Magnetic Colloidal Supraparticles: Design, Fabrication and Biomedical Applications
Dedicated to the 20 Anniversary of Department of Macromolecular Science of Fudan University
Magnetic nanoparticles (MNPs) bear many intriguing properties such as superparamagnetism, high specifi c surface area, remarkable colloidal stability and biocompatibility, which evoke great interest and desire of exploration in biomedical applications. For the use in the complicated physiological environment, MNPs are still being developed to have the enhanced perfor-mances and down-to-earth practicality. Engineering of MNPs into hierarchical structures is thus proposed to create a new family of magnetic materials, magnetic colloidal supraparticles (MCSPs), which exhibit collective properties and unique nanomaterial characters. From a biomedical point of view, appli-cability of MCSPs is somewhat more distinctive in contrast to their primary MNPs, because MCSPs are amenable to modulation of secondary structure, promotion of magnetic responsiveness and ease of function design. As a result, MCSPs have been subject to intense researches in recent years, with the aim to develop outstanding composite materials for biomedical applica-tions. In this review, we embark on an overview of foundational topics that detail the design and fabrication of MCSPs by evaporation-induced emulsion and solvothermal techniques, and continue with a guideline for modifi cation of MCSPs with inorganic oxides and organic polymers. Particular focus is then placed on the biomedical applications of modifi ed MCSPs. Many exam-ples illustrate the latest progress in design of MCSP-based microspheres for magnetic resonance imaging, targeted drug delivery, sensing, and harvesting of peptides/proteins. After these detailed accounts, the current challenges and future development of researches and applications are discussed as a conclusion to the review.
1. Introduction
Magnetic nanoparticles (MNPs), [ 1 , 2 ] especially iron oxides, have been subjected to extensive studies in the past few dec-ades owing to their unique size-dependent magnetic proper-ties and promising potentials in biomedical applications. [ 3–6 ]
Dr. J. Guo, Prof. W. L. Yang, Prof. C. C. WangState Key Laboratory of Molecular Engineering of PolymersDepartment of Macromolecular ScienceLaboratory of Advanced MaterialsFudan UniversityShanghai 200433, ChinaE-mail: [email protected]
DOI: 10.1002/adma.201301896
Adv. Mater. 2013, DOI: 10.1002/adma.201301896
To date, many chemical preparation methods have been developed, such as chemical coprecipitation, [ 7 , 8 ] thermal decomposition, [ 9–11 ] sol-gel synthesis, [ 12 ] hydrothermal reaction, [ 13 ] electrochemical method, [ 14 , 15 ] supercritical fl uid method [ 16 ] and so on. Based on these methods, the uniform MNPs (size distribution is less than 5%) with tunable sizes, controllable shapes and various compositions can be prepared. Recently, many reviews have elaborately delineated the state-of-the-art progress of the preparation and applica-tion of MNPs. [ 17 − 20 ]
For biomedical applications, consid-erable attention is routinely paid on the magnetic properties of MNPs, which is expected to exhibit rapid magnetic respon-siveness and low remnant magnetic moment after removal of applied magnetic fi eld. This pivotal performance of MNPs is greatly dependent on their single-domain sizes. From the point of view of theory, the relaxation of magnetic moment ori-entation of each particle is determined by τ = τ 0 e KV/2kT , wherein τ is relaxation time at one orientation, K is the particle’s ani-sotropy constant, V is particle volume, k is the Boltzmann’s constant, and T is temperature. [ 21 , 22 ] When the particle size decreases to a small value, KV becomes comparable to the thermal energy kT, and
the magnetic moment starts to fl uctuate from one direction to another quickly. Thus the net magnetic moment of the MNP is randomized to zero, leading to the so-called superparamagnetic characteristic. In this status, because the free MNPs are not susceptible to strong magnetic interaction on each other, their colloidal stability in physiological solution is thus improved and benefi cial in biomedical applications. [ 23 , 24 ] In order to yield the superparamagnetism, the diameters of MNPs should be lim-ited to less than 30 nm. The small-size MNPs with appropriate functionalities and tailored surface properties are suitable for many biomedical applications, but the weak magnetic respon-siveness in solution constrains their practical use in some areas, such as DNA/protein separation, cell sorting and target drug delivery. With the aim to increase the magnetic responsiveness in a controllable manner while retain their superparamagnetic
in macromolecular chem-istry and physics in 2007 at the Fudan University, China. Under the support of JSPS postdoctoral fellow-ship program, he continued to conduct research in the Institute for Molecular Science, Japan, where he investigated the synthesis of covalent organic frameworks.
In 2009, he returned to China and became a lecture at the Fudan University. His scientifi c interests are the design and construction of porous organic polymers and organic-inorganic hybrid nanomaterials for catalytic and biomedical applications.
Wuli Yang received his BS degree in 1995, MS degree in 1998 and PhD degree in 2001 from Fudan University. He joined the Department of Macromolecular Science at Fudan University in 2001 as a Lecturer and was promoted to Associate Professor in 2003. In 2010 he was pro-moted to Full Professor. His research focuses on emulsion
polymerization, functional inorganic nanoparticles, func-tional polymeric composite microspheres and biomedical nanoparticles for diagnostic and drug delivery.
Changchun Wang received his PhD in Polymer Chemistry and Physics from Fudan University 1996. He was a visiting scientist 1996-1998 at Eastern Michigan University. He is now the Professor of Department of Macromolecular Science at Fudan University. His research interests are in design and synthesis of
various polymer microspheres and magnetic composite nanomaterials with controlled structure and properties for DNA and protein enrichment, biomarker detection and targeting drug delivery.
characteristic, the strategy of assembly of MNPs into suprapar-ticles has been developed.
Recently, a great progress has been made in the development of formulation of secondary structures of colloidal supraparti-cles. Their uniqueness in microstructure results in prominent properties as a result of a preferable combination of exclusive natures of individual nanoparticles and integrated functionali-ties of nanoparticle aggregates, which promises great potentials for the development of advanced nanomaterials. [ 25 ] In light of the distinctive characters of hierarchical microstructures, mag-netic colloidal supraparticles (MCSPs) have attracted mounting interests due to promotion of magnetic responsiveness as well as preservation of the superparamagnetism from the primary MNPs of supraparticles. Moreover, more diverse functionalities are capable of being accomplished by the accurate control over compositions and reactivity of surface functional groups. [ 26 ]
This review focuses on recent progresses in the design, fab-rication and surface modifi cation of the MCSPs and their appli-cations in biomedical fi elds. The fi rst perspective concerned is relevant to the synthetic tricks of MCSPs, enabling controlla-bility of secondary microstructures with specifi city in porosity and surface areas. Some promising technologies, for example, the microwave-assisted synthesis, have been involved in this part. Afterwards, the advancement in surface modifi cation and functionalization of the MCSPs are discussed elaborately. Then a particular emphasis will be given to their applications in mag-netic resonance imaging, biomarker sensing, targeted drug delivery, and enrichment and detection of proteins/peptides.
2. Design and Fabrication of Magnetic Colloidal Supraparticles (MCSPs)
Magnetic colloidal supraparticles (MCSPs) are featured with high regularity in clustering structure and superior stability as colloids behave in aqueous solution. The synthetic strategies of MCSPs can fall into a multiple-step evaporation-induced route and a one-step solvothermal route. The following discus-sion will be detailed on this topic, to shed light on the recent advancement in controlled synthesis of MCSPs.
2.1. Evaporation-Induced Preparation of MCSPs from Magnetic Emulsions
The evaporation-induced preparation of MCSPs has been widely accepted but adopted just in recent years. The earliest report could be traced back to 1993 by Bibette. [ 27 ] Also, Elas-sari et al. did intense studies in this area during the early stage of the related researches. [ 28–30 ] The classic preparation process could be described as follows: (1) synthesis of a stable disper-sion of MNPs in organic solvent (ferrofl uid), (2) preparation of emulsions with submicron-sized magnetic droplets by control-lably shearing a mixture of ferrofl uids and surfactants in dis-persion medium, and (3) evaporation of solvent in droplets of emulsion if needed. Thanks to a complete repartition of MNP dispersion, the magnetic droplets were a homogeneous single phase and exhibited the regular spherical shape. Their average diameters were around 200 nm with 10% of standard deviation
measured by quasi-elastic light scattering, implying that a narrow size polydispersity could be obtained in the emulsion.
As the evaporation-induced emulsion method has uni-versality, tremendous efforts have been dedicated to inves-tigate the construction of monodisperse supraparticles with
Figure 1 . (A) Schematic illustration of evaporation-induced synthesis of MCSPs in emulsion; (B) TEM images of MCSPs with controllable compositions, sizes, and shapes: (a) BaCrO 4 , (b) Ag 2 Se, (c) PbS, and (d) Fe 3 O 4 . Reproduced with permission. [ 31 ]
different sizes, shapes and properties by using the dispersible nanoparticles (NPs) as building blocks. [ 31 ] A variety of NPs (BaCrO 4 , Ag 2 Se, PbS and Fe 3 O 4 ) could be gathered, assembled and fi xed simultaneously in the emulsion droplets through the hydrophobic interaction of surfactants and ligands of NPs ( Figure 1 A). Upon evaporation of low-boiling solvent in droplets, the colloidal supraparticles were formed in the size range of 50 nm to 2 μ m. Also, the shapes, compositions and surface charges of colloidal supraparticles could be tuned by the pre-designed experiment parameters (Figure 1 B), such as surfactant species, concentrations, organic solvents, tempera-tures, stirring and ultrasonic conditions. Compared with the early methods, this approach possesses two main advantages: (1) low-boiling solvent in oil-in-water (O/W) emulsion can be easily removed; (2) ligand-stabilized NPs are well dispersed in nonpolar medium as a consequence of phase stability. In addi-tion, the modifi ed method based on O/W emulsion is available for the further modifi cation of supraparticles.
Within this context, an exciting result has recently been reported, which concerned anisotropy-driven self-assembly of semiconducting nanorods into highly ordered colloidal suprapar-ticles with well-defi ned supercrystalline domains. [ 32 ] The prepa-ration process includes two steps: (1) synthesis of water-soluble nanorod micelles by mixing a chloroform solution of nanorods with an aqueous solution of dodecyl trimethylammonium bro-mide and sequentially evaporating chloroform from mixtures, and (2) growth of supraparticles from nanorod micelles in an aqueous solution of ethylene glycol. Varying the total number (N) of nanorods could fl exibly control the confi gurations and sizes of multiple supercrystalline domains in the supraparticle. When N was less than ∼ 80,000, the shape of supraparticles was double-domed cylinders, wherein the cylindrical domain was
larger than those of the two-end domes. When N was more than ∼ 80,000, the supraparticles appeared as either irregular-multidomain particles or double-domed cylinders. Fol-lowing this line, they prepared MCSPs using Fe 3 O 4 nanocubes under thermodynamic con-trol ( Figure 2 ). [ 33 ] The resulting MCSPs with unique shapes had exceptional stability upon the arrival of minimized Gibbs free energy. The simple cubic superlattice structure was acquired, and the shape of MCSPs could be tuned between spheres and cubes by varying relative contributions of surface and bulk free energies (Figure 2 B). Also, the size-dependent hydration effect played an important role in the formation of highly ordered suparparti-cles. LCW theory could be applied to predict the formation processes of sphere- and cube-shaped MCSPs. This is the fi rst example for engineering of a well-oriented MCSP through the thermodynamic equilibrium.
