Advances in Colloid and Interface Science 207 (2014) 155–163
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Lipid, protein and poly(NIPAM) coated mesoporous silica nanoparticles for biomedical applications Yang Yang a,⁎, Junbai Li a,b,⁎⁎ a
National Center for Nanoscience and Technology, Beijing 100190, China Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
b
a r t i c l e
i n f o
Available online 8 November 2013 Keywords: Lipids Protein Poly(NIPAM) Mesoporous silica nanoparticles Biomedical application
a b s t r a c t In the past decade, mesoporous silica nanoparticles (MSNs) as nanocarriers have showed much potential in advanced nanomaterials due to their large surface area and pore volume. Especially, more and more MSNs based nanodevices have been designed as efficient drug delivery systems (DDSs) or biosensors. In this paper, lipid, protein and poly(NIPAM) coated MSNs are reviewed from the preparation, properties and their potential application. We also introduce the preparative methods including physical adsorption, covalent binding and self-assembly on the MSNs' surfaces. Furthermore, the interaction between the aimed cells and these molecular modified MSNs is discussed. We also demonstrate their typical applications, such as photodynamic therapy, bioimaging, controlled release and selective recognition in biomedical field. © 2013 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Preparation and properties of the functional molecules coated MSNs 2.1. MSNs . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Lipid coated MSNs . . . . . . . . . . . . . . . . . . . . . 2.3. Protein coated MSNs . . . . . . . . . . . . . . . . . . . . 2.4. Poly(NIPAM) coated MSNs . . . . . . . . . . . . . . . . . 3. Potential applications and outlooks . . . . . . . . . . . . . . . . . 3.1. In photodynamic therapy . . . . . . . . . . . . . . . . . . 3.2. In cell imaging . . . . . . . . . . . . . . . . . . . . . . . 3.3. In controlled release . . . . . . . . . . . . . . . . . . . . 3.4. In selective recognition . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
⁎ Correspondence to: Y. Yang, National Center for Nanoscience and Technology, Zhong Guan Cun, Bei Yi Tiao No. 11, Beijing 100190, China. Tel.: +86 10 8254 5540; fax: +86 10 8261 2629. ⁎⁎ Correspondence to: J. Li, Institute of Chemistry, Chinese Academy of Sciences, Zhong Guan Cun, Bei Yi Jie No. 2, Beijing 100190, China. Tel.: +86 10 8261 4087; fax: +86 10 8261 2629. E-mail addresses: [email protected] (Y. Yang), [email protected] (J. Li). 0001-8686/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cis.2013.10.029
After the discovery of highly ordered mesoporous silica materials by the Mobil Corporation in 1992 [1,2], the preparation of mesoporous silica-based materials has become highly attractive due to their potential applications in the fields of catalysis, lasers, sensors and environmental application [3–7]. In the past decade, more and more mesoporous silica based composite materials were designed to apply in biomedical fields [8–11] since they were first reported as drug delivery systems (DDSs)
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[12]. Several features result in mesoporous silica materials as good carriers in biomedical application. Firstly, they have the ordered pore network and homogeneous size for the drug loading; secondly, high pore volume and surface area will host the required amount of drug molecules; most importantly, a silanol-containing functionalized surface allows them to be modified easily to control over drug loading and release. Mesoporous silica materials made by the original procedures are mainly mesoporous silica sheets with disorganized morphologies. For increasing their biocompatibility, significant research efforts have been made towards the smaller size and mono-dispersity. In 2001, Cai et al. firstly obtained MCM-41 typed mesoporous silica nanoparticles (MSNs) with 100 nm by using a dilute surfactant solution [13]. Later, various MSNs with well-defined and controllable particle morphology were developed by Lin's and other research groups in the pursuit of biocompatible materials used in controlled release and drug delivery systems [14–16]. These mesoporous silica materials with nanosizes are more valuable in biological application. Nano-sized particles are particularly interesting in medical and biological fields. They are often used as cell markers, gene transfection reagents or MRI contrast agents to realize the maximization of cellular uptake [17,18]. Several studies have been done on their biocompatibility, cytotoxicity [19–21], blood compatibility [22–24], biodegradability [25,26], biodistribution and excretion [27–30]. The downsides of MSNs, we think they are rigid and exhibit a little aggregation after modification. Furthermore, despite the MSNs' degradability has been reported, they still need more than days for degradation thoroughly. Up to date, there are many reports on MSNs based nanomaterials used in biomedical field. Some popular systems, such as cap systems, have been recently attracted great attentions. Inorganic nanoparticles [31–33] or large molecules (cyclodextrins or rotaxanes) [34–37] have been used as the cap of the pores on the surface of MSNs. These caps are able to release entrapped guest molecules inside MSNs by using diverse physical and chemical stimuli. Some relevant review articles have been also reported [8,10,16]. Here we will mainly introduce the lipids, protein or poly(N-isopropylacrylamide) (poly(NIPAM)) modified MSNs used in biomedical field based on the recent works. These molecules are soft and biocompatible with the unique property, chemical conformation and their own functions [38]. Our group has taken these species to assemble many different biomimetic systems as micro/nano materials towards the biological applications [15,39–48]. Herein, we briefly summarize the above mentioned molecules coated MSN nanocomposites from preparation, property to potential applications. In the preparation section, layer-by-layer assembly, covalent linkage and the electrostatic interaction are described, respectively. Subsequently, biocompatibility, autofluorescence and stimuli responsibility of these nanocomposites are introduced in detail. It demonstrates that these assembled molecules coated MSNs are suitable for the photodynamic therapy, cell imaging, controlled release or selective recognition. 