In situ synthesis of magnetic poly(N-tert-butyl acrylamide-co-acrylic acid)/Fe₃O₄ nanogels for magnetic resonance imaging

Abstract

Poly(N-tert-butylacrylamide-co-acrylic acid)/Fe₃O₄ (P(TBA-co-AA)/Fe₃O₄) nanogels have been synthesized successfully by two steps: firstly, poly(N-tert-butylacrylamide-co-acrylic acid) (P(TBA-co-AA)) nanogels were synthesized through emulsion precipitation polymerization by using N-tert-butylacrylamide (TBA) and acrylic acid (AA) as reactive monomers; then P(TBA-co-AA)/Fe₃O₄ nanogels were synthesized by in situ oxidation of Fe²⁺ into Fe₃O₄ nanoparticles inside the networks of the P(TBA-co-AA) nanogels. The prepared P(TBA-co-AA)/Fe₃O₄ nanogels were about 70 nm in dry state, and 118.9 nm in aqueous solution with a low polydispersity index (PDI) of 0.124. The small size and low PDI of the magnetic nanogels could be applied in living body. Besides, the nanogels had good superparamagnetic property, and high r₂ relaxivity value of about 512.01 mM⁻¹ s⁻¹. These magnetic properties endowed the nanogels with a new ability to be applied in T₂-style imaging. In short, this work would provide the possibility of using the P(TBA-co-AA)/Fe₃O₄ nanogels as contrast agents for high resolution of magnetic resonance imaging (MRI).

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1. Introduction

Magnetic nanoparticles (MNPs) have been used incrementally in the biomedical field,^1–5 among which iron oxide is the most commonly used because of its stability and low level of toxicity.⁶ For example, Fe₃O₄ nanoparticles have been researched^7,8 and applied as magnetic resonance imaging (MRI) contrast agents because they can improve r₂ relaxivity.^9–11 However, they may aggregate easily owing to their tiny sizes and high surface energy, and cause blockages in blood vessels when they are injected into veins.^12,13 Moreover, the surfaces of commonly used Fe₃O₄ nanoparticles are hydrophobic^6,12 which is incompatible with the human environment. And there is growing evidence that iron accumulation in brain may associate with Parkinson's disease.¹⁴ All these defects above of Fe₃O₄ nanoparticles limit their applications in practice.

Nanogels are in the range of 10–1000 nm with three-dimensional network nanostructures which have good biocompatibility and stimuli-responsive.^15,16 Therefore, many researchers used nanogels to encapsulate the Fe₃O₄ nanoparticles to avoid their aggregation and form protective layers to improve their hydrophilicity.^14,17,18 The traditional method to synthesize magnetic hybrid nanogels is prepare Fe₃O₄ nanoparticles first, and then coat Fe₃O₄ nanoparticles with nanogel shells.^18,19 However, that method could easily cause aggregation and low encapsulation efficiency of Fe₃O₄.²⁰ In situ synthesis of Fe₃O₄ nanoparticles in the interior of nanogels is an alternative approach for the magnetic nanogels,²¹ in which the nanogels could be used as nanoreactors to avoid the aggregation, and maintain the structure and content of Fe₃O₄ nanoparticles.²²

N-tert-Butylacrylamide (TBA) is an important material to synthesize copolymer nanogels, which can enhance the rigidity of molecular chain and then decrease the phase transition temperature and salt resistance.^23,24 TBA-based nanogels have good biocompatibility and their mean size is smaller than other isonomic nanogels under the same reactive situation for hydrophobicity of TBA, thus they are more suitable as an intravenous injection for living body. Therefore, we described a novel magnetic nanogels preparation technology. In this work, TBA and acrylic acid (AA) were used as monomers to synthesize P(TBA-co-AA) nanogels, in which TBA was used as nanogels frameworks and ionized AA was used to attract more Fe²⁺ inside the networks of the nanogels. Then Fe²⁺ was oxidized to form Fe₃O₄ nanoparticles in networks of the nanogels, in which FeSO₄·7H₂O was used as iron source to provide Fe²⁺.²⁵ The whole synthesis process was showed in Fig. 1. The prepared magnetic nanogels had a small size, a low polydispersity index (PDI) value, a good superparamagnetic property, and a high r₂ relaxivity value by using P(TBA-co-AA) nanogels as polymer shell to in situ synthesize Fe₃O₄ nanoparticles.

Fig. 1 The synthesis process of P(TBA-co-AA) and P(TBA-co-AA)/Fe₃O₄ nanogels, Fe₃O₄.