Since the stabilizers used for MNPs (e.g. oleic acid) prefer to the hydrophobic drop-lets and are compatible to the surfactants or polymers, thereby, the MNPs can be well dis-persed in O/W emulsion or micelles for gen-eration and manipulation of MCSPs. Com-
paratively, one-step preparation of MCSPs needs specifi c stabi-lizers to control evolution of MCSPs not only in geometry, but also in surface and interior properties such as charge density, dangling group species, and porosity, which will be discussed in the following part.
2.2. One-Step Preparation of MCSPs by Solvothermal Method
One-step solvothermal reaction is considered as a potent, facile and time-saving method for preparation of MCSPs. [ 26 ] Li and coworkers described the solvothermal approach to synthesizing MCSPs by reduction of FeCl 3 with ethylene glycol ( Figure 3 ). [ 34 ] The as-prepared MCSPs had narrowly distributed sizes that could be tuned in the range of 200–800 nm, and the standard deviation of size distribution was lower than 5%. Albeit without any stabilizers used, the obtained MCSPs could be dispersed in water for more than 1 h, implying their nice dispersibility in solution. The solvothermally synthesized MCSPs offer sev-eral important advantageous features for biomedical appli-cations: (1) excellent magnetic responsiveness, conducive to conveniently manipulate MCSPs by an external magnetic fi eld; (2) controllability of particle sizes at the submicrometer scale; (3) inexpensive raw materials and high product yields, ame-nable to large-scale production for industrial demand. Since the solvothermal technique was proposed, much attention has been gained for modulation in secondary structures and func-tionalities of MCSPs, and this method extends to many other research areas as well.
As expected, poly(acrylic acid) (PAA) was used as stabilizer to decorate the MCSPs resulting in the fi rst hydrophilic MCSPs with abundant carboxyl groups on the surface. [ 35 ] Zhao and
3wileyonlinelibrary.comeim
www.advmat.dewww.MaterialsViews.com
REV
IEW
Figure 2 . (A) Representation of self-assembly of iron oxide nanocubes into MCSPs by processes of (i) embryo growth, (ii) crystallization, and (iii) assembly: (a) nanocube micelles, (b, c) MCSP embryos, (d) spherical MCSPs, and (e) cubic MCSPs; (B) TEM images of (a) spherical MCSP, (b) a spherical MCSP viewed along the [001] facet, (c) cubic MCSPs, and (d) a cubic MCSP viewed along the [001] facet. Reproduced with permission. [ 33 ] Copyright 2012, American Chemical Society.
coworkers adopted the identical route but utilized sodium citrate instead of PAA to stabilize the MCSPs in aqueous solution. [ 36 ] The magnetic responsiveness of the citrate-modifi ed MCSPs was greatly improved compared with the primary MNPs, but the large surface area was sacrifi ced. Moreover, as the secondary structure was assembled very toughly without any voids, it was thus not suitable for drug storage and delivery. Thereafter, devel-oping a facile way to fabricate MCSPs with large surface area or inner cavity became a big challenge. With these all in mind, we designed and prepared the hollow-core-structured MCSPs by replacing the electrostatic stabilizer NaOAc with NH 4 OAc in the recipe ( Figure 4 ). [ 37 ] Under solvothermal conditions, we found that the NH 4 OAc played a key role in structural control. The function of NH 4 OAc could be summarized in the following three aspects: (1) NH 4 OAc acted as electrostatic stabilizer to
Figure 3 . TEM and SEM images of MCSPs of (a, b) Fe 3 O 4 , (c, d) MnFe 2 Ocorresponding electron diffraction patterns. Reproduced with permission
prevent particle agglomeration; (2) NH 4 OAc assisted ethylene glycol to reduce Fe(OH) 3 partially into Fe(OH) 2 for the succes-sive dehydration reaction of Fe 3 O 4 nanoparticles; (3) NH 4 OAc served as structure-directing agent to take effect in the structure transformation from solid to hollow aggregates. The possible reason might lie in that NH 4 OAc decomposed to HOAc and NH 3 , and the gaseous NH 3 bubbles worked as soft template to manage the evolution of hollow-core structure. Also, the intersti-tial space was formed during this process in the shells of hollow MCSPs, which was favorable for drug storage and sustained release. [ 38 ]
As poly( γ -glutamic acid) (PGA) was added in the recipe for preparation of MCSPs, an interesting mesoporous structure was constructed in our consecutive research. [ 39 ] As shown in Figure 5 A, NH 3 bubbles generated in decomposition of
GmbH & Co. KGaA, Weinheim
4 , (e, f) ZnFe 2 O 4 , and (g, h) CoFe 2 O 4 . The insets of TEM images show the . [ 34 ]
Adv. Mater. 2013, DOI: 10.1002/adma.201301896
www.advmat.dewww.MaterialsViews.com
REV
IEW
Figure 4 . (a) TEM and (b) SEM images of hollow-core-structured MCSPs, (c) enlarged TEM image, and (d) HR TEM image for the marked rectangular area in (c) and SAED pattern of the hollow MCSPs (inset). Reproduced with permission. [ 37 ] Copyright 2010, Royal Society of Chemistry.
NH 4 OAc allowed the PGA globules to reside at the liquid/gas interface, leading to PGA-stabilized bubbles. Then they were engineered with the PGA-capped MNPs to form the initial col-
Figure 5 . (A) Proposed mechanism of formation of mesoporous MCSPs; (B) Representative TEthesized with addition of PGA of (a, b) 0.1 g, (c, d) 0.5 g, and (e, f)1.0 g. All scale bars are 200 American Chemical Society.
Adv. Mater. 2013, DOI: 10.1002/adma.201301896
loidal clusters. As the reaction progressed, the entrapped NH 3 bubbles in the initial MCSPs were forced out and left the internal accessible channels, eventually resulting in a distinct mesoporous structure. The obtained MCSPs had large surface areas comparable or superior to that of individual MNPs, as well as high magnetization given by multi ple NMPs. It is worth emphasizing that the conjugated PGA stabilizers render the mesoporous MCSPs to have prominent bio-compatibility, superior dispersibility and sta-bility, and abundant carboxylate groups avail-able to further surface modifi cation. More importantly, PGA could effectively manipu-late the particle sizes of MCSPs as well as the surface areas and pore sizes by varying the feeding amount of PGA (Figure 5 B). Besides, the similar mesoporous structures could be obtained for MCSPs using other bio-macro-molecules, such as agarose, casein, and soy-bean protein. [ 40b ]
Although the one-pot solvothermal method is advanced in preparation of
MCSPs, the synthetic conditions are severe, usually requiring a long reaction time (more than 10 h) and high tempera-ture (up to 200 ° C) in the autoclave. To further develop the
5wileyonlinelibrary.comheim
M and SEM images of the mesoporous MCSPs syn-nm. Reproduced with permission. [ 39 ] Copyright 2011,
6
www.advmat.dewww.MaterialsViews.com
REV
IEW
high-throughout technique, a rapid and environmentally-
friendly microwave assistant reaction has been investigated toward increased output of MCSPs in our group. [ 41a ] In contrast to the traditional solvothermal method, the microwave-aided growth of MCSPs was dramatically speeded up and completed within minutes. The as-prepared MCSPs showed narrow size distribution from 200 to 400 nm. When a structure-directing agent, casein, was added to the reaction, hollow-core-structured MCSPs were formed within 10 min under microwave condi-tions as well. [ 41b ]
3. Fabrication of Composite Microspheres from MCSPs
Since evaporation-induced method for preparation of MCSPs was reported, the exploration of MCSP-based composite microspheres has been performed coincidently. Investiga-tion of this subject covers issues concerning tunability in microstructure and composition of microspheres and diversity in functionality and applicability.
Encapsulation of the MCSPs with inorganic oxides is one of the important modifi cation strategies. Using the sol-gel tech-nique, inorganic coatings, i.e. SiO 2 , [ 42 ] mesoporous SiO 2 [ 43 ] and TiO 2 , [ 44 ] could be readily implemented on the surface of MCSPs. Zhao and coworkers reported a surfactant-templating approach to synthesis of sandwich-structured composite microspheres
Figure 6 . (A) Synthetic procedure of MCSP@SiO 2 @mSiO 2 microspheremSiO 2 , and (f) SEM image of MCSP@SiO 2 @mSiO 2 . Reproduced with pe
with a MCSP@SiO 2 core and a mesoporous SiO 2 shell that had perpendicularly oriented channels ( Figure 6 ). [ 45 ] The obtained microspheres possessed superparamagnetism, high magnetiza-tion (53.3 emu g − 1 ), uniform mesopore (2.3 nm), high surface area (365 m 2 g − 1 ) and large pore volume (0.29 cm 3 g − 1 ). The synthetic procedure is illustrated in Figure 6 A. The solvother-mally synthesized MCSPs were coated with a thin silica layer through a sol-gel approach, leading to a nonporous MCSP@SiO 2 microsphere. Cetyltrimethylammonium bromide (CTAB) was used as a template to yield a mesostructured CTAB/silica composite on the periphery of MCSP@SiO 2 microspheres. Then mesoporous SiO 2 shell was generated by acetone extrac-tion of CTAB templates, ultimately resulting in a well-defi ned core-shell Fe 3 O 4 @SiO 2 @mSiO 2 microsphere.
Apart from the inorganic coatings, polymer modifi cation have been extensively investigated as well. [ 20 ] In this context, organic polymers such as polystyrene (PSt), [ 46 ] poly(styrene- b -(acrylic acid)), [ 47 ] poly(styrene- b -( N -isopropylacryl amide)) [ 48 ] and so on, have been applied to modify the MCSPs for the different intended uses. The prime factors in determining magnetic con-tents are polymerization procedure and intrinsic interaction between MCSPs and polymer components. Gu and coworkers synthesized the uniform high-magnetic-content composite microsphere consisting of a Fe 3 O 4 /PSt core and a silica shell by using the double-miniemulsion-emulsion technique. [ 46b ] The miniemulsion containing roughly 100-nm droplets of oleic acid-stabilized Fe 3 O 4 NP aggregates was mixed with the
GmbH & Co. KGaA, Weinheim
s; (B) TEM images of (a) MCSPs, (b) MCSP@SiO 2 , (c–e) MCSP@SiO 2 @rmission. [ 45 ] Copyright 2008, American Chemical Society.
