Tailored dual coating of magnetic nanoparticles for enhanced drug loading

Abstract

A novel type of superparamagnetic iron oxide nanoparticles (SPIONs) with dual coatings was reported to address the problem of poor loading capability of traditional SPIONs. It was found that the loading of a model drug, paclitaxel, in such SPIONs increased up to ca. 190 times.

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As a major life-threatening disease, cancer ranks top in terms of both mortality and morbidity globally.¹ In recent years, the employment of nanoparticles for cancer treatment and diagnosis has been prevalent aiming for improved efficacy and reduced toxicity; a few products have been approved for clinical use with many still in clinical trials or under development.² Ideal anti-cancer nanotherapy should integrate the imaging, tumor targeting, and drug delivery into one platform. Finely engineered magnetic nanoparticles is an excellent example to combine the above three features together.³

Among the diverse range of magnetic nanomaterials, superparamagnetic iron oxide nanoparticles (SPIONs), composed of a magnetic core and a surface coating, provide a flexible solution to effective cancer therapies due to their intrinsic appealing characteristics.⁴ SPIONs exhibit a biodegradable superparamagnetic iron core rendering them suitable as T₂ contrast agents for non-invasive magnetic resonance imaging (MRI), which demonstrates the advantage of high spatial resolution and superior contrast differentiation between soft tissues.⁵ The surface coating not only affords a protective layer to maintain the physical stability of SPIONs, but also enables the loading of therapeutic agents and the attachment of targeting ligands, fluorescent probes, and other functional moieties.⁶ Therefore, surface engineering is crucial for developing multifunctional anticancer SPIONs; various types of coating materials have been previously utilized including proteins, polysaccharides, fatty acids, and various types of polymers.⁷

The superparamagnetism of SPIONs only occurs for particles below a threshold core size (ca. 25 nm for Fe₃O₄), when the thermal energy outcompetes the energy barrier of magnetic flipping.⁸ However, such size restriction of SPIONs led to the very limited particle surface area, slim surface coating, and thus poor drug loading. For example, the loading of a model anti-cancer agent, 17-DMAG, in SPIONs with monolayer surface coating was reported less than 1% (w/w).⁹ Increasing the thickness of SPIONs surface coating is a common approach to solve the loading problem. It was reported that the method of sequential deposition of multi-layer different coating polymers could increase the loading of the model drug, doxorubicin, to ca. 6% (w/w). Nevertheless, the layer-by-layer process is tedious, laborious and less productive. An alternative approach was the generation of hybrid systems where Fe₃O₄ was loaded in polymeric nanoparticles, but such systems would suffer from premature Fe₃O₄ release and non-uniform magnetic responsiveness.¹⁰ In the current study, the aim was to design novel “proof-of-concept” SPIONs with tailored dual coating for enhanced drug loading (Fig. 1). The Fe₃O₄ core was covered with an inner SiO₂ layer to enlarge the core surface area. Then the modified core was further coated with an outer polymer layer, i.e. methoxy poly(ethylene glycol)–poly(lactic acid) (mPEG–PLA), where the model drug, paclitaxel, was located.

Fig. 1 The schematic illustration of SPIONs with dual coating for enhanced drug loading.

The production details of a Fe₃O₄ core, SPIONs with a single silica coating (i.e. Fe₃O₄–SiO₂), SPIONs with both silica and polymer dual coating (i.e. Fe₃O₄–SiO₂–Polym) were provided in ESI (Fig. S1–S3†). Fe₃O₄ was generated by the simple coprecipitation method with citric acid as the stabilizer to prevent oxidation and aggregation. The deposition of silica was based on the in situ hydrolysis and condensation of tetraethyl orthosilicate, a sol–gel precursor. Then an amine group was introduced to the Fe₃O₄–SiO₂ surface followed by the conjugation with activated mPEG–PLA to build up the outer polymer layer of SPIONs. The same type of polymer was employed for SPIONs coating. The selection of silica as the conjoining layer is due to the superb capability of the surface silanol group to easily react with various ligands, which facilitates the further coating beyond SiO₂ with different materials.¹¹ In the current study, the SiO₂ coating not only protects the Fe₃O₄, but also acts as a cushion layer enlarging the surface area of SPIONs, and introducing sufficient APS and hence terminal amino groups to facilitate the conjugation with polymer.