2. Preparation and properties of the functional molecules coated MSNs 2.1. MSNs Up to now, the preparation and characterization of MSNs are already recognized. Typically, they are prepared using a base-catalyzed sol-gel method reported by Cai and Lin's group, respectively [13,49]. As shown in Fig. 1A, the porous structure consists of a series of parallel channels with an average pore diameter of 3 nm that are packed in a 2D hexagonal geometry. These characteristics can also be proved by powder X-ray diffraction. The ordered porous structures enable MSNs to become good drug carriers due to their high surface areas (900– 1500 cm2 g−1) and large pore volumes (0.5–1.5 cm3 g− 1). Furthermore, Slowing et al. also reported that MCM-41 typed MSNs with the sizes ranging from 20 to 500 nm, and with pore sizes ranging from 2 to 6 nm could be synthesized according to the modified methods [50].
MSN materials with various particle morphologies, such as spheres, ellipsoids and rods could also be prepared (Fig. 1B–D) [51]. Zhang et al. also prepared different helical morphological MSN particles by the cocondensation of TEOS and hydrophobic organoalkoxysilane such as 3-mercaptopropyltrimethoxysilane (MPTS), using achiral surfactants as templates [14]. They synthesized the surfactant-extracted samples by using surfactants (either C16TAB or C18TAB) as a template. The morphology and pitch of helical mesostructured silica can be controlled by simply varying the amount of added organoalkoxysilane MPTS. The size and porosity are important for their different biomedical applications. When MSN is used as drug carriers, the adsorption of molecules in MSN is determined by size of inner pores. The mesopore diameters can be tuned from 2 nm to 6 nm which make MSNs to host different sized drug molecules. The pore size also controls the drugrelease rate which has been researched systemically by M. Vallet-Regi et al. [52]. The diameter of MSNs can also affect the interaction between MSNs and cells. Zhao et al. reported interaction of different sized MSNs with human red blood cell (RBC) membranes [53]. They found that small MSNs (ca. 100 nm) were adsorbed to the surface of RBCs without disturbing the membrane while large MSNs (ca. 600 nm) to RBCs induced a strong local membrane deformation leading to eventual hemolysis. They believe that size of MSNs is decisive in attractive interaction between MSNs and RBCs and the bending of the cell membrane. Only small MCM-41-type MSN materials (100–200 nm) may be considered as potentially safe candidates for intravascular drug delivery. In the following introduction, we will mainly consider ca. 100 nm sized spherical mesoporous silica nanoparticles as MSNs except were mentioned especially. 2.2. Lipid coated MSNs Supported lipid bilayers that mimic a cell membrane are well popular model systems for fundamental research and also are critical for the development of new types of biosensor, biodevices and functional materials. On the supported surfaces, lipid bilayer not only can improve the biocompatibility of nanocomposites, but also can be used as a means to attach more biological functionality for them. For instance, due to the amphiphilic character of lipids, on the one hand, hydrophilic compounds can be adsorbed on bilayer surface via hydrophilic interactions, and on the other hand, hydrophobic molecules can be inserted in the bilayer hydrocarbon chain region via hydrophobic effects. Therefore, various micro-/nanomaterials, such as microcapsules [54–56], colloidal particles [57], nanotubes [42,58], and nanopatterns [59,60], were used as supported surface of lipid bilayers for more biofunctions. Predictably, lipid bilayer modified MSNs will not only improve their biocompatibility and biofunctions, but also maintain the properties of MSNs as ideal nanocarrier systems. Therefore, we prepared lipid coated MSNs as photosensitive drug carriers in our recent work [19]. After being calcined, MSNs were dispersed into hypocrellin B (HB, a kind of photosensitive drug) solution for adsorption. Then, vesicles consisting of mixed phospholipids were coated on the surfaces of MSNs. TEMs (Fig. 2 B,D) showed the differences before and after coating phospholipid vesicles. For further investigation, confocal laser scanning microscopy (CLSM) was used to observe the nanocomposites (Fig. 3). From the results, phospholipid mixture can be adsorbed on the surface of MSNs through the higher fluidity of the lipid vesicles, and a subsequent easier fusion with the support [61,62]. The adsorption depends weakly on electrostatic attraction. As drug carriers, lipid coated MSNs also make hydrophobic drug dispersing in water very well. As we anticipate, the lipid layers can improve the cell compatibility of MSN materials. The result from flow cytometry measurement proves that lipid coated MSNs can be more readily identified and internalized by cancer cells than bare MSNs. Similarly, taking advantage of the amphiphilic character of lipids bilayer, we have assembled the thrombin binding aptamer (TBA), anti-cancer drug docetaxel as well as a hydrophilic PEG into the lipids
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Fig. 1. TEM images of MSNs with different morphology prepared by Trewyn et al. (A) spheres, (B) ellipsoids, (C) rods, and (D) tubes. Reprinted with permission from ref. 51 (Copyright 2004 American Chemical Society).
