μm.

Conclusions

In this study, we designed and synthesized a dual-modality magnetic resonance and optical imaging nanoprobe via the surface engineering of NaGdF4 nanoparticles using a polymer having chemico-fluorescent responsive properties. The DBM-based monomer was synthesized and incorporated into the polymer via RAFT polymerization, making it possible to achieve concomitant DBM conjugation-induced fluorescence and display facile and specific conjugation characteristics through a copper-free “click” reaction between an azide linker and a BCN terminated single-chain antibody. The bisphosphonic acid and PEG in the copolymer serving as surface anchoring and anti-biofouling compartments endowed NaGdF4 nanoparticles with stability and biocompatibility. Then, further conjugation of the single-chain antibody with the nanoparticles confers the probe with targeting ability towards activated GPIIb/IIIa receptors. The in vitro experiments demonstrated that the DBM containing PEGylated NaGdF4 nanoprobe displayed excellent biocompatibility, glowing effect and targeting efficiency. Therefore, we propose that a polymer design strategy based on DBM functionality and targeting ligand will offer a powerful tool for platelet targeting and thrombosis imaging.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (Project No. CE140100036), the National Natural Science Foundation of China (81571746, 51722307 and 51673179) and the National Health and Medical Research Council (Project Grant 1078118 and Senior Research Fellowship 1154270 to C. E. H.). Y. L. thanks Dr Changkui Fu from The University of Queensland, Australia, for the characterization of the relaxivity of NaGdF4 nanoparticles.

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Footnote

Electronic supplementary information (ESI) available: Supporting Fig. S1–S9. See DOI: 10.1039/c9py01568j

This journal is © The Royal Society of Chemistry 2020