Adv. Mater. 2013, DOI: 10.1002/adma.201301896
www.advmat.dewww.MaterialsViews.com
REV
IEW
Figure 7 . (A) Schematic representation of fabrication of (a) MCSP-PMMA microspheres, (b) MCSP-PSt microspheres, (c) MCSP-PMMA core/PSt shell microspheres, and (d) PSt core/MCSP-PMMA shell microspheres; (B) Photographs of (a1) MCSP-PMMA latex, (c1) MCSP-PSt latex, and (a2, c2) core/shell-structured microspheres by polymerization of second monomer, in the absence and presence of a magnet, respectively; TEM images of (b) MCSP-PMMA core/PSt shell microspheres (the inset is the enlarged view of TEM image) and (d) PSt core/MCSP-PMMA shell microspheres (the inset is a TEM image of the ultrathin cross section with a thickness of ∼ 50 nm). Reproduced with permission. [ 49 ]
other emulsion made of styrene (St) monomer droplets with a uniform size of 3.7 μ m prepared by membrane emulsifi cation equipment. Since the surfactant concentration (sodium dodecyl sulfate) was limited below the critical micelle concentration and the population of Fe 3 O 4 droplets was four orders of mag-nitude larger than that of St droplets, the St monomers could diffuse through the water to the Fe 3 O 4 droplets, and, in turn, the polymerization locally occurred upon addition of initiators. The formed Fe 3 O 4 /PSt particles were monodispersed and could be coated by silica via a modifi ed Stöber method, resulting in a core/shell-structured magnetic composite microsphere.
We employed the modifi ed evaporation-induced method to prepare core-shell MCSP-based microspheres with site-specifi c placement of MNPs in either the core or the shell. [ 49 ] As displayed in Figure 7 , the feeding order of two mono-mers, St and methyl methacrylate (MMA), were modulated to construct variant core/shell structures via the emulsion polymerization (Figure 7 A). When MMA was used as the fi rst monomer and St was added sequentially in the pres-ence of Fe 3 O 4 micelles, the two-step polymerization resulted in a well-defi ned core-shell structure consisting of a MCSP@PMMA core and an encapsulating PSt shell (Figure 7 B-a,b). In the opposite way, when St was used as the fi rst monomer and MMA was polymerized successively in the presence of Fe 3 O 4 micelles, the anchored Fe 3 O 4 NPs on the surface of PSt cores could be successfully intercalated into the outer PMMA shell to form a unique polymer-magnetite hybrid shell (Figure 7 B-c). TEM images confi rmed that the Fe 3 O 4 NPs were incorporated selectively into the PMMA shell, but were not present in the PSt core (Figure 7 B-d). The main reason for the structure control was that the oleic acid stabilizers of Fe 3 O 4 NPs were miscible with PMMA; this was conducive to accommodation of NPs in PMMA matrix. In contrast, because of the poor miscibility with PSt, Fe 3 O 4 NPs could not be implanted in PSt moiety.
To our knowledge, the weak interaction between MNPs and organic polymers possibly impede conjunction with each
other. Thus an intermediate layer of SiO 2 is introduced to build a functionalized surface for polymer deposition. A commonly used path to fabrication of polymer-covered MCSP micro-spheres includes four steps, [ 42 ] (1) solvothermal synthesis of MCSPs, (2) silica encapsulation of MCSPs, (3) modifi cation of silica shells with vinyl groups using silane coupling agents, and (4) coating of polymers on the surface of MCSP@SiO 2 particles by seed precipitation polymerization. Alternatively, direct covering of the silane coupling agents also enabled the modifi ed MCSPs to have the pendent vinyl groups for connec-tion of polymer shells. [ 50 ] More recently, we found the phenol-formaldehyde condensation polymerization that could be performed on the surface of MCSPs through the use of micro-wave assistance. [ 51 ] The obtained MCSP@PF microspheres were uniformly sized and characterized with defi nitely core-shell microstructure.
Distillation–precipitation polymerization is a potent syn-thetic approach to preparation of varied hydrophilic polymer shells on the basis of silica-modifi ed MCSPs. In our group, the double hydrophilic polymer shell was successfully prepared on the MCSP cores recently ( Figure 8 A). [ 52 ] The approximately 300-nm MCSPs stabilized by citrate were synthesized by a modifi ed solvothermal reaction. Hydration–condensation reac-tion of silane coupling agent ( γ -methacryloxypropyl trimethox-ysilane) was undergone on the surface of MCSPs, to give the available vinyl groups. Following two-step distillation-precipi-tation polymerization, a poly(methylacrylic acid) (PMAA) shell was formed on the modifi ed MCSPs, and sequentially EGMP (ethylene glycol methacrylate phosphate) was polymerized on PMAA shells. We deemed that the interim PMAA layer was crucial for the formation of the outer PEGMP shell; it could shield the interaction between the phosphate group of EGMP and Fe 3 O 4 and thus stabilize the reaction system. Meanwhile, the strong hydrogen bonds between �COOH and–H 2 PO 4 groups facilitated the coating of PEGMP layer over PMAA. The as-prepared MCSP@PMAA@PEGMP composite microspheres
7wileyonlinelibrary.commbH & Co. KGaA, Weinheim
www.advmat.dewww.MaterialsViews.com
REV
IEW
Figure 8 . (A) Preparation of Ti 4 + -immobilized MCSP@PMAA@PEGMP core/shell/shell microspheres; (B) Representative TEM images of (a) MCSPs, (b) MCSP@PMAA, and (c) MCSP@PMAA@PEGMP, respectively (all scale bars are 200 nm); (d) hydrodynamic diameter distributions of (i) MCSP, (ii) MCSP@PMAA, and (iii) MCSP@PMAA@PEGMP. Reproduced with permission. [ 52 ]
could coordinate numerous Ti 4 + ions, which showed remark-able selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/nonphosphopeptides (1:500).
4. Biomedical Applications of MCSPs
4.1. MCSP-Based Probes for MR Imaging
Exploration of innovative magnetic resonance (MR) contrast agents has aroused considerable interest since MR imaging has become a ubiquitous tool for biomedical imaging and clinic diagnosis. Paramagnetic molecular complex (e.g. Gd III che-lates) is the representative MR contrast agent, which exhibits an increase in signal intensity and appear bright in T 1 -weighted images (“positive” imaging agents). [ 53 ] The other class of MR contrast agents is based on nanostructures including para-magnetic-complexes-doped framework nanostructure [ 54 ] and inorganic magnetic nanoparticles. [ 55 ] Of those members, super-paramagnetic Fe 3 O 4 NPs are most commonly used as MR con-trast agents in biomedicine. [ 56 ] They can generate a decrease in signal intensity and appear dark in T 2 -weighted images (“nega-tive” imaging agents). Although the recent advancements wit-ness the rapid development on the fabrication and application
of MNPs with controllable physic-chemical properties such as size, shape, and modifi ed surface nature, the rational design of Fe 3 O 4 MR contrast agents with high relaxivity and sensitivity as well as reduced toxicity is still in high demand thus so far.
In comparison to a single-domain MNP, MCSPs are distinc-tive in relaxivity because their peculiar nanostructures change the proton relaxation effect. [ 57 ] As far as is known, two inde-pendent relaxation processes, namely, longitudinal and trans-verse relaxation, are responsible for generating MR imaging. Because of close agglomeration of numerous MNPs, the hier-archical structure of MCSPs is conductive to alter longitudinal ( r 1 ) and transverse relaxivities ( r 2 ) together. [ 58 ] On one hand, the clustering architecture decreases the surface of magnetic nanoparticles in contact with water and hence impairs the longitudinal relaxation effect of the interior MCSPs. On the other hand, the number of MNPs in an assembly and the mag-netic moment are both proportional to r 2 . The overall effects of MCSPs on r 1 and r 2 thereby lead to the increased ratio of r 2 / r 1 and, in turn, yield the high-effi ciency T 2 contrast agents. In contrast to a large-size MNP that possesses increased r 2 , the advantage of MCSPs is that the superparamagnetic character is retained as a result of their inclusion of small-size MNPs ( < 30 nm), while bearing superior dispersibility in the medium.
Gazeau et al . demonstrated that MCSPs could optimize the r 1 and r 2 together in order to improve detection sensitivity in MR
sensing and imaging. [ 59 ] Fe 3 O 4 supraparticles were synthesized by thermal hydrolysis of FeCl 3 and FeCl 2 in mixed solvents of diethylene glycol and N -methyl diethanolamine at 220 ° C for 12 h, and then were fully oxidized into Fe 2 O 3 supraparticles with controllable sizes. The r 1 value could culminate at 600 mM − 1 s − 1 for 37-nm MCSPs at low frequency fi eld ( ∼ 0.01 MHz) as a result of their slowing dynamics of magnetic moment. The r 2 value reached 365 mM − 1 s − 1 at elevated frequency fi eld (9.25 MHz); it was increased by a factor of 1.8 with respect to a single Fe 2 O 3 NP. Porosity of MCSPs has gained considerable concern for the sake of integrating different functionalities such as MR imaging, magnetic hyperthermia, and drug storage. Wang et al. reported that the mesoporous MCSPs (72.3 m 2 g − 1 ) possessed a high r 2 of 417.4 mM − 1 s − 1 ; the feature would provide possibility for enhancing sensitivity in T 2 -weighted imaging and, simulta-neously, reducing toxicity by decreasing the agent dosage. [ 60 ]
Hydrophilic stabilizers are very useful in synthesis of a series of water-soluble MCSPs for high-quality MR imaging. [ 61 ] The reaction containing the mixture of Fe(acac) 3 , HOOC − PEG − COOH, and oleylamine was allowed to proceed in phenyl ether for 24 h at 260 ° C. PEG served as stabilizer and hence endowed the resultant MCSPs with prominent biocom-patibility and colloidal stability. The cluster sizes of MCSPs were tuned to 42, 30 and 19 nm, and the corresponding r 2 values were measured at 148, 238 and 126 mM − 1 s − 1 under the clinic conditions (1.5 T, 60 MHz), respectively. T 2 -weighted MR images revealed the fact that the change trend of r 2 domi-nantly relied on the synergistic effect of the size and number of primary MNPs in one MCSP. The r 2 value of 30-nm MCSPs (238 mM − 1 s − 1 , 1.5 T) was 2.3 times higher than that of the com-mercial Feridex (104 mM − 1 s − 1 , 1.5 T). [ 62 ] Along this line, the amine-functionalized MCSPs were synthesized using the same solvothermal approach. [ 63 ] The better relaxivities were obtained. The amine-terminated MCSPs gave r 2 of up to 314.6 mM − 1 s − 1 , which was 60% higher than that of 6-nm Fe 3 O 4 nanoparticles and 2.7 times higher than that of the commercial Ferumox-ytol. Promotion of detection sensitivity for MR imaging is of signifi cance since MR sensitivity for tracking small amount of cells is still relatively low in use of commercial MNP contrast agents. [ 64 ] In this regard, Zhang et al . demonstrated that 63-nm PAA-modifi ed MCSPs had r 2 value of as high as 630 mM − 1 s − 1 , which is among the most sensitive MNP contrast agents ever reported. [ 65 ] Compared with commercially available SHU555A (Resovist), the effi ciency and sensitivity of MCSPs were better both in vitro and in vivo, wherein the detection limits arrived at 3000 and 10000 labeled cells, respectively.