bilayer, and then coated them on the surfaces of MSNs [63]. Such assembled complexes can be used to suppress tumor cell proliferation and release anticancer drug into cell after cell uptake. 2.3. Protein coated MSNs Protein is another native biomolecule which is often used for constructing biomaterials. As we know, for fragile protein, it is difficult to keep them stable during the entire process of assembling biomaterials. Up to now, various methods have been used to construct stable protein films including the Langmuir–Blodgett (LB) technique, the sol-gel method, physical adsorption, and covalent cross-linking strategies, etc [64–67]. In covalent cross-linking strategy, glutaraldehyde (GA) is a common protein-immobilized agent [68]. In our previous work, we made use of GA as a covalent cross-linker to fabricated hemoglobin and glucose oxidase (GOD) microcapsules via the layer-by-layer (LbL) assembly technique [69,70]. Recently, we used this strategy for coating protein, hemoglobin and GOD, on the surface of MSNs with GA via the LbL method. The thickness of protein can be controlled by the number of assembly layers (Fig. 4). As we know, GOD catalyzes the oxidation and hydrolysis of β-Dglucose into gluconic acid and H2O2. In the presence of hemoglobin, H2O2 can react with the fluorogenic reagent Amplex Red to produce fluorescent compound resorufin. Therefore, when Amplex Red and β-D-glucose were added into an MSN@protein dispersed solution, we can detect the enzymatic activity and glucose sensitivity of coupled proteins through the adsorption/emission peak of resorufin at ~ 570/585 nm. The results also proved the hypothesis through real-time monitoring of glucose catalysis at different concentrations
within MSN@protein using fluorescence spectrofluorometer (Fig. 5). More interestingly, the MSN@protein particles presented the feature of autofluorescence without any external fluorochromes. The protein layers were constructed on the surface of MSNs via Schiff's bases reaction between GA and protein. The autofluorescence can be attributed to the n − π* transition of C_N bonds in the Schiff's bases formed during the crosslinking reaction between the amino groups of proteins and the aldehyde groups of GA [71,72]. Since we have proved that the mixture of hemoglobin and GOD cannot present autofluorescence without the cross-linker GA under the same conditions.
2.4. Poly(NIPAM) coated MSNs Poly(NIPAM) is well known as thermosensitive polymer which will undergo phase transition when temperature is changed. Its lower critical solution temperature (LCST) at about 32 °C is close to physiological temperature [73–75]. Several groups have reported that poly(NIPAM) does not present any sign of acute toxicity for some cell lines [76]. Therefore, poly(NIPAM) has often been designed smart biosensors used as a drug delivery material, cell attachment/detachment matrix, and hemostatic agent [77–80]. “Grafting to” and “grafting from” are two typical methods to attach poly(NIPAM) onto substrate by chemical covalent interaction. “Grafting to” method means that end-functionalized polymer chains are attached directly to an appropriate substrate. While in “grafting from” approach, an initiator is modified on the surface of aimed materials. Then, the initiator-modified materials can initiated the polymerization of monomers through living/controlled polymerization, such as the surface initiated atom transfer radical polymerization (ATRP).
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Fig. 2. SEM (A) and TEM (B) of MSN after calcination; TEM (C) of vesicles of lipid mixture; TEM (D) of lipid-MSN-HB. The samples of C and D were stained with 1% phosphotungstic acid. Reprinted with permission from ref. 19 (Copyright 2010 Royal Society of Chemistry).