2. Experiment

2.1. Materials

N-tert-Butylacrylamide (TBA, 97%), acrylic acid (AA, 99%), sodium dodecyl sulfate (SDS, ≧99%), N,N′-methylenebisacrylamide (BIS, 99%), and ammonium persulfate (APS, ≧98%) were purchased from Sigma-Aldrich. NaOH, FeSO₄·7H₂O, NaNO₂, NH₃·H₂O (28 wt%), and pure Fe₃O₄ nanoparticles (nm) were A.R. grade and purchased from Sinopharm Chemical Reagent Co., Ltd. All reagents were used without further purification. Ultrapure water (UPW) was 18.2 MΩ cm and prepared by Direct-Q 3UV System from Shanghai Tongdi Co., Ltd.

2.2. Preparation of P(TBA-co-AA) nanogels

Approximately 1.50 g of TBA, 0.10 g of AA, 0.08 g of SDS, and 0.04 g of BIS were dissolved in 145 mL of UPW, and stirred the system at 400 rpm for 2 h at room temperature by electric-heated thermostatic water bath under a N₂ atmosphere. Then the solution was maintained at 60 °C for 30 min, 5 mL of APS at a concentration of 0.008 g mL⁻¹ was injected into the system. The reaction was kept for 4 h, and the nanogels were purified by dialysis bag (molecular weight cutoff of 8000–14 000 Da) with plenty of deionized water at room temperature for a week. The deionized water was replaced with fresh water three times a day.

2.3. Preparation of P(TBA-co-AA)/Fe₃O₄ nanogels

0.2 M of NaOH was used to regulate the pH of P(TBA-co-AA) nanogels to 6.2 ± 0.2. Then 0.0695 g of FeSO₄·7H₂O was mixed with 50 mL of the nanogels above, and stirred at 200 rpm at room temperature for 12 h by mechanical stirrer under a N₂ atmosphere. After dialyzing the nanogels for 3 h, 0.052 g of NaNO₂ was mixed with the solution under the N₂ atmosphere. 0.5 h later, the stirring rate was improved to 600 rpm and 5 mL of NH₃·H₂O was quickly injected into the system. The reaction was kept for 3 h at room temperature. The whole process was operated in the dark condition and the resulting magnetic nanogels were purified by the same method as the dialysis of P(TBA-co-AA) nanogels.

2.4. Characterizations

The hydrodynamic particle size and monodispersity of the samples were measured with dynamic light scattering (DLS, BI-200SM, Brookhaven Co., Ltd., USA) equipped with a standard 633 nm laser, the test temperature was 25 °C, the pH was 7.4 ± 0.1, and the concentration was 0.1 g mL⁻¹. The chemical functional groups of the samples were determined using Fourier transform infrared (FTIR, Nicolet6700, Thermo Fisher Technology Co., Ltd., USA), the samples were freeze dried and KBr was mixed before the testing. The morphology and size of the particles were characterized with transmission electron microscope (TEM, JEM-2100, JEOL Co., Ltd., JPN), the samples were diluted to 50 mg mL⁻¹ and tested in dry state with 200 kV. The Fe concentration of the magnetic sample was analyzed using inductively coupled plasma-atomic emission spectroscopy (ICP-AES, Prodigy, Leeman Co., Ltd., USA). The crystalline structure of samples were determined using X-ray diffraction (XRD, D/Max-2550 PC, Rigaku Co., Ltd., JPN), the scan range (2θ) was from 20 to 70°. The magnetic properties were examined using vibrating sample magnetometer (VSM, Lakeshore7407, Trend Limited Co., Ltd., USA) at 300 K. T₂ relaxometry was performed by a 0.5 T NMI20-Analyst NMR Analyzing and Imaging system (Shanghai Niumag Co., Ltd., CHN) using a wrist receiver coil with a Carr–Purcell–Meiboom–Gill (CPMG) sequence, the P(TBA-co-AA)/Fe₃O₄ nanogels were diluted in UPW with Fe concentration in the range of up to 0.05 mM. The instrumental parameters were set at point resolution = 156 mm × 156 mm, section thickness = 0.6 mm, TR = 5500 ms, TE = 0.2 ms, and number of excitation = 1. The r₂ relaxivity was calculated by a linear fit of the inverse T₂ (1/T₂) relaxation time as a function of Fe concentration.