Silica encapsulation of MCSPs endows the resultant mag-netic composites with reactivity of surface functional groups as well as biocompatibility. Shi et al . prepared the silica-coated MCSPs for MR imaging. [ 66 ] Poly( ε -caprolactone)- b -poly(acrylic acid) was applied as template to assemble MNPs into one micelle with tunable sizes. Silica shells were then formed upon MCSPs by the typical sol-gel route, and, in turn, Rhodamine B was labeled on the amine-modifi ed MCSP@SiO 2 micro-spheres. The bio-distribution experiments revealed that the MCSP@SiO 2 -RhB microspheres were dominantly harvested in liver. A maximum r 2 value was 320.7 mM − 1 s − 1 , much higher than that of the commercial MNPs (Feridex). [62] Also, Shi et al . explored the PEGlated MCSP@SiO 2 microspheres with the
identical T 2 -weighted MR imaging feature. [ 67 ] Although the r 2 value was reduced to 215.3 mM − 1 s − 1 , decoration of PEG was benefi cial to reduce the phagocytic capture of nanoparticles from the immune system.
A facile route has been developed to synthesize MCSP-based micelles with the assistance of amphiphilic block copolymers for enhancing transverse relaxivities. Jeong and Kim et al. pre-pared the micelle-like MCSPs consisting of cationic poly(amino acid)s and clustered MNPs. [ 68 ] Poly(amino acid)s, poly- α , β -( N -2-dimethylaminoethyl L-aspartamide), were modifi ed with octa-decyl chains by simple aminolysis of poly(succinimide) with octadecylamine and N,N′ -dimethylethylenediamine sequentially. The MCSP micelles were formed by self-assembly of amphiph-ilic poly(amino acid)s and hydrophobic MNPs in the emulsion. In comparison with a commercial MRI contrast agent (Feridex I. V.), the r 2 of MCSP micelles was up to 333 mM − 1 s − 1 , which was twice as much as that of Feridex I. V. (166 mM − 1 s − 1 ). Riffl e et al . modifi ed the MNPs with the PAA block of an amino-terminated poly(ethylene oxide- b -acrylate) (NH 2 -PEO- b -PAA), and then the PEO diacrylate was added to yield MCSP micelles through the reaction with the NH 2 -PEO moiety of polymer-modifi ed nano-particles. [ 69 ] With an increase in size, the r 2 could be correspond-ingly increased from 190 to 604 mM − 1 s − 1 measured at 1.4 T. Also, the other copolymers, such as poly(ethylene glycol)- b -poly( ε -caprolactone) (PEG- b -PCL), [ 70 ] poly(trimethylammonium ethylacrylate methyl sulfate)- b -poly(acrylamide) (PTEA- b -PAM), [ 71 ] poly(L-actide)- b -poly (ethylene glycol) (PLA- b -PEG) [ 72 ] and poly(acrylic acid)- b -polystyrene(PAA- b -PS), [ 73 ] have been used as building templates for fabricating MCSP micelles with improved MR imaging properties.
The effect has been studied of different modifi ed compo-nents for MCSPs on MR sensitivity and cell labeling effi cacy. Gu et al . applied a miniemulsion, sol-gel and polyol approaches to synthesizing MCSPs with coating of silica, polyethyl-eneimine (PEI) and citric acid, respectively. [ 74 ] As those func-tionalized MCSPs with similar sizes ( ∼ 200 nm) were detected on a 3T MRI scanner, the r 2 values calculated were up to 299 mM − 1 s − 1 for MCSP@SiO 2 , 124 mM − 1 s − 1 for MCSP@PEI, and 360 mM − 1 s − 1 for citric acid-modifi ed MCSPs. The pri-mary reason might lie in that the volume fractions of primary MNPs were different. Additionally, since silica covering was more preferable to label cells (RAW264.7), the detection limit of the MCSP@SiO 2 -labeled cell concentration was as low as 10000 cells mL − 1 on a 3T MRI scanner.
4.2. Targeted Drug Delivery
One very intriguing application of MCSPs is the encapsula-tion and controlled release of drugs. In comparison with an individual MNP, hierarchically structured MCSPs prepared by using self-assembly or solvothermal techniques can be subjected to fl exible modulation of secondary structures and interface natures, giving rise to features of high surface area or inherent hollow core. Thus drugs can be impregnated into “intersti-tial space” of MCSPs or by chemical conjugation to functional groups located on the large surface. Besides, the superior perfor-mance in magnetically targeting delivery is another signifi cant advantage given by MCSP vehicles. A rapid response to applied
9wileyonlinelibrary.commbH & Co. KGaA, Weinheim
10
www.advmat.de
REV
IEW
magnetic fi eld can occur within minutes for the solution-dis-
persed MCSPs, while the enrichment/redispersion of MCSPs is operated reversibly due to their superparamagnetic character.
Although most large-size particles are easily cleared by the reticuloendothelial system (RES) or mononuclear phagocytic system, modifi ed MNPs smaller than 100 nm in diameter have been suggested to be ideal for cancer treatment because of their favorable bio-distribution and clearance/accumulation behavior. [ 75 ] Apart from particle sizes, other properties such as composition, hydrophilic-lipophilic balance and surface charge, have a profound infl uence in RES-related clearance and bio-distribution in body and, therefore, are essential for design of MCSPs. Another effect to consider is the enhanced permeability retention (EPR) effect that is able to enhance the permeability of nanoparticles and macromolecules to tumor vessels compared with normal vessels, and the impaired clearance of these objects from the tiny space of tumor. The EPR effect allows for improve-ment of accumulated concentrations of nanoparticles or macro-molecules in the tumor, as much as fi ve to ten times higher than in normal tissue within 1-2 days. [ 76 ] In order to develop the EPR-related drug-MCSP conjugates, the vehicle sizes should be accurately controlled with range of pore cut-off sizes. Also, both the surface functionalization and the drug conjugation need to be conceived ahead because their behaviors in medium will have a remarkable infl uence on drug delivery and release. As the matter of fact, it has been rarely studied thus so far, but the ongoing investigation to MCSP-based drug delivery systems for cancer is underway based on these principles.
Taking porosity and surface natures of MCSPs into account, a variety of model drugs have been conjugated with modifi ed MCSPs. Polarity of guest molecules is different resulting in the elegantly designed MCSPs with preferable affi nity to entrapped drugs. Our group reported a biopolymer-controlled synthetic approach to preparation of mesoporous PGA-coated MCSPs with characters of tunable surface area and pore volume, acid degradation, and hydrophilicity. [ 39 ] As depicted in Section 2.2, PGA could serve as structure-directed agent to modulate the assembling sizes and patterns of MCSPs and render abun-dant carboxylic acid groups and negatively charged surface. Hydrophobic paclitaxel (TXL) of up to 35% was loaded into the mesoporous PGA-MCSPs by a modifi ed nano-precipitation method. [ 77 ] The embedded TXL could be controllably released by the acid degradation of PGA-MCSPs at pH of 4.5-5.5 within 48 h. This is an excellent property for a drug delivery system since the release behavior of drugs is pH dependent and the car-riers are “degradable”, with no harm to normal cells and organs. The cytotoxicity experiments for the HeLa cells demonstrated that the improved inhibitory effect of the TXL-loaded MCSPs was likely originated from the sustained release of TXL from mesopores of MCSPs, in a sharp contrast to free TXL. Conse-quently, we utilized different biopolymers (including soybean protein, casein, PGA, agarose and chitosan) to investigate the biopolymer-controlled evolution of high-surface-area MCSPs under solvothermal conditions. [ 40b ] Proteins and poly(amino acid)s were both capable to construct the mesoporous MCSPs with high surface areas of 100–200 m 2 /g and pore sizes of 6–13 nm. Therapeutic docetaxel and ceramide as a couple of model drugs were simultaneously encapsulated into the struc-turally optimized MCSPs via the hydrogen binding interaction,
and they showed a pronounced enhancement of the inhibitory and apoptotic effects in PC-3 cells for treatment of prostate cancer. Ha et al. employed the sodium citrate as stabilizer to undergo the similar solvothermal synthesis, leading to a water-dispersed, highly porous MCSP (141 m 2 g − 1 ). [ 78 ] Ibuprofen (IBU) was deposited into the pores of MCSPs and could exhibit sustained release upon pH variation. Approximately 80% of adsorbed IBU were released at pH of 5.0 and 7.4 within 48 h.
Hollow-core MCSPs have been created for storage and con-trolled release of drugs in our group. [ 37 ] Solvothermal condi-tions facilitated the evolution of Ostwald ripening that caused different crystallization processes for the outer and inner components of MCSPs, and thus the hollow-core porous-shell MCSPs were formed. TXL was penetrated into the hollow core through the slim channels of nanoparticle-attached shells as the TXL-containing solution was evaporated. ∼ 20.3% of TXL was loaded and released with response to pH due to acid deg-radation of MCSPs. They showed the dose-dependent cytotox-icity against HeLa cells, and the higher growth inhibition effect was obtained as compared with an equivalent does of free TXL. To achieve the aim of receptor-targeting delivery, we exchanged sodium citrate with folate-grafting PAA to functionalize the hollow-core MCSPs. [ 38 ] The hollow chamber was charged with doxorubicin (DOX), and the DOX conjugates showed pH-dependent release property and higher cytotoxicity to HeLa cells than free DOX. Covalently conjugated drugs within porous MCSPs have also been studied in our group ( Figure 9 ). [ 40a ] Ago-rose-capped MCSPs were modifi ed with vinyl groups, followed by a click reaction with mercaptoacetyl hydrazine to form the terminal hydrazide groups. DOX was then covalently bound to MCSPs through a hydrazone bond, which is acid cleavable, thereby providing a pH-sensitive drug release capability. Com-pared with the free DOX, DOX-conjugated MCSPs exhibited a comparable inhibitory effect for the gastric carcinoma cell line SGC7901, but a signifi cantly lower cytotoxicity in the normal cell line HEK 293T. Cai et al. prepared a biodegradable polymer reservoir using MCSPs as switching carriers for reversible pulsed drug release. [ 79 ] The micro-device substrate containing multi-reservoirs was made from biodegradable poly-DL-lactide, which was charged with drugs and MCSPs ( ∼ 200 nm). A bio-degradable porous polycarbonate membrane with average pore size of ca. 125 nm was utilized to seal the reservoirs. When a magnetic fi eld was added on the upper reservoirs, the MCSPs moved to fi ll in the pores of membranes and switched off the drug release. When the magnetic fi eld was moved to the oppo-site side, the MCSPs were guided to the bottom of the reser-voirs, and the drug release was thus switched on.
Modifi ed MCSPs are susceptible to surface polymerization or self-assembly leading to a well-defi ned core-shell structure. The functionalized shells are applicable to capture/release objective drugs for target delivery. Crosslinked PAA-covered MCSPs were enabled to load DOX of up to 44.6% via ionic interaction between abundant �COOH groups and DOX, and a pH-responsive release of entrapped DOX was presented. [ 80 ] MTT assay for assessment of the cellular cytotoxicity of the drug conjugates revealed that the PAA-MCSPs allowed the sus-tained release of DOX so as to improve the inhibitory effect in HeLa cell growth and proliferation. Zhang et al. adopted the coprecipitation-calcination strategy to prepare the MCSPs-based
Figure 9 . (A) Synthesis of pH-sensitive DOX-conjugated MCSPs; (B) (a) UV-vis absorbance spectra of Fe 3 + ions as MCSPs are degraded at 37 ° C in acid buffer solution, (b) release of Fe 3 + ions and remaining portion of MCSPs (%) as a function of incubation time, and (c) pH-dependent release of DOX from the MCSP-DOX conjugates at 37 ° C. Reproduced with permission. [ 40a ]
microspheres comprising the layered double hydroxides (LDH) shells. [ 81 ] Doxifl uridine (DFUR) was implanted within interlayer galleries of LDH by exchange reaction, and the quasi pulsatile drug release was implemented by the magnetic-force-induced rearrangement of MCSPs.