For instance, You et al. grafted poly(NIPAM) to the surface of the preformed, thiol-functionalized MSN nanoparticles by the “grafting-to” method [81]. At the same time, we reported MSN@poly(NIPAM) core– shell structured nanomaterials by ATRP and the “grafting-from” method [15]. The synthetic process and typical sample are showed in Fig. 6. The synthesized composite nanomaterial has both a mesoporous silica core and a thermosensitive poly(NIPAM) shell. The ATRP technique can incorporate more condensed polymers onto the substrate surfaces compared to the “grafting-to” method. Polymer layers on the particle surface are more uniform with controllable thickness, while the inner channels remained. The measured LCST of MSN@poly(NIPAM) material is about 32 °C, which is consistent with pure poly(NIPAM) in water. 3. Potential applications and outlooks As mentioned above, such functional molecular/MSN nanocomposites provide new type of intelligent materials with core–shell structures. The soft organic layer endows MSNs with a novel functionality, while polyporus inorganic core makes nanocomposite more stable and also adsorbs more drugs when being used as drug carriers. Therefore, these organic–inorganic nanomaterials can be looked as novel nanotanks or nanocarriers in biomedical field. The application of such nanocomposites
might be a favorable resolution to some novel challenges in controlled drug delivery in intelligent therapeutic system, molecular recognition, bioimaging and so on.
3.1. In photodynamic therapy Photodynamic therapy (PDT) is an effective and selective means of suppressing diseased tissues without altering the surrounding healthy tissue. It is based on the systemic or topical administration of a photosensitive drug which is also known as a photosensitizer (PS) [82,83]. Light activated PS can generate reactive oxygen species which can irreversibly damage the treated tissues [84]. However, most PS molecules are hydrophobic and can aggregate easily in aqueous media, where PS aggregation will result in a decrease of their quantum yield and damage to healthy cells [85]. MSNs may be ideal PS carriers for PDT because they are hydrophilic and mono-dispersed. Nano-sized particles can also penetrate deep into tissues and be taken up efficiently by cancer cells. Especially for MSNs, they possess ordered porous structures and molecular oxygen can diffuse through the pores and interact with the PS loaded into the MSN carriers. The photogenerated reactive oxygen can diffuse out of the particle to generate the cytotoxic effect.
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Fig. 3. CLSM of lipid-MSN-HB particles in solution. The corresponding images of the HB (red) (A); NBD-PC in lipid mixture (green) (B); the overlapped image (C) and the pseudo-bright field image (D). Reprinted with permission from ref. 19 (Copyright 2010 Royal Society of Chemistry).
To take one example, lipid coated MSNs have been used as photosensitive drug (HB) carriers for photodynamic experiment in vitro [19]. With the lipid modification, the carriers can be internalized more easily by MCF-7 cells (human breast carcinoma cells) through an endocytic mechanism (Fig. 7). These intracellular HB-loaded lipid– MSNs show biocompatibility under darkness and high cytotoxicity after irradiation which provides the possibility of controlled delivery and release of drugs. The results indicate MSN based materials can be used as good photosensitizer carriers in photodynamic therapy. There will be further opportunity to use these materials for photodetection.
MSNs were found to be either adsorbed or embedded into Hela cell membranes (Fig. 8 A–D). When another cell line (ESF cells) was selected, we found that MSN@protein also presented to be cell membrane friendly (Fig. 8 E–H). As is well known, cell membranes consist of lipid bilayers and membrane proteins. In this work, the system of MSN@protein might be more favorable to the structure of the cell membrane and internalized by the cell membrane. These unique features make this nanocomposite a good material as cell marker for cell imaging.
3.2. In cell imaging
A facile functionalized surface of MSNs combined with the ATRP method provides a new strategy to construct stimuli-responsive polymer/MSN nanocomposites for controlled release. Recently, Yu and Sun et al. prepared pH sensitive poly(N,N-dimethylaminoethyl methacrylate) coated MSNs through the ATRP method for pH-responsive controlled release [86,87]. Another instance, poly(NIPAM) coated MSNs reported by us can also be used in thermosensitive controlled release system [15]. In detail, a fluorescent molecule, FITC, was used as model guest molecule to test the encapsulation ability of the MSN@ Poly(NIPAM) particles. FITC can be entrapped by the stretched polymer chains at 20 °C and be locked inside MSN at 40 °C. After that, FITC will
As we introduced before, nano-sized particles were particularly significant when they were used as cell markers to realize the maximization of cellular uptake. In our several work about biomolecules modified MSNs, most of composite MSNs presented good cell biocompatibility [15,19,20,61]. They could be readily attached onto the cell surface or internalized into the cells according to the uptake mechanism. For example, GA crosslinked protein (hemoglobin/GOD) coated MSNs present autofluorescent properties which make them become good carrier candidate to be used in biological tracing. Furthermore, this protein coated
3.3. In controlled release
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Fig. 4. TEM images of protein coated MSNs by different assembly layer numbers. (A) MSN; (B) MSN@(hemoglobin/GOD); (C) MSN@(hemoglobin/GOD)2; (D) MSN@(hemoglobinx/GOD)3. Reprinted with permission from ref. 20 (Copyright 2011 Royal Society of Chemistry).