The FTIR analysis confirmed the presence of both the silica and polymer coatings (Fig. 2A). The band at 580 cm⁻¹ corresponds to the stretching vibration of Fe–O from the core; the bands at 800 cm⁻¹ and 1092 cm⁻¹ are assigned to the symmetric and asymmetric stretching vibration of Si–O–Si from the inner silica coating, respectively; the bands at 1751 and 2934 cm⁻¹ are due to the stretch of C=O ester and methine from the outer mPEG–PLA layer in turn.¹² The TEM examination revealed the size of Fe₃O₄–SiO₂ (I and II) at 63 ± 11 nm and 86 ± 14 nm, respectively (Fig. 2B), which was around 20 nm smaller compared to their corresponding hydrodynamic diameters assayed by dynamic light scattering (DLS) (Table S1†). This might be a result of the smoothed hydration of Fe₃O₄–SiO₂ owing to the existence of abundant surface hydroxyl groups. Similarly, the size of the dual layer-coated SPIONs were 117 ± 14 nm and 156 ± 15 nm for Fe₃O₄–SiO₂–Polym (I and II), individually. Due to the manifestation of the hydrophilic PEG segment, the hydrodynamic size of Fe₃O₄–SiO₂–Polym (I and II) was ca. 60 nm larger than their matching practical size. Besides the external magnetic field-guided SPIONs targeting, the appropriate size of double layer-coated SPIONs (100–200 nm) would enable their passive tumor targeting via the “enhanced permeability and retention (EPR) effect”.¹³ In addition, the polymer surface could be conjugated with a diverse range of targeting molecules (e.g. folate, transferrin, epidermal growth factor) that was over-expressed in tumor cell surface; this would endow SPIONs the active targeting ability, generating multiple-targeting SPIONs.¹ As the control, the size of Fe₃O₄ was only 10 ± 1 nm (Fig. S4†). Such small unmodified SPIONs are subject to rapid renal clearance and significant uptake in the liver and spleen upon in vivo administration.¹⁴ In the current study, surface engineering can avoid this size-dependent limitation by covering Fe₃O₄ with fitted dual layers to produce customized SPIONs.

Fig. 2 The characterization of dual layer-coated SPIONs: (A) FTIR (Fourier transform infrared spectroscopy) spectra of Fe₃O₄, Fe₃O₄–SiO₂, and Fe₃O₄–SiO₂–Polym; (B) TEM (transmission electron microscopy) images of Fe₃O₄–SiO₂ (I), Fe₃O₄–SiO₂ (II) (a and b), and Fe₃O₄–SiO₂–Polym (I), Fe₃O₄–SiO₂–Polym (II) (c and d); (C) XRD (X-ray diffraction) spectra of Fe₃O₄, Fe₃O₄–SiO₂–Polym (I), and Fe₃O₄–SiO₂–Polym (II); (D) magnetization curves of Fe₃O₄–SiO₂–Polym (I) and Fe₃O₄–SiO₂–Polym (II). Polym represents mPEG–PLA polymer and the thickness of SPIONs coating increases from type I to type II, correspondingly.