4.3. MCSP-Based Sensors for Biomarker Detection
Early detection is one of the key challenges in disease con-trol and prevention. Traditional screening methods such as biopsy, blood detection and clinical imaging, however, are not very powerful at early stages of some diseases as well as quite costly. [ 82 ] Conceptually, a disease biomarker is an indicator of a biological state or condition, which implies a change in expres-sion or state of a protein, DNA/RNA, or an organic chemical that correlates with the risk or evolution of a disease. [ 83 ] Bio-marker detection is emerging as one of the most promising strategies for early disease management.
Nanotechnology is playing an important role in the improve-ment of early detection. [ 84 ] Generally, nanosensors are conjugated
with a targeting ligand to fi nd the specifi c biomarkers of interest, thus giving the nanosensor specifi city, while they also act as the generator or detector of a signal, assigning them sensitivity. To date, extensive studies have been conducted on exploring high-sensitivity nanosensors for biomarkers in response to optical, magnetic, mechanical, or electric signals. [ 85 ] As for MCSP-related sensors, some pioneering work has been well presented to eluci-date their advantages in sensitivity and specifi city.
MR imaging shows great value in early cancer diagnosis due to its distinct advantages including non-invasive imaging, deep tissue penetration, and high spatial resolution. Design of magnetic nanosensors has been pursued for biomarker detection by virtue of MR imaging technique. Superparamag-netic nanoparticles can shorten transverse relaxation time ( T 2 ) and elicit negative contrast. Also, as depicted in Section 4.1, as long as the MNPs or MCSPs are aggregated through the interaction of affi nity ligands and biomarkers, their T 2 relaxa-tion time is altered correspondingly. It is hence useful in sen-sitive and accurate detection of disease biomarkers with the change of T 2 . Perez et al . reported the MR assay method to sense oligonucleotides, proteins, and enzyme activity in view of
11wileyonlinelibrary.commbH & Co. KGaA, Weinheim
12
www.advmat.dewww.MaterialsViews.com
REV
IEW
the high sensitivity and selectivity of MCSP-based sensors. [ 86 ]
Firstly Fe 3 O 4 NPs were conjugated with the oligonucleotide. Upon the hybridization with complementary oligonucleotides, the T 2 relaxation time was reduced due to formation of MNP aggregates. In contrast, the reverse magnetic nanosensors have been designed to detect caspase-3 activities. Caspase-3 is a family of intracellular cysteine proteases that is known as a specifi c mediator of the apoptotic process. DEVD (a amino acid sequence Asp-Glu-Val-Asp) acted as structure-directed agent to organize the MNPs into MCSPs. Since DEVD can be rec-ognized and cleaved by caspase-3, the DEVD-blended MCSPs were dissembled when using caspase-3; this process could be detected with an increase in T 2 . Additionally, Yoon and Lee’s group studied the change trend of T 2 when the as-prepared MCSPs were assembled through the streptavidin-biotin interac-tion. [ 87 ] A series of PAA-stabilized MCSPs were solvothermally synthesized and showed the controlled particle sizes from 15, 30 to 50 nm, which corresponded to an increase in r 2 from 247, 340 and 364 mM − 1 s − 1 , respectively. Biotinylated MCSPs were then prepared through the amidation of biotin hydrazide and residual �COOH groups of PAA moiety on MCSPs. Upon cou-pling of biotinylated MCSPs and streptavidin, a notable increase in T 2 was observed and the detection limit of the streptavidin could reach as low as 0.01 nM (5% Δ T 2 ) when the Fe concentra-tion was 2 μ M. Also, it was likely that detection limit was fur-ther decreased using smaller amount of biotinylated MCSPs.
Mechanical nanosensors are based on the ultrasensitive detection of extremely small mechanical forces occurring on the molecular scale, wherein the main component is the micro-cantilever. [ 88 ] When the analyte molecules bind to the immobi-lized receptors on the surface of a cantilever, the microcanti-lever can make response to the change of surface stress and mass by the cantilever bending and the resonance frequency shifting. [ 89 ] Also, detection specifi city can be improved by selec-tive biochemical reactions like coating the cantilever with DNA probes, antibodies or peptides. [ 90 ] Jeon et al . developed a novel gravimetric immunoassay for sensitive detection of multiple protein biomarkers through the use of silicon microcantilever arrays and MCSP-based microspheres ( Figure 10 ). [ 91 ] MCSP@SiO 2 microspheres were synthesized according to the reported methods and were covered by a highly crystalline TiO 2 shell using a solvothermal reaction. Prior to mechanical immuno-assay, antibody-functionalized MCSP@SiO 2 @TiO 2 were used to concentrate the antigens in human serum, and silicon canti-levers were also modifi ed with antibodies toward multiple target biomarkers (interleukin-6, interferon- γ and α -fetoprotein). Since the modifi ed MCSP@SiO 2 @TiO 2 microspheres were connected to the specifi c cantilevers and were consequently subjected to photocatalytic deposition of silver, the increasing mass extremely enhanced the sensitivity of microcantilevers. Frequency changes could be calculated to estimate the detec-tion limit of as low as ∼ 0.1 pg mL − 1 better than the clinical threshold of the biomarkers.
4.4. Enrichment and Detection of Proteins/Peptides
Proteomics is an interdisciplinary subject formed on the basis of the research and development of the Human
Genome Project, and also is of great pertinence to functional genomics. [ 92 ] In this research fi eld, it is highly required to develop the high-throughput separation and identifi cation of proteins/peptides for insight into intracellular protein com-position, structure, and its own unique activity patterns. MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-fl ight mass spectrometry) is the commonly used mass spectrometric tool for identifying proteins/peptides, [ 93 ] but it cannot give suffi cient information for the low-abundance pep-tides/proteins extracted from real biological tissues. Moreover, non-specifi c peptides/proteins mixed in protein digests also suppresses signals of target biomolecules. Therefore, prior to MS analysis, enrichment and separation of proteins/peptides become a very important step. Immobilized metal ion affi nity chromatography (IMAC) is a traditional technique applied for work-up of peptides from proteolytic digest mixtures. However, this chromatography could capture a large number of peptides in the column due to porosity of resin beads, and thus the signal intensity of MS is largely restricted. [ 94 ] As an alternative to IMAC, magnetic microspheres, which can be isolated readily from the sample solutions by employing a magnetic fi eld, is superbly useful in pre-concentration of peptides/proteins. Only a short time is needed to acquire massive proteins/peptides for MALDI-MS analysis. Moreover, synthesis and functionaliza-tion strategies for MCSP-based microspheres are versatile and fl exible, providing a promising view for selective enrichment and detection of low-abundance peptides/proteins.
4.4.1. Metal Ions Immobilized into MCSP-Based Microspheres
In the pioneering work, Zhang and Deng's group immobilized Fe 3 + complexes on MCSP-based microspheres for enrichment of phosphopeptides. [ 95 ] The MCSP@SiO 2 composite micro-spheres were modifi ed with silane coupling agents to bind abundant carboxylic acid groups on silica shells for anchoring Fe 3 + ions. Through the analysis of MALDI-TOF MS technique, the MCSP@SiO 2 -Fe 3 + microspheres exhibited the selective enrichment capability for the phosphopeptides, and 0.2 nM of bovine β -casein digest could be treated to attain the explicit MS signals.
Zou et al . adopted atomic transfer radical polymerization to graft PEG brushes onto the surface of MCSP@SiO 2 micro-spheres for anchoring the Ti 4 + ions with reactive hydroxyl groups ( Figure 11 ). [ 96 ] The hydrophilic PEG brushes decreased the nonspecifi c adsorption of nonphosphopeptides as well as enhanced binding capacity of phosphopeptide by a high den-sity of terminal functional groups. Enrichment results dem-onstrated that the MCSP@SiO 2 @PEG-Ti 4 + microspheres had a high phosphopeptide recovery ( > 70%), low detection limit (0.5 fmol), and great specifi city in binding of phosphopeptides from a tryptic digest mixture (the molar ratio of BSA/ α -casein is 2000:1). In analysis of real biological samples (Arabidopsis), the MCSP@SiO 2 @PEG–Ti 4 + microspheres displayed fan-tastic performances in determining phosphopeptides (2447), far better than those of MCSP@SiO 2 -Ti 4 + (1186) and com-mercial TiO 2 (961). In our group, we performed the two-step distillation-precipitation polymerization upon MCSPs to pre-pare the magnetic polymer microspheres consisting of a PMAA interim layer and a PEGMP outer layer, as described in Section
Figure 10 . (A) Illustration of a MCSP@TiO 2 microsphere-assisted microcantilever bioassay with improved sensitivity by photocatalytic formation of silver; (B) Changes in resonance frequency of (a) the antigen-binding cantilever array (I), the antigen-MPNP-fi xed cantilever array (II) and the silver-enhanced antigen-MPNP-linked cantilever array (III), and of (b) each cantilever after binding of various protein biomarkers (1 pg/mL in serum) and photocatalytic silver enhancement. The insets in (a) and (b) show the IL6-antibody-functionalized microcantilever array and the multiple antibody-functionalized microcantilever array, respectively. Reproduced with permission. [ 91 ] Copyright 2012, American Chemical Society.
3. [ 52 ] Numerous Ti 4 + ions were immobilized on the MCSP@PMAA@PEGMP microspheres and were utilized to selectively enrich phosphopeptides from complex biological samples. It was proved that the composite microspheres enabled selective enrichment of phosphopeptides in mixtures containing phos-phopeptides/nonphosphopeptides with the molar ratio of 1:500. Also, they could fi sh the low-abundance phosphopeptides from the tryptic digests of drinking milk and human serum, indica-tive of their outstanding selectivity and specifi city in phospho-proteome analysis.