release again slowly from MSN@Poly(NIPAM) at 20 °C, but not at 40 °C (Fig. 9). The polymer chains on the surface of MSN will be stretched below LCST which let guest molecule go into pores freely. However, the chains will collapse and block guest molecule from being release
when raising the temperature above LCST. This process is reversible which proves that the systems can be used in DDSs. What's more, the composite materials can be internalized into MCF-7 cells easily which make it a promising material for application in drug carriers.
Fig. 5. (A) Real-time monitoring of glucose catalysis at different concentrations within MSN@protein to analyze glucose consumption with respect to time using fluorescence spectrofluorometer. (B) Fluorescence intensity increased with the concentration of glucose at 585 nm after 10 min for glucose consumption. Reprinted with permission from ref. 20 (Copyright 2011 Royal Society of Chemistry).
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Fig. 6. A schematic illustration of the surface functionalization for MSN and the fabrication of MSN@Poly(NIPAM) and TEM of MSN@Poly(NIPAM) through the “graft-from” method. Reprinted with permission from ref. 15 (Copyright 2008 Royal Society of Chemistry).
Fig. 7. CLSM of MCF-7 cells and lipid-MSN-HB suspensions after 12 h of co-culturing. The corresponding images show the NBD-PC in lipid mixture (green) (A) and the HB (red) (B) taken up by the cells; the overlapped image (C) and the pseudo-bright field image (D). (E) MCF-7 cells after incubation in lipid-MSN in the presence of the FM 4-64. The main image shows the area of xy section, while the white and yellow lines indicate the area of the xz and yz sections, respectively. The image shows the lipid-MSN particles surrounded by cell membrane (red). Reprinted with permission from ref. 19 (Copyright 2010 Royal Society of Chemistry).
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Fig. 8. CLSM images of Hela cells (A–D) and ESF cells (E–H) stained with FM 4-64 and MSN@protein suspensions after 12 h co-culturing. The corresponding images of the MSN@protein nanoparticles (green) (A, E); FM 4-64 labeled cell membranes (red) (B, F); the overlapped image (C, G) and the pseudo-bright field image (D, H). (in Fig. E–G, blue fluorescence come from cell nucleus marker, Hoechst 33342). Reprinted with permission from ref. 20 (Copyright 2011 Royal Society of Chemistry).
3.4. In selective recognition Recent attention has been devoted to the design of nanodevices with highly efficient ligands that selectively recognize tumor-associated or tumor-specific antigens [88]. Nucleic acid ligand (aptamer) is a novel class of targeting molecules for therapeutic and diagnostic applications [89]. For example, 15-mer thrombin binding aptamer (TBA) is such targeting molecules which can inhibit the enzymatic function of thrombin [90–92]. The significant neoplastic biological effect of thrombin involves clotting-dependent mechanism and protease-activated receptor-1 (PAR-1) related signaling; this leads to several tumor functions, specifically proliferation and angiogenesis [93–96]. In our work, we used TBA-tethered lipid-coated MSN composite as an extra and intracellular anticancer nanocarrier [63]. The composites comprise MSN as support core, mixed lipid layers with the incorporation of thrombin
binding aptamer and anticancer drug docetaxel as well as a hydrophilic PEG shell. Two approaches are involved in the whole tumor cell proliferation suppression process. Firstly, TBAA15 recombined on the bioconjugate selectively recognizes thrombin and inhibits its proteolytic capacity in the extracellular surroundings which results in interference of signal transduction pathways activated by the interaction of thrombin and PAR-1. Furthermore, incorporated docetaxel releases into cytoplasm where it triggered higher cellular cytotoxicity. The hybrid system constructed will be extended for combined anticancer treatment related to PAR-1 overexpressed. Conclusions We have provided a brief overview of the immobilization of the functional molecules on the MSNs' surfaces by layer-by-layer methods, covalent linkage or electrostatic interaction. The composite nanomaterials create various possibilities where both sophisticated functions of the molecules shells and mechanical stability of MSN cores are fulfilled. It is clear from the introduced work that functional molecules coated MSNs are stable and biocompatible. Moreover, such nanocomposites display much potential applications in biomedical field, such as photodynamic therapy, cell imaging, selective recognition and so on. These strategies might be universal methods for constructing hybrid organic–inorganic nanomaterials which can be widely applied in biomedical field. Acknowledgments The authors acknowledge the financial support of this work by the National Basic Research Program of China (973 Program 2009CB930100, 2013CB932800) and the National Nature Science Foundation of China (21003027, 21273055). References
Fig. 9. FITC release from MSN@Poly(NIPAM) at different cycle times which was recorded by UV absorbance at 480 nm. The fresh water was replaced and FITC loaded MSN@ Poly(NIPAM) particles were redispersed at 20 °C (▲) or 40 °C (■) for 15 min. The inset image illustrates the whole process. Reprinted with permission from ref. 15 (Copyright 2008 Royal Society of Chemistry).