The XRD spectra demonstrated that the Fe₃O₄ core of dual layer-coated SPIONs maintained a face-centered cubic crystal structure (Fig. 2C). The broad peak at ca. 22 degree (2θ) for Fe₃O₄–SiO₂–Polym (I and II) was due to the contribution of the silica layer. This can be explained by the low rotational energy for the Si–O bond and the open Si–O–Si bond angle, which led to a flexible and mobile molecular structure of silica.¹⁵ As a result of the shielding effect of dual coating, the intensity of the characteristic peaks for Fe₃O₄ decreased markedly. However, two peaks (311 and 440) remained clear after silica and polymer coating, while another peak (511) was also noticeable. The obtained SPIONs with tailored dual layers exhibited superparamagnetic behavior without magnetic hysteresis (Fig. 2D). Despite being much lower than the control Fe₃O₄ (at about 60 emu g⁻¹), the degree of the saturation magnetization of Fe₃O₄–SiO₂–Polym decreased when increasing the coating thickness from ca. 50 nm (type I) to 70 nm (type II). With the help of inductively coupled plasma atomic emission spectroscopy (ICP-AES), the mass of the coating layer was subtracted and the weight-normalized saturation magnetization values for both types of Fe₃O₄–SiO₂–Polym were consistent with that of the control Fe₃O₄. In terms of the threshold of saturation magnetization, it was reported that the value greater than 7 emu g⁻¹ was sufficient for biomedical applications.¹⁶ Although the Fe₃O₄–SiO₂–Polym nanoparticles obtained in the current study exhibited a lower saturation magnetization compared to the threshold, the level of the saturation magnetization could be finely tuned and optimized to suit in vivo tumor theranostic application via the manipulation of coating material and thickness.¹⁷ Moreover, the amphiphilic nature of mPEG–PLA would extend the systemic circulation of SPIONs, thus avoiding elimination prior to reaching the target tissue.

The paclitaxel loading in Fe₃O₄–Polym, Fe₃O₄–SiO₂–Polym (I), and Fe₃O₄–SiO₂–Polym (II) was 44 ± 2, 3092 ± 10, and 8369 ± 33 μg per mg Fe, respectively (Fig. 3). The employment of the dual coating strategy considerably increased the loading of paclitaxel in Fe₃O₄–SiO₂–Polym up to nearly 190 times compared to that with only single layer of coating (i.e. Fe₃O₄–Polym). In addition, the thicker polymer coating in Fe₃O₄–SiO₂–Polym (II) in comparison to Fe₃O₄–SiO₂–Polym (I) gave rise to a relatively higher loading. It was postulated that the polymer layer that held the majority of the encapsulated drug mainly via the hydrophobic interaction considering the hydrophobicity of both PLA and paclitaxel. Based on the TEM particle size analysis, the volume ratio of the outer polymer layer was calculated to be ca. 2.6 times between Fe₃O₄–SiO₂–Polym (II) and Fe₃O₄–SiO₂–Polym (I), which agreed well with the ratio of paclitaxel loading (i.e. 2.7) in these two SPIONs. It is the increased particle surface area and PLA content that contribute to the enhanced drug loading. A previous study employed poly(beta-amino ester) to generate single-layer SPIONs (ca. 80 nm by DLS) and the loading of the active agent (doxorubicin) and polymer was ca. 700 and 7000 μg per mg Fe, respectively.¹⁸ In spite of the divergence of the utilized polymer and model drug, the dual layer method employed in the current study efficiently enlarged the “apparent” core of SPIONs without losing their superparamagnetic feature. This leads to a drug loading capability 10 times higher than the above reported value at the cost of increased mPEG–PLA packing (ca. 77 824 μg per mg Fe) for Fe₃O₄–SiO₂–Polym (II). The SPIONs with the highest loading in the current study were chosen for the drug release experiment that was carried out in sink conditions. The profile exhibited a rapid drug release at the beginning and the curve started getting plateaued after 8 h; over 24 h nearly 80% of the total drug was released. Although PLA is biodegradable, the release was thought to be primarily controlled by diffusion as the SIPONs's disintegration induced by PLA degradation is negligible within a short time at a neutral condition.

Fig. 3 The drug (paclitaxel) loading of Fe₃O₄–Polym and Fe₃O₄–SiO₂–Polym (top); the drug release profile from Fe₃O₄–SiO₂–Polym (II) (bottom). Fe₃O₄–Polym was coated only by the mPEG–PLA polymer with APS as the linker. Fe₃O₄–SiO₂–Polym was coated by both silica and polymer, and the coating thickness of Fe₃O₄–SiO₂–Polym (II) was higher than that of Fe₃O₄–SiO₂–Polym (I); the polymer volume in single-layer Fe₃O₄–Polym nanoparticles was two orders of magnitude smaller compared to that in Fe₃O₄–SiO₂–Polym (I and II).