In light of coordination interaction of Cu 2 + ions with pep-tides/proteins, Deng et al. synthesized the Cu 2 + -immobilized MCSP porous microspheres for enrichment of peptides and
size exclusion of high-molecular-weight proteins. [ 97 ] The mesoporous silica shells covered the MCSPs by a surfactant templating approach, and the chelating groups (�COOH) were grafted onto the inner channels of mesoporous silica shells for immobilizing Cu 2 + ions. The resulting MCSP@mSiO 2 -Cu 2 + microspheres were applied to enrich hydrophobic angiotensin II and hydrophilic microcystin-LR at a very low concentration (5 nM), respectively. As revealed by MS analysis, the S/N ratios for two peptides were both increased greatly upon treatment with the Cu 2 + -immobilized composite microspheres. Based on the size exclusion effect of mesopores, peptides captured from human serum could be limited at a mass range from 800 to 3500 Da. Meanwhile, the MCSP@mSiO 2 -Cu 2 + microspheres
13wileyonlinelibrary.commbH & Co. KGaA, Weinheim
1
www.advmat.dewww.MaterialsViews.com
REV
IEW
Figure 11 . (A) Synthesis of MCSP@SiO 2 @PEG-Ti 4 + microspheres; (B) (a) the number of identifi ed unique phosphopeptides from the tryptic digest of Arabidopsis lysate and the corresponding enrichment specifi city, and (b) the percentage and overlap of the identifi ed unique phosphopeptides by MCSP@SiO 2 @PEG-Ti 4 + , MCSP@SiO 2 -Ti 4 + and TiO 2 microspheres, respectively. (Total number: 3441.) Reproduced with permission. [ 96 ] Copyright 2012, Royal Society of Chemistry.
could largely adsorb bovine hemoglobin (BHb) (418.6 mg g − 1 ) and remove BHb from bovine blood. [ 98 ] Besides, more specifi c metal ions have been utilized to modify MCSP-based micro-spheres for proteomics research, for example, immobilized Zr 4 + for selective enrichment of phosphopeptides [ 99 ] and Ni 2 + for separation of histidine-tagged proteins. [ 100 , 101 ]
4.4.2. Inorganic Nanomaterials Supported on MCSP-Based Microspheres
In addition to MCSP-supported metal ions complexes for binding peptides/proteins, metal oxides have been demon-strated to be another excellent candidates in trapping biological molecules through the reversible chemisorption of functional groups on their surfaces. Our group formulated a design to combine MCSPs with mesoporous TiO 2 with high surface area (167.1 m 2 g − 1 ) and appropriate pore size (8.6–16.4 nm) ( Figure 12 ). [ 44a ] Examined by using a mixture of phosphopep-tides/nonphosphopeptides with molar ratio of 1:1000, the MCSP@mTiO 2 microspheres exhibited remarkable selectivity for phosphopeptides, large enrichment capacity (225 mg g − 1 ), extremely low detection limit (fmol level), short collecting time ( < 5 min), and high recovery of phosphopeptides (93%).
Zhang and coworkers also synthesized MCSP@Carbon@TiO 2 microspheres by using the different method. [ 102 ] MCSPs were coated with a thin layer of carbon by the polymerization and carbonization of glucose under hydrothermal conditions. Tetrabutyltitanate was pre-hydrolyzed and deposited onto the MCSP@Carbon microspheres, and then a TiO 2 shell was formed by calcination under nitrogen. The obtained MCSP@Carbon@TiO 2 microspheres could selectively harvest phos-phopeptides from a tryptic digest of casein mixture (composed of α -S1 and α -S2 units, and β -casein) at a low concentration (5 ng μ L − 1 ). Besides, core-shell-structured composites of Al 2 O 3 , [ 103 ] Ga 2 O 3 , [ 104 ] NiO, [ 105 ] and SnO 2 [ 106 ] with MCSPs have also been synthesized and equally show improved function in concentration of peptides/proteins.
Zeolite nanoparticles have been proved to be prominent bio-materials for various applications including preconcentration of low-abundance proteins/peptides. [ 107 ] Zhao et al . synthesized the core-shell magnetic microspheres consisting of MCSPs cores and zeolite shells, showing a strong affi nity to trypsin with a high adsorption capacity (62 μ g mg − 1 ). [ 108 ] Trypsin-immobilized MCSP@Zeolite improved the effi ciency of protein digestion with the assistance of microwave irradiation. Also, they could be conveniently recovered by the external magnetic
Figure 12 . (A) (a) preparation of core-shell-structured mesoporous MCSP@mTiO 2 microspheres and (b) their application for enrichment of phospho-rylated peptides; (B) MALDI MS analysis for evaluation of (a) enrichment capacity of MCSP@mTiO 2 and MCSP@TiO 2 and (b) selective enrichment of the phosphopeptides from a tryptic digest mixture of β -casein and BSA with a molar ratio of 1:1000 by using MCSP@mTiO 2 . Reproduced with permission. [ 44a ] Copyright 2012, American Chemical Society.
fi eld and be reused at least seven times without any reduction in digestion effi ciency.
Rare-earth phosphate (LnPO 4 ) nanomaterials have specifi c affi nity to phosphopeptides. [ 109 ] The precursor particles were synthesized fi rstly by deposition of rare-earth basic carbonate [Ln(OH)CO 3 ] on MCSPs through a urea-based chemical precipitation method, and then they were subjected to ion-exchange reaction with (NH 4 ) 2 HPO 4 under hydrothermal con-ditions, yielding the resultant MCSP@LnPO 4 (Ln = Eu, Tb, Er) microspheres. In comparison with the commercial TiO 2 , high-surface-area MCSP@ErPO 4 microspheres displayed the better affi nity ability in enrichment of phosphopeptides from a mixture of β -casein and BSA (1:50 molar ratio), and nonfat milk.
Wei and coworkers synthesized a novel core-shell-struc-tured MCSP@SiO 2 @LDH microspheres for selective adsorp-tion of the histidine-tagged protein from Escherichia coli lysate. [ 110 ] This nanostructure was composed of a MCSP@SiO 2 core and a layered double hydroxide (LDH) nanoplatelet shell. It was formed using a two-step growth route including a layer-by-layer deposition on MCSP@SiO 2 cores and a sub-sequent in-situ crystallization ( Figure 13 ). The sizes and compositions of LDH shells could be precisely controlled, resulting in a series of MCSP@SiO 2 @M(II)Al-LDH (M = Ni, Co, Zn, Mg) microspheres. Of those members, the MCSP@SiO 2 @NiAl-LDH microspheres had the best binding ability in adsorption of the histidine-tagged green fl uorescent protein (GFP). The loading content of histidine-tagged GFP in MCSP microspheres was as high as 239 μ g mg − 1 , and the enriching
level could be retained after the microspheres were reused in fi ve cycles.
In our group, selective enrichment of glycopeptides from complex biological samples has been investigated using MCSP-supported Ag nanoparticles by virtue of multivalent interaction. [ 111 ] A PMAA shell was formed on MCSP by the distillation-precipitation polymerization and entrapped Ag + ions using the pendent carboxyl groups on the particle sur-face. Then Ag nanoparticles were locally prepared via reduc-tion reaction, resulting in an Ag-nanoparticle-attached layer on the MCSP@PMAA microspheres. In view of the multi-valent interaction between Ag and glycan, the composite microspheres could effi ciently capture low-abundance glyco-peptides in the mixture of glycopeptides/nonglycopeptides with the molar ratio of 1:100. In analysis of a real biological sample (rat serum) that contains the 245 non-redundant gly-copeptides, 127 unique glycopeptides mapped to 51 different glycoproteins could be harvested from only 1 μ L of rat serum sample, implying their excellent selectivity in glycoproteomics analysis.
4.4.3. Hydrophobically Modifi ed MCSP-Based Microspheres
Hydrophobic enrichment from low-abundance proteins/peptides is also of critical signifi cance in a real proteomic analysis, as these peptides/proteins have extremely low con-centrations ( < 1 nM), and their MS signals suffer strong interference from those of high abundant proteins as well as contaminants generated during pretreatment procedures.
15wileyonlinelibrary.commbH & Co. KGaA, Weinheim
16
www.advmat.dewww.MaterialsViews.com
REV
IEW
Figure 13 . (A) Fabrication of MCSP@SiO 2 @LDH microspheres; (B) Photographs for showing the separation of histidine-tagged GFP by MCSP@SiO 2 @NiAl-LDH in 30 min; (C) (a) Recycling tests of separation of histidine-tagged GFP using MCSP@SiO 2 @NiAl-LDH, and (b) SDS-PAGE evalu-ation of reusability of MCSP@SiO 2 @NiAl-LDH (lanes 1-5) in the cell lysate containing histidine-GFP (lane L). Lane M is a molecular weight marker. Reproduced with permission. [ 110 ] Copyright 2012, American Chemical Society.
Therefore, hydrophobically modifi ed MCSP microspheres have been designed as affi nity probes toward concentration and identifi cation of proteins/peptides in biological tissues. To this end, Zhang and Deng's group synthesized the C 8 -functi-noalized MCSP microspheres. The carbon-covered MCSPs prepared by a two-step hydrothermal process were modifi ed with chlorodimethyloctylsilane, resulting in MCSP@Carbon-C 8 microspheres. [ 112 ] Then peptide analysis was studied using Angiotensin II as a model. They demonstrated that the as-synthesized hydrophobic MCSP microspheres possessed more superior ability in enrichment of Angiotensin II, and the MS analysis gave a S/N ratio of up to 1103.82 at the very low con-centration of peptides (5 fmol μ L − 1 ). The value was seven times higher than that of the commercially available MB-C 8 (a mag-netic-bead-based C 8 -hydrophobic chromatography resin). In the protein analysis, after BSA digest was treated by the hydro-phobic MCSP microspheres, the S/N ratios were pronouncedly
increased and 23 peaks with a sequence coverage of 32% were identifi ed in pure BSA digest. Again, it was proved that a real proteomic analysis could be performed. Following the sim-ilar line, their group encapsulated the core-shell-structured MCSP@SiO 2 with linear PMMA chains via a seeded poly-merization. [ 113 ] It was confi rmed that the hydrophobic property of PMMA moiety was preferable to most peptides, which could be enriched effectively and detected on MS with a pronounced S/N ratio.
Since graphene and graphene oxide both have a strong affi nity for biomolecules, it is thus desirable that they are combined with MCSPs for the concentration and magnetic separation of proteins/peptides. [ 114 ] Jiang and coworkers synthe-sized a novel graphene-functionalized MCSP microspheres. [ 115 ] Negatively charged graphene oxide was fi rstly deposited onto the surface of amine-functionalized MCSP@SiO 2 micro-spheres via the electrostatic interaction, and the reduction of
graphene oxide was conducted by using hydrazine, leading to a core-shell-structured MCSP@SiO 2 @Graphene microspheres. Basic (Cyt c ), neutral (Myo) and acidic ( β -Lac) proteins as well as tryptic digest of BSA were tested. The adsorption capacity (20.02 mg g − 1 ), recovery (58.2–101.2%) and detection limits (3.8–68 fmol) of the composite microspheres were all better than those of the commercial C 18 adsorbent.
5. Conclusions
Advancement in self-assembly chemistry of supermolecular nanostructures simulates the advent and development of engi-neering inorganic nanomaterial into supraparticles, thereby generating more comprehensive and superior performances for the cross-domain applications. In this context, magnetic colloidal supraparticles (MCSPs) originated from assem-bled magnetic nanoparticles appeal mounting attention not only owing to largely enhanced magnetic responsiveness and preserved superparamagnetism but a hierarchical and func-tional integration that renders diverse applicability of MCSPs in biomedical fi elds. One-step solvothermal recipe allows the synthesized MCSPs to controllably form intrinsic mes-oporosity and hollow void in core, which notably improve the loading capacity of drugs and enable sustained release in response to environmental stimuli. Also, assembling dimen-sion is under control, and the optimized sizes are benefi cial to enhance the T 2 -weighted contrast in MR imaging. Simultane-ously, ligands anchored on MCSPs confer a number of reactive function groups, capable of being conjugated with drugs, tar-geting agents, labeling probes, or non-biofouling polymers, as well as giving remarkable dispersibility in biological medium. Multiple-step modifi cation by coating of organic polymers and inorganic nanomaterials results in a variety of defi nitely core-shell-structured MCSP composite microspheres, opening up an ideal avenue in enrichment and identifi cation of low-abun-dance proteins/peptides. A series of breakthrough in specifi city, sensitivity, reusability, and convenience demonstrates that the design of MCSP-based composite microspheres entail rationale and superiority in this regard.