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Lipid, protein and poly(NIPAM) coated mesoporous silica nanoparticles for biomedical applications Yang Yang a,⁎, Junbai Li a,b,⁎⁎ a
National Center for Nanoscience and Technology, Beijing 100190, China Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
b
a r t i c l e
i n f o
Available online 8 November 2013 Keywords: Lipids Protein Poly(NIPAM) Mesoporous silica nanoparticles Biomedical application
a b s t r a c t In the past decade, mesoporous silica nanoparticles (MSNs) as nanocarriers have showed much potential in advanced nanomaterials due to their large surface area and pore volume. Especially, more and more MSNs based nanodevices have been designed as efficient drug delivery systems (DDSs) or biosensors. In this paper, lipid, protein and poly(NIPAM) coated MSNs are reviewed from the preparation, properties and their potential application. We also introduce the preparative methods including physical adsorption, covalent binding and self-assembly on the MSNs' surfaces. Furthermore, the interaction between the aimed cells and these molecular modified MSNs is discussed. We also demonstrate their typical applications, such as photodynamic therapy, bioimaging, controlled release and selective recognition in biomedical field. © 2013 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Preparation and properties of the functional molecules coated MSNs 2.1. MSNs . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Lipid coated MSNs . . . . . . . . . . . . . . . . . . . . . 2.3. Protein coated MSNs . . . . . . . . . . . . . . . . . . . . 2.4. Poly(NIPAM) coated MSNs . . . . . . . . . . . . . . . . . 3. Potential applications and outlooks . . . . . . . . . . . . . . . . . 3.1. In photodynamic therapy . . . . . . . . . . . . . . . . . . 3.2. In cell imaging . . . . . . . . . . . . . . . . . . . . . . . 3.3. In controlled release . . . . . . . . . . . . . . . . . . . . 3.4. In selective recognition . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
⁎ Correspondence to: Y. Yang, National Center for Nanoscience and Technology, Zhong Guan Cun, Bei Yi Tiao No. 11, Beijing 100190, China. Tel.: +86 10 8254 5540; fax: +86 10 8261 2629. ⁎⁎ Correspondence to: J. Li, Institute of Chemistry, Chinese Academy of Sciences, Zhong Guan Cun, Bei Yi Jie No. 2, Beijing 100190, China. Tel.: +86 10 8261 4087; fax: +86 10 8261 2629. E-mail addresses: [email protected] (Y. Yang), [email protected] (J. Li). 0001-8686/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cis.2013.10.029
After the discovery of highly ordered mesoporous silica materials by the Mobil Corporation in 1992 [1,2], the preparation of mesoporous silica-based materials has become highly attractive due to their potential applications in the fields of catalysis, lasers, sensors and environmental application [3–7]. In the past decade, more and more mesoporous silica based composite materials were designed to apply in biomedical fields [8–11] since they were first reported as drug delivery systems (DDSs)
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[12]. Several features result in mesoporous silica materials as good carriers in biomedical application. Firstly, they have the ordered pore network and homogeneous size for the drug loading; secondly, high pore volume and surface area will host the required amount of drug molecules; most importantly, a silanol-containing functionalized surface allows them to be modified easily to control over drug loading and release. Mesoporous silica materials made by the original procedures are mainly mesoporous silica sheets with disorganized morphologies. For increasing their biocompatibility, significant research efforts have been made towards the smaller size and mono-dispersity. In 2001, Cai et al. firstly obtained MCM-41 typed mesoporous silica nanoparticles (MSNs) with 100 nm by using a dilute surfactant solution [13]. Later, various MSNs with well-defined and controllable particle morphology were developed by Lin's and other research groups in the pursuit of biocompatible materials used in controlled release and drug delivery systems [14–16]. These mesoporous silica materials with nanosizes are more valuable in biological application. Nano-sized particles are particularly interesting in medical and biological fields. They are often used as cell markers, gene transfection reagents or MRI contrast agents to realize the maximization of cellular uptake [17,18]. Several studies have been done on their biocompatibility, cytotoxicity [19–21], blood compatibility [22–24], biodegradability [25,26], biodistribution and excretion [27–30]. The downsides of MSNs, we think they are rigid and exhibit a little aggregation after modification. Furthermore, despite the MSNs' degradability has been reported, they still need more than days for degradation