3. Results and discussion

3.1. Chemical and crystal structures

FTIR characterization was applied to characterize the chemical structures of the prepared P(TBA-co-AA)/Fe₃O₄ nanogels. And FTIR spectra of AA, TBA, the P(TBA-co-AA) nanogels, pure Fe₃O₄ nanoparticles were used as control samples. As was shown in Fig. 2a, the bands at 2964–2968 cm⁻¹ could be attributed to stretching and bending vibrations of C–H groups.²⁶ The characteristic band at 1705 cm⁻¹ was assigned to the C=O stretching of AA. The bands at 1651–1658 cm⁻¹ and 1546–1560 cm⁻¹ were regarded as amide I (C=O stretching) and amide II (N–H bending), respectively.^26,27 The characteristic bands at 1627–1636 cm⁻¹ and 984–994 cm⁻¹ assigned to C=C of AA and TBA, which could not be found in the spectra of the P(TBA-co-AA) and P(TBA-co-AA)/Fe₃O₄ nanogels, indicating that the AA and TBA monomers totally formed the P(TBA-co-AA) and P(TBA-co-AA)/Fe₃O₄ nanogels in the process of polymerization.^28,29 Two characteristic bands for Fe–O stretching observed at 587–593 cm⁻¹ and 464–467 cm⁻¹ were found in the Fe₃O₄ spectra. Moreover, the two characteristic bands of Fe₃O₄ were found in the P(TBA-co-AA)/Fe₃O₄ nanogels spectra but didn't appear in the P(TBA-co-AA) nanogels spectra which confirmed presence of Fe₃O₄ in the magnetic P(TBA-co-AA)/Fe₃O₄ nanogels.^27,30

Fig. 2 FTIR spectra of AA, TBA, P(TBA-co-AA), pure Fe₃O₄ and P(TBA-co-AA)/Fe₃O₄ (a). XRD patterns of Fe₃O₄, P(TBA-co-AA) and P(TBA-co-AA)/Fe₃O₄ nanogels (b).

Meanwhile, XRD spectra also confirmed the encapsulation of crystal Fe₃O₄. Fig. 2b were the XRD patterns of pure Fe₃O₄ nanoparticles, the P(TBA-co-AA) and P(TBA-co-AA)/Fe₃O₄ nanogels. The P(TBA-co-AA) nanogels revealed an amorphous structure and did not show any crystal peaks. And the pure Fe₃O₄ nanoparticles exhibited strong diffraction peaks and indexed to (220), (311), (400), (422), (511) and (533) planes of spinel cubic structure of Fe₃O₄ (Joint Committee on Powder Diffraction Standards (JCPDS) card number 88-0315). In contrast, the P(TBA-co-AA)/Fe₃O₄ nanogels exhibited apparent diffraction peaks at 2θ = 30.081 (220), 35.598 (311), 43.205 (400), 57.200 (511) and 62.761 (400).^27,31 This evidence indicated that the crystal structures of Fe₃O₄ particles were kept in the magnetic nanogels.

3.2. Morphology and size characterizations

Fig. 3a and b were TEM photos of the P(TBA-co-AA) and P(TBA-co-AA)/Fe₃O₄ nanogels, respectively. The P(TBA-co-AA) nanogels were spherical, and the P(TBA-co-AA)/Fe₃O₄ nanogels were near-spherical. Moreover, the dry mean sizes of P(TBA-co-AA) nanogels and P(TBA-co-AA)/Fe₃O₄ nanogels were measured to be about 50 nm and 70 nm, respectively. Apparently, the mean size of P(TBA-co-AA)/Fe₃O₄ nanogels was bigger than the other one, this was probably because the Fe₃O₄ nanoparticles were synthesized and contained in the interior of the nanogels. And from the higher magnification TEM photo in Fig. 3b, the P(TBA-co-AA)/Fe₃O₄ nanogels were filled with Fe₃O₄ nanoparticles. However, the magnetic nanogels with 70 nm in diameter were still small sizes which could be applied for more tissues imaging in vivo. Besides, the P(TBA-co-AA) nanogels could improve the hydrophilicity and stability of magnetic nanoparticles (MNPs), thus they could not be identified easily by macrophage when they were injected into vein, which could increase the half-time of phagocytosis and the time of contract imaging.³²

Fig. 3 TEM photos of P(TBA-co-AA) (a) and P(TBA-co-AA)/Fe₃O₄ nanogels (b). Particle size distribution of P(TBA-co-AA) and P(TBA-co-AA)/Fe₃O₄ nanogels (c).

Fig. 3c showed the hydrodynamic mean sizes and PDI values of the P(TBA-co-AA) and P(TBA-co-AA)/Fe₃O₄ nanogels which were characterized by DLS. They were measured to be 82.1 nm and 118.9 nm, respectively. And the PDI values were 0.085 and 0.124 accordingly. Meanwhile, magnetic nanogels had a quite small PDI value, indicating that the nanogels had uniform size distribution. And the hydrodynamic mean size of the P(TBA-co-AA)/Fe₃O₄ nanogels was bigger than the other one, this result was in consistent with the dates measured by TEM. Besides, the particle size distribution of P(TBA-co-AA) nanogels (red parabola) and P(TBA-co-AA)/Fe₃O₄ nanogels (black parabola) in Fig. 3c, showed both nanogels had a very narrow size distribution range. And photos (Fig. 3c, inset) showed the P(TBA-co-AA) nanogel sample was light blue and the P(TBA-co-AA)/Fe₃O₄ nanogel sample was puce.