Although promising from the view of achievements in pri-mary studies, the use of MCSPs for practical bio-applications is, however, hampered by the complex biological issues in vivo. Solvothermal method currently has no potent way to limit the sizes of MCSPs to be less than 100 nm, which has been proved to favor the bio-distribution and EPR-enhanced target delivery. Besides, tunability of secondary structure of MCSPs still remains challenges. Mesopores stemming from the ligand-con-trolled agglomeration of primary MNPs are expected to have open, oriented, uniform channels perpendicular to the MCSP’s surface, with the aim of screening and immobilizing proteins/peptides effi ciently; the better understanding and substantial experiment evidences for the forming mechanism of porosity is also in high demand. In comparison to severe solvothermal conditions, microwave assistance technique is advantageous in many facets, but few of reports has been presented to realize the transformation from solid, to hollow, to porous structures of MCSPs, as well as the controllability of composi-tions, sizes and surface properties. Toxicity issue of MCSPs is extremely important in clinic use, which is directly associated
with the administrated dose and route, chemical composition, size, structure, surface chemistry, biodegradability, and so on. Although it is found that mesoporous MCSPs are susceptible to acid degradation in vitro, intense studies to comprehensively estimate the toxicity and biocompatibility of MCSPs are still urgently needed. Along this line, presumably coating of bio-macromolecules or functional materials is the better alterna-tive to pristine MCSPs that are unknown in biocompatibility, also accompanied with integration of drug delivery, sensing and imaging. In addition, it is questionable whether there is the best technology for the setup of external magnetic fi elds that can be deeply penetrated into the tissues and reside at the intended disease sites with suffi cient safety. Overall, we are aware that much progress has been made recently, but there is still a long way to go for tackling numerous challenges so as to gradually implement the real application of MCSPs in the diagnosis and treatment of disease.
Acknowledgements This work was supported by National Science and Technology Key Project of China (Grant No. 2012AA020204), and National Science Foundation of China (Grant Nos. 21034003, 21128001 and 51073040).
Received: April 28, 2013 Revised: June 3, 2013
Published online:
[ 1 ] X. Wang , J. Zhuang , Q. Peng , Y. D. Li , Nature 2005 , 437 , 121 . [ 2 ] S. H. Sun , C. B. Murray , D. Weller , L. Folks , A. Moser , Science 2000 ,
287 , 1989 . [ 3 ] Y. H. Deng , D. W. Qi , C. H. Deng , X. M. Zhang , D. Y. Zhao , J. Am.
Chem. Soc. 2008 , 130 , 28 . [ 4 ] X. Q. Xu , C. H. Deng , M. X. Gao , W. J. Yu , P. Y. Yang , X. M. Zhang ,
Adv. Mater. 2006 , 18 , 3289 . [ 5 ] J. Shin , R. M. Anisur , M. K. Ko , G. H. Im , J. H. Lee , I. S. Lee , Angew.
Chem., Int. Ed. 2009 , 48 , 321 . [ 6 ] K. Cheng , S. Peng , C. J. Xu , S. H. Sun , J. Am. Chem. Soc. 2009 , 131 ,
10637 . [ 7 ] R. Massart , IEEE Trans. Magn. 1981 , 17 , 1247 . [ 8 ] J. H. Wu , S. P. Ko , H. L. Liu , S. Kim , J. S. Ju , Y. K. Kim , Mater. Lett.
2007 , 61 , 3124 . [ 9 ] J. Park , K. An , Y. Hwang , J. G. Park , H. J. Noh , J. Y. Kim , J. H. Park ,
N. M. Hwang , T. Hyeon , Nature Mater. 2004 , 3 , 891 . [ 10 ] J. Cheon , N. J. Kang , S. M. Lee , J. H. Lee , J. H. Yoon , S. J. Oh , J.
Am. Chem. Soc. 2004 , 126 , 1950 . [ 11 ] H. Zeng , P. M. Rice , S. X. Wang , S. H. Sun , J. Am. Chem. Soc.
2004 , 126 , 11458 . [ 12 ] H. Itoh , T. Sugimoto , J. Colloid Interface Sci. 2003 , 265 , 283 . [ 13 ] F. Chen , Q. Gao , G. Hong , J. Ni , J. Magn. Magn. Mater. 2008 , 320 ,
1775 . [ 14 ] L. Cabrera , S. Gutierrez , N. Menendes , M. P. Morales , P. Herrasti ,
Electrochem. Acta 2008 , 53 , 3436 . [ 15 ] R. F. C. Marques , C. Garcia , P. Lecante , J. L. Ribeiro , L. Noe ,
N. J. O. Silva , V. S. Amaral , A. Millan , M. Verelst , J. Magn. Magn. Mater. 2008 , 320 , 2311 .
[ 16 ] U. Teng-Lam , R. Mammucari , K. Suzuki , N. R. Foster , Ind. Eng. Chem. Res. 2008 , 47 , 599 .
17wileyonlinelibrary.commbH & Co. KGaA, Weinheim
18
www.advmat.dewww.MaterialsViews.com
REV
IEW
[ 17 ] L. H. Reddy , J. L. Arias , J. Nicolas , P. Couvreur , Chem. Rev. 2012 ,
112 , 5818 . [ 18 ] M. Colombo , S. Carregal-Romero , M. F. Casula , L. Gutierrez ,
M. P. Morales , I. B. Bohm , J. T. Heverhagen , D. Prosperi , W. J. Parak , Chem. Soc. Rev. 2012 , 41 , 4306 .
[ 19 ] R. Hao , R. J. Xing , Z. C. Xu , Y. L. Hou , S. Gao , S. H. Sun , Adv. Mater. 2010 , 22 , 2729 .
[ 20 ] a) J. K. Oha , J. M. Park , Prog. Polym. Sci. 2011 , 36 , 168 ; b) D. L. Shi , N. M. Bedford , H. S. Cho , Small 2011 , 7 , 2549 .
[ 21 ] A. H. Morrish , The Physical Principles of Magnetism , Wiley , New York 1965 , Ch. 7 .
[ 22 ] S. H. Sun , Adv. Mater. 2006 , 18 , 393 . [ 23 ] Q. A. Pankhurst , J. Connolly , S. K. Jones , J. Dobson , J. Phys. D:
Appl. Phys. 2003 , 36 , R167 . [ 24 ] T. Neuberger , B. Schopf , H. Hofmann , M. Hofmann ,
B. von Rechenber , J. Magn. Magn. Mater. 2005 , 293 , 483 . [ 25 ] Y. S. Xia , Z. Y. Tang , Chem. Commun. 2012 , 48 , 6320 . [ 26 ] Z. D. Lu , Y. D. Yin , Chem. Soc. Rev. 2012 , 41 , 6874 . [ 27 ] J. Bibette , J. Magn. Magn. Mater. 1993 , 122 , 37 . [ 28 ] F. Montagnea , O. Mondain-Monvalb , C. Pichota , H. Mozzanegac ,
A. Elassari , J. Magn. Magn. Mater. 2002 , 250 , 302 . [ 29 ] R. Veyret , A. Elaissari , P. Marianneau , A. A. Sall , T. Delair , Anal.
Biochem. 2005 , 346 , 59 . [ 30 ] R. Veyret , T. Delair , C. Pichot , A. Elaissari , J. Magn. Magn. Mater.
2005 , 295 , 155 . [ 31 ] F. Bai , D. S. Wang , Z. Y. Huo , W. Chen , L. P. Liu , X. Liang , C. Chen ,
X. Wang , Q. Peng , Y. D. Li , Angew. Chem. Int. Ed. 2007 , 46 , 6650 . [ 32 ] T. Wang , J. Q. Zhuang , J. Lynch , O. Chen , Z. L. Wang , X. R. Wang ,
D. LaMontagne , H. M. Wu , Z. W. Wang , Y. C. Cao , Science 2012 , 338 , 358 .
[ 33 ] T. Wang , X. R. Wang , D. LaMontagne , Z. L. Wang , Z. W. Wang , Y. C. Cao , J. Am. Chem. Soc. 2012 , 134 , 18225 .
[ 34 ] H. Deng , X. L. Li , Q. Peng , X. Wang , J. P. Chen , Y. D. Li , Angew. Chem. Int. Ed. 2005 , 44 , 2782 .
[ 35 ] J. P. Ge , Y. X. Hu , M. Biasini , W. P. Beyermann , Y. D. Yin , Angew. Chem. Int. Ed. 2007 , 46 , 4342 .
[ 36 ] J. Liu , Z. K. Sun , Y. H. Deng , Y. Zou , C. Y. Li , X. H. Guo , L. Q. Xiong , Y. Gao , F. Y. Li , D. Y. Zhao , Angew. Chem. Int. Ed. 2009 , 48 , 5875 .
[ 37 ] B. Luo , S. Xu , W. F. Ma , W. R. Wang , S. L. Wang , J. Guo , W. L. Yang , J. H. Hu , C. C. Wang , J. Mater. Chem. 2010 , 20 , 7107 .
[ 38 ] D. Li , J. Tang , C. Wei , J. Guo , S. L. Wang , D. Chaudhary , C. C. Wang , Chem. Eur. J. 2012 , 18 , 16517 .
[ 39 ] B. Luo , S. Xu , A. Luo , W. R. Wang , S. L. Wang , J. Guo , Y. Lin , D. Y. Zhao , C. C. Wang , ACS Nano 2011 , 5 , 1428 .
[ 40 ] a) D. Li , J. Tang , C. Wei , J. Guo , S. L. Wang , D. Chaudhary , C. C. Wang , Small 2012 , 8 , 2690 ; b) S. Xu , C. Sun , J. Guo , K. Xu , C. C. Wang , J. Mater. Chem. 2012 , 22 , 19067 .
[ 41 ] a) S. Xu , Z. M. Luo , Y. J. Han , J. Guo , C. C. Wang , RSC Adv. 2012 , 2 , 2739 ; b) S. Xu , B. R. Yin , J. Guo , C. C. Wang , J. Mater. Chem. B 2013 , DOI:10.1039/C3TB20238K.
[ 42 ] B. Luo , X. J. Song , F. Zhang , A. Xia , W. L. Yang , J. H. Hu , C. C. Wang , Langmuir 2010 , 26 , 1674 .