thoroughly. Up to date, there are many reports on MSNs based nanomaterials used in biomedical field. Some popular systems, such as cap systems, have been recently attracted great attentions. Inorganic nanoparticles [31–33] or large molecules (cyclodextrins or rotaxanes) [34–37] have been used as the cap of the pores on the surface of MSNs. These caps are able to release entrapped guest molecules inside MSNs by using diverse physical and chemical stimuli. Some relevant review articles have been also reported [8,10,16]. Here we will mainly introduce the lipids, protein or poly(N-isopropylacrylamide) (poly(NIPAM)) modified MSNs used in biomedical field based on the recent works. These molecules are soft and biocompatible with the unique property, chemical conformation and their own functions [38]. Our group has taken these species to assemble many different biomimetic systems as micro/nano materials towards the biological applications [15,39–48]. Herein, we briefly summarize the above mentioned molecules coated MSN nanocomposites from preparation, property to potential applications. In the preparation section, layer-by-layer assembly, covalent linkage and the electrostatic interaction are described, respectively. Subsequently, biocompatibility, autofluorescence and stimuli responsibility of these nanocomposites are introduced in detail. It demonstrates that these assembled molecules coated MSNs are suitable for the photodynamic therapy, cell imaging, controlled release or selective recognition. 2. Preparation and properties of the functional molecules coated MSNs 2.1. MSNs Up to now, the preparation and characterization of MSNs are already recognized. Typically, they are prepared using a base-catalyzed sol-gel method reported by Cai and Lin's group, respectively [13,49]. As shown in Fig. 1A, the porous structure consists of a series of parallel channels with an average pore diameter of 3 nm that are packed in a 2D hexagonal geometry. These characteristics can also be proved by powder X-ray diffraction. The ordered porous structures enable MSNs to become good drug carriers due to their high surface areas (900– 1500 cm2 g−1) and large pore volumes (0.5–1.5 cm3 g− 1). Furthermore, Slowing et al. also reported that MCM-41 typed MSNs with the sizes ranging from 20 to 500 nm, and with pore sizes ranging from 2 to 6 nm could be synthesized according to the modified methods [50].
MSN materials with various particle morphologies, such as spheres, ellipsoids and rods could also be prepared (Fig. 1B–D) [51]. Zhang et al. also prepared different helical morphological MSN particles by the cocondensation of TEOS and hydrophobic organoalkoxysilane such as 3-mercaptopropyltrimethoxysilane (MPTS), using achiral surfactants as templates [14]. They synthesized the surfactant-extracted samples by using surfactants (either C16TAB or C18TAB) as a template. The morphology and pitch of helical mesostructured silica can be controlled by simply varying the amount of added organoalkoxysilane MPTS. The size and porosity are important for their different biomedical applications. When MSN is used as drug carriers, the adsorption of molecules in MSN is determined by size of inner pores. The mesopore diameters can be tuned from 2 nm to 6 nm which make MSNs to host different sized drug molecules. The pore size also controls the drugrelease rate which has been researched systemically by M. Vallet-Regi et al. [52]. The diameter of MSNs can also affect the interaction between MSNs and cells. Zhao et al. reported interaction of different sized MSNs with human red blood cell (RBC) membranes [53]. They found that small MSNs (ca. 100 nm) were adsorbed to the surface of RBCs without disturbing the membrane while large MSNs (ca. 600 nm) to RBCs induced a strong local membrane deformation leading to eventual hemolysis. They believe that size of MSNs is decisive in attractive interaction between MSNs and RBCs and the bending of the cell membrane. Only small MCM-41-type MSN materials (100–200 nm) may be considered as potentially safe candidates for intravascular drug delivery. In the following introduction, we will mainly consider ca. 100 nm sized spherical mesoporous silica nanoparticles as MSNs except were mentioned especially. 