In summary, both silica and polymer coated SPIONs successfully addressed the poor drug loading issue of conventional SPIONs. The precise manipulation of the thickness of dual layers would balance the requirement of drug loading and saturation magnetization, which is particularly true for potent active agents. Such an approach could be further manipulated to engineer multifunctional nanotheranostics with imaging, therapeutics loading and targeting for improved tumor therapy.

We gratefully acknowledge the funding support from Tianjin Research Program of Application Foundation and Advanced Technology (11JCZDJC20600; 13JCQNJC13300) and National Natural Science Foundation of China (11001197). Prof. Tonglei Li from Purdue University kindly went through this manuscript and made valuable suggestions.

Notes and references

1. Z. L. Cheng, A. Al Zaki, J. Z. Hui, V. R. Muzykantov and A. Tsourkas, Science, 2012, 338, 903.
2. M. Ferrari, Nat. Rev. Cancer, 2005, 5, 161.
3. Y. Lalatonne, C. Paris, J. M. Serfaty, P. Weinmann, M. Lecouvey and L. Motte, Chem. Commun., 2008, 2553; Y. Pan, X. Du, F. Zhao and B. Xu, Chem. Soc. Rev., 2012, 41, 2912.
4. C. Rumenapp, B. Gleich and A. Haase, Pharm. Res., 2012, 29, 1165.
5. O. Veiseh, J. W. Gunn and M. Zhang, Adv. Drug Delivery Rev., 2010, 62, 284.
6. M. V. Yigit, A. Moore and Z. Medarova, Pharm. Res., 2012, 29, 1180; S. A. Corr, A. O'Byrne, Y. K. Gun'ko, S. Ghosh, D. F. Brougham, S. Mitchell, Y. Volkov and A. Prina-Mello, Chem. Commun., 2006, 4474.
7. Q. M. Quan, J. Xie, H. K. Gao, M. Yang, F. Zhang, G. Liu, X. Lin, A. Wang, H. S. Eden, S. Lee, G. X. Zhang and X. Y. Chen, Mol. Pharmaceutics, 2011, 8, 1669; M. H. El-Dakdouki, D. C. Zhu, K. El-Boubbou, M. Kamat, J. J. Chen, W. Li and X. F. Huang, Biomacromolecules, 2012, 13, 1144.
8. K. M. Krishnan, IEEE Trans. Magn., 2010, 46, 2523.
9. P. Zou, Y. Yu, Y. Wang, Y. Zhong, A. Welton, C. Galban, S. Wang and D. Sun, Mol. Pharmaceutics, 2010, 7, 1974.
10. N. Schleich, P. Sibret, P. Danhier, B. Ucakar, S. Laurent, R. Muller, C. Jerome, B. Gallez, V. Preat and F. Danhier, Int. J. Pharm., 2013, 447, 94.
11. Q. Liu, J. A. Finch and R. Egerton, Chem. Mater., 1998, 10, 3936.
12. R. Al-Oweini and H. El-Rassy, J. Mol. Struct., 2009, 919, 140; S. K. Choi and D. Kim, J. Appl. Polym. Sci., 2002, 83, 435.
13. H. Maeda, J. Wu, T. Sawa, Y. Matsumura and K. Hori, J. Controlled Release, 2000, 65, 271.
14. I. J. Majoros, T. P. Thomas, C. B. Mehta and J. R. Baker, J. Med. Chem., 2005, 48, 5892.
15. J. Zhang, M. Sun, A. Fan, Z. Wang and Y. Zhao, Int. J. Pharm., 2013, 441, 389.
16. X. He, X. Wu, X. Cai, S. Lin, M. Xie, X. Zhu and D. Yan, Langmuir, 2012, 28, 11929.
17. F. M. Kievit and M. Zhang, Acc. Chem. Res., 2011, 44, 853.
18. F. Chen, F. M. Kievit, O. Veiseh, Z. R. Stephen, T. Wang, D. Lee, R. G. Ellenbogen and M. Zhang, J. Controlled Release, 2012, 162, 233.

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Footnote

† Electronic supplementary information (ESI) available: Details of SPIONs generation and characterization. See DOI: 10.1039/c3ra42861c

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