[ 43 ] Y. X. Hu , L. He , Y. D. Yin , Small 2012 , 8 , 3795 . [ 44 ] a) W. F. Ma , Y. Zhang , L. L. Li , L. J. You , P. Zhang , Y. T. Zhang ,
J. M. Li , M. Yu , J. Guo , H. J. Lu , C. C. Wang , ACS Nano 2012 , 6 , 3179 ; b) W. Li , J. P. Yang , Z. X. Wu , J. X. Wang , B. Li , S. S. Feng , Y. H. Deng , F. Zhang , D. Y. Zhao , J. Am. Chem. Soc. 2012 , 134 , 11864 .
[ 45 ] Y. H. Deng , D. W. Qi , C. H. Deng , X. M. Zhang , D. Y. Zhao , J. Am. Chem. Soc. 2008 , 130 , 28 .
[ 46 ] a) F. Montagne , O. Mondain-Monval , C. Pichot , A. Elaissari , J Polym. Sci.: Polym. Chem. 2006 , 44 , 2642 ; b) H. Xu , L. Cui , N. Tong , H. Gu , J. Am. Chem. Soc. 2006 , 128 , 15582 .
[ 47 ] L. P. Ramirez , K. Landfester , Macromol. Chem. Phys. 2003 , 204 , 22 .
[ 48 ] F. Sauzedde , A. Elaissari , C. Pichot , Colloid Polym. Sci. 1999 , 277 , 846 .
[ 49 ] A. Xia , J. H. Hu , C. C. Wang , D. L. Jiang , Small 2007 , 3 , 1811 . [ 50 ] W. F. Ma , S. Xu , J. M. Li , J. Guo , Y. Lin , C. C. Wang , J. Polym. Sci.
Polym. Chem. 2011 , 49 , 2725 . [ 51 ] L. J. You , S. Xu , W. F. Ma , D. Li , Y. T. Zhang , J. Guo , J. Hu ,
C. C. Wang , Langmuir 2012 , 28 , 10565 . [ 52 ] W. F. Ma , Y. Zhang , L. L. Li , Y. T. Zhang , M. Yu , J. Guo , H. J. Lu ,
C. C. Wang , Adv. Funct. Mater. 2013 , 23 , 107 . [ 53 ] P. Caravan , J. J. Ellison , T. J. McMurry , R. B. Lauffer , Chem. Rev.
1999 , 99 , 2293 . [ 54 ] a) P. J. Endres , T. Paunesku , S. Vogt , T. J. Meade , G. E. Woloschak ,
J. Am. Chem. Soc. 2007 , 129 , 15760 ; b) K. M. L. Taylor , J. S. Kim , W. J. Rieter , H. An , W. Lin , W. Lin , J. Am. Chem. Soc. 2008 , 130 , 2154 ; c) Y. Song , X. Xu , K. W. MacRenaris , X. Q. Zhang , C. A. Mirkin , T. J. Meade , Angew. Chem. Int. Ed. 2009 , 48 , 9143 ; d) L. M. Manus , D. J. Mastarone , E. A. Waters , X. Q. Zhang , E. A. Schultz-Sikma , K. W. MacRenaris , D. Ho , T. J. Meade , Nano Lett. 2010 , 10 , 484 .
[ 55 ] H. B. Na , I. C. Song , T. Hyeon , Adv. Mater. 2009 , 21 , 2133 . [ 56 ] R. C. Semelka , T. K. Helmberger , Radiology 2001 , 218 , 27 . [ 57 ] S. Laurent , D. Forge , M. Port , A. Roch , C. Robic , L. Vander Elst ,
R. N. Muller , Chem. Rev. 2008 , 108 , 2064 . [ 58 ] a) A. Roch , Y. Gossuin , R. N. Muller , P. Gillis , J. Magn. Magn.
Mater. 2005 , 293 , 532 ; b) K. C. Barick , M. Aslam , Y. P. Lin , D. Bahadur , P. V. Prasad , V. P. Dravid , J. Mater. Chem. 2009 , 19 , 7023 .
[ 59 ] L. Lartigue , P. Hugounenq , D. Alloyeau , S. P. Clarke , M. Lévy , J. C. Bacri , R. Bazzi , D. F. Brougham , C. Wilhelm , F. Gazeau , ACS Nano 2012 , 6 , 10935 .
[ 60 ] J. C. Jia , J. C. Yu , X. M. Zhu , K. M. Chan , Y. X. Wang , J. Colloid Inter-face Sci. 2012 , 379 , 1 .
[ 61 ] F. Hu , K. W. MacRenaris , E. A. Waters , E. A. Schultz-Sikma , A. L. Eckermann , T. J. Meade , Chem. Commun. 2010 , 46 , 73 .
[ 62 ] W. S. Seo , J. H. Lee , X. Sun , Y. Suzuki , D. Mann , Z. Liu , M. Terashima , P. C. Yang , M. V. McConnell , D. G. Nishimura , H. Dai , Nat. Mater. 2006 , 5 , 971 .
[ 63 ] K. C. Barick , M. Aslam , P. V. Prasad , V. P. Dravid , D. Bahadur , J. Magn. Magn. Mater. 2009 , 321 , 1529 .
[ 64 ] F. Hu , H. M. Joshi , V. P. Dravid , T. J. Meade , Nanoscale 2010 , 2 , 1884 .
[ 65 ] M. Li , H. Gu , C. Zhang , Nanoscale Res. Lett. 2012 , 7 , 204 . [ 66 ] D. Niu , Z. Zhang , S. Jiang , Z. Ma , X. Liu , Y. Li , L. Zhou , C. Liu ,
Y. Li , J. Shi , J. Mater. Chem. 2012 , 22 , 24936 . [ 67 ] D. Niu , X. Liu , Y. Li , Z. Ma , W. Dong , S. Chang , W. Zhao , J. Gu ,
S. Zhang , J. Shi , J. Mater. Chem. 2011 , 21 , 13825 . [ 68 ] H. J. Lee , K. S. Jang , S. Jang , J. W. Kim , H. M. Yang , Y. Y. Jeong ,
J. D. Kim , Chem. Commun. 2010 , 46 , 3559 . [ 69 ] N. Pothayee , S. Balasubramaniam , N. Pothayee , N. Jain , N. Hu ,
Y. Lin , R. M. Davis , N. Sriranganathan , A. P. Koretsky , J. S. Riffl e , J. Mater. Chem. B 2013 , 1 , 1142 .
[ 70 ] H. Ai , C. Flask , B. Weinberg , X. Shuai , M. D. Pagel , D. Farrell , J. Duerk , J. M. Gao , Adv. Mater. 2005 , 17 , 1949 .
[ 71 ] J.-F. Berret , N. Schonbeck , F. Gazeau , D. El Kharrat , O. Sandre , A. Vacher , M. Airiau , J. Am. Chem. Soc. 2006 , 128 , 1755 .
[ 72 ] A. Bakandritsos , G. Mattheolabakis , R. Zboril , N. Bouropoulos , J. Tucek , D. G. Fatouros , K. Avgoustakis , Nanoscale 2010 , 2 , 564 .
[ 73 ] R. J. Hickey , A. S. Haynes , J. M. Kikkawa , S. J. Park , J. Am. Chem. Soc. 2011 , 133 , 1517 .
[ 74 ] F. Xu , C. Cheng , F. Xu , C. Zhang , H. Xu , X. Xie , D. Yin , H. Gu , Nanotechnology 2009 , 20 , 405102 .
[ 75 ] a) M. E. Davis , Z. Chen , D. M. Shin , Nat. Rev. Drug Discovery 2008 , 7 , 771 ; b) E. Gullotti , Y. Yeo , Mol . Pharmaceutics 2009 , 6 , 1041 ; c) H. S. Cho , Z. Y. Dong , G. M. Pauletti , J. M. Zhang , H. Xu ,
[ 96 ] L. Zhao , H. Qin , Z. Hu , Y. Zhang , R. Wu , H. Zou , Chem. Sci. 2012 , 3 , 2828 .
[ 97 ] S. Liu , H. Chen , X. Lu , C. Deng , X. Zhang , P. Yang , Angew. Chem. Int. Ed. 2010 , 49 , 7557 .
[ 98 ] M. Zhang , D. Cheng , X. He , L. Chen , Y. Zhang , Chem. Asian J. 2010 , 5 , 1332 .
[ 99 ] X. S. Li , J. H. Wu , Y. Zhao , W. P. Zhang , Q. Gao , L. Guo , B. F. Yuan , Y. Q. Feng , J. Chromatogr. A 2011 , 1218 , 3845 .
[ 100 ] Z. Liu , M. Li , X. Yang , M. Yin , J. Ren , X. Qu , Biomaterials 2011 , 32 , 4683 .
[ 101 ] Y. Zhang , Y. Yang , W. Ma , J. Guo , Yao Lin , C. C. Wang , ACS Appl. Mater. Interfaces 2013 , 5 , 2626 .
[ 102 ] Y. Li , J. Wu , D. Qi , X. Xu , C. Deng , P. Yang , X. Zhang , Chem. Commun. 2008 , 564 .
[103] Y. Li, Y. Liu, J. Tang, H. Lin, N. Yao, X. Shen, C. Deng, P. Yang, X. Zhang, J. Chromatogr. A , 2007 , 1172 , 57.
[ 104 ] Y. Li , H. Lin , C. Deng , P. Yang , X. Zhang , Proteomics 2008 , 8 , 238 .
[ 105 ] Z. Liu , M. Li , F. Pu , J. Ren , X. Yang , X. Qu , J. Mater. Chem. 2012 , 22 , 2935 .
[ 106 ] D. Qi , J. Lu , C. Deng , X. Zhang , J. Phys. Chem. C 2009 , 113 , 15854 .
[ 107 ] Y. Zhang , X. Wang , W. Shan , B. Wu , H. Fan , X. Yu , Y. Tang , P. Yang , Angew. Chem. Int. Ed. 2005 , 44 , 615 .
[ 108 ] Y. Deng , C. Deng , D. Qi , C. Liu , J. Liu , X. Zhang , D. Zhao , Adv. Mater. 2009 , 21 , 1377 .
[ 109 ] Z. G. Wang , G. Cheng , Y. L. Liu , J. L. Zhang , D. H. Sun , J. Z. Ni , Small 2012 , 8 , 3456 .
[ 110 ] M. Shao , F. Ning , J. Zhao , M. Wei , D. G. Evans , X. Duan , J. Am. Chem. Soc. 2012 , 134 , 1071 .
[ 111 ] W. F. Ma , L. L. Li , Y. Zhang , Q. An , L. J. You , J. M. Li , Y. T. Zhang , S. Xu , M. Yu , J. Guo , H. J. Lu , C. C. Wang , J. Mater. Chem. 2012 , 22 , 23981 .
[ 112 ] H. Chen , C. Deng , Y. Li , Y. Dai , P. Yang , X. Zhang , Adv. Mater. 2009 , 21 , 2200 .
[ 113 ] H. Chen , C. Deng , X. Zhang , Angew. Chem. Int. Ed. 2010 , 49 , 607 .
[ 114 ] L. A. L. Tang , J. Z. Wang , K. P. Loh , J. Am. Chem. Soc. 2010 , 132 , 10976 .
[ 115 ] Q. Liu , J. Shi , M. Cheng , G. Li , D. Cao , G. Jiang , Chem. Commun. 2012 , 48 , 1874 .