3.3. Magnetic properties

Photos of the different states of the P(TBA-co-AA)/Fe₃O₄ nanogels which clearly showed their magnetic behavior were shown in Fig. 4a. The nanogel dispersion was puce and turbid (left), when magnets were placed near to the sample after 1–2 h, the suspension became clear and transparent (right). And the magnetic nanogels were collected at the vial wall because of the attraction from the magnets. However, when the magnets were removed and ultrasonic was afforded, the sample turned back to its original state. The process above was repeatable which showed the P(TBA-co-AA)/Fe₃O₄ nanogels kept a good magnetic performance and good dispersion at aqueous media. What's more, the synthesized Fe₃O₄ nanoparticles can be encapsulated by the network of the P(TBA-co-AA) nanogels. And the content of Fe element of the nanogels was 1.73 μg mL⁻¹.

Fig. 4 Photos of the P(TBA-co-AA)/Fe₃O₄ nanogels (a) and magnetization curves for Fe₃O₄ and P(TBA-co-AA)/Fe₃O₄ nanogels (b).

The magnetization curves of the pure Fe₃O₄ nanoparticles (blue line) and the prepared P(TBA-co-AA)/Fe₃O₄ nanogels (black line) were shown in Fig. 4b. Yang Yudong³³ reported that the superparamagnetic magnetic particles had a huge magnetism in a weak external magnetic field but had no magnetism without the outer magnetic field. As shown in Fig. 4b, no hysteresis loop was observed, and both coercivity (H_c) and remanence (M_r) were measured to be zero, meaning these samples had a superparamagnetic property.^34,35 Thus the magnetic nanogels could be used to improve the clarity of T₂-style magnetic resonance imaging (MRI). Besides, the magnetization values of Fe₃O₄ and P(TBA-co-AA)/Fe₃O₄ were found to be 83.3 emu g⁻¹ and 10.1 emu g⁻¹. These values were similar to previously reported work of nanogels-based MNPs.^27,36

3.4. r₂ relaxivity measurements

Fig. 5 showed the transverse relaxation time (T₂) of water protons in an aqueous solution of the P(TBA-co-AA)/Fe₃O₄ nanogels. From the T₂-weighted MRI (Fig. 5a), it showed that the P(TBA-co-AA)/Fe₃O₄ sample was able to induce the decrease of the MR signal intensity with the increase of Fe concentration. By linear fitting of the T₂ relaxation rate (1/T₂) as a function of Fe concentration (Fig. 5b), the r₂ relaxivity of the P(TBA-co-AA)/Fe₃O₄ nanogels was estimated to be 512.01 mM⁻¹ s⁻¹. It is easy to find that the value is higher than that of other Fe₃O₄ nanoparticles coated with polymer shells,^37,38 which was essential for the prepared P(TBA-co-AA)/Fe₃O₄ nanogels to be used as good T₂ negative contrast agent for sensitive MRI applications with small dose.

Fig. 5 T₂-weighted MRI was characterized under different Fe concentrations of 0.05, 0.025, 0.0125, 0.00625, and 0.003125 mM (a) and linear fitting of 1/T₂ (b) of P(TBA-co-AA)/Fe₃O₄ at the Fe concentrations in range of 0.05–0.003125 mM with transverse relaxation time of 38.0665, 79.2508, 139.3062, 273.6175, and 475.7778 ms, accordingly.

4. Conclusions

In summary, a new method was developed to prepare the P(TBA-co-AA)/Fe₃O₄ nanogels which had a very low PDI value of 0.124, the hydrodynamic and dry mean size were 118.9 nm and 70 nm, respectively. The magnetization value and the r₂ relaxivity of the P(TBA-co-AA)/Fe₃O₄ nanogels were 10.1 emu g⁻¹ and 512.01 mM⁻¹ s⁻¹. Moreover, the P(TBA-co-AA)/Fe₃O₄ nanogels had a superparamagnetic property and a significant contrast enhancement in T₂-weighted MRI, meaning the magnetic nanogels had high sensitivity and clear imaging as MRI contrast agent.

Acknowledgements

The authors thank for financial supports by National Nature Science Foundation of China (51473032, 51503034), Fundamental Research Funds for the Central Universities (CUSF-DH-D-2016024, 2232015D3-12) and Science and Technology Commission of Shanghai Municipality for Yangfan Program (15YF1400700).

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