2.2. Lipid coated MSNs Supported lipid bilayers that mimic a cell membrane are well popular model systems for fundamental research and also are critical for the development of new types of biosensor, biodevices and functional materials. On the supported surfaces, lipid bilayer not only can improve the biocompatibility of nanocomposites, but also can be used as a means to attach more biological functionality for them. For instance, due to the amphiphilic character of lipids, on the one hand, hydrophilic compounds can be adsorbed on bilayer surface via hydrophilic interactions, and on the other hand, hydrophobic molecules can be inserted in the bilayer hydrocarbon chain region via hydrophobic effects. Therefore, various micro-/nanomaterials, such as microcapsules [54–56], colloidal particles [57], nanotubes [42,58], and nanopatterns [59,60], were used as supported surface of lipid bilayers for more biofunctions. Predictably, lipid bilayer modified MSNs will not only improve their biocompatibility and biofunctions, but also maintain the properties of MSNs as ideal nanocarrier systems. Therefore, we prepared lipid coated MSNs as photosensitive drug carriers in our recent work [19]. After being calcined, MSNs were dispersed into hypocrellin B (HB, a kind of photosensitive drug) solution for adsorption. Then, vesicles consisting of mixed phospholipids were coated on the surfaces of MSNs. TEMs (Fig. 2 B,D) showed the differences before and after coating phospholipid vesicles. For further investigation, confocal laser scanning microscopy (CLSM) was used to observe the nanocomposites (Fig. 3). From the results, phospholipid mixture can be adsorbed on the surface of MSNs through the higher fluidity of the lipid vesicles, and a subsequent easier fusion with the support [61,62]. The adsorption depends weakly on electrostatic attraction. As drug carriers, lipid coated MSNs also make hydrophobic drug dispersing in water very well. As we anticipate, the lipid layers can improve the cell compatibility of MSN materials. The result from flow cytometry measurement proves that lipid coated MSNs can be more readily identified and internalized by cancer cells than bare MSNs. Similarly, taking advantage of the amphiphilic character of lipids bilayer, we have assembled the thrombin binding aptamer (TBA), anti-cancer drug docetaxel as well as a hydrophilic PEG into the lipids
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Fig. 1. TEM images of MSNs with different morphology prepared by Trewyn et al. (A) spheres, (B) ellipsoids, (C) rods, and (D) tubes. Reprinted with permission from ref. 51 (Copyright 2004 American Chemical Society).
bilayer, and then coated them on the surfaces of MSNs [63]. Such assembled complexes can be used to suppress tumor cell proliferation and release anticancer drug into cell after cell uptake. 2.3. Protein coated MSNs Protein is another native biomolecule which is often used for constructing biomaterials. As we know, for fragile protein, it is difficult to keep them stable during the entire process of assembling biomaterials. Up to now, various methods have been used to construct stable protein films including the Langmuir–Blodgett (LB) technique, the sol-gel method, physical adsorption, and covalent cross-linking strategies, etc [64–67]. In covalent cross-linking strategy, glutaraldehyde (GA) is a common protein-immobilized agent [68]. In our previous work, we made use of GA as a covalent cross-linker to fabricated hemoglobin and glucose oxidase (GOD) microcapsules via the layer-by-layer (LbL) assembly technique [69,70]. Recently, we used this strategy for coating protein, hemoglobin and GOD, on the surface of MSNs with GA via the LbL method. The thickness of protein can be controlled by the number of assembly layers (Fig. 4). As we know, GOD catalyzes the oxidation and hydrolysis of β-Dglucose into gluconic acid and H2O2. In the presence of hemoglobin, H2O2 can react with the fluorogenic reagent Amplex Red to produce fluorescent compound resorufin. Therefore, when Amplex Red and β-D-glucose were added into an MSN@protein dispersed solution, we can detect the enzymatic activity and glucose sensitivity of coupled proteins through the adsorption/emission peak of resorufin at ~ 570/585 nm. The results also proved the hypothesis through real-time monitoring of glucose catalysis at different concentrations
within MSN@protein using fluorescence spectrofluorometer (Fig. 5). More interestingly, the MSN@protein particles presented the feature of autofluorescence without any external fluorochromes. The protein layers were constructed on the surface of MSNs via Schiff's bases reaction between GA and protein. The autofluorescence can be attributed to the n − π* transition of C_N bonds in the Schiff's bases formed during the crosslinking reaction between the amino groups of proteins and the aldehyde groups of GA [71,72]. Since we have proved that the mixture of hemoglobin and GOD cannot present autofluorescence without the cross-linker GA under the same conditions.
2.4. Poly(NIPAM) coated MSNs Poly(NIPAM) is well known as thermosensitive polymer which will undergo phase transition when temperature is changed. Its lower critical solution temperature (LCST) at about 32 °C is close to physiological temperature [73–75]. Several groups have reported that poly(NIPAM) does not present any sign of acute toxicity for some cell lines [76]. Therefore, poly(NIPAM) has often been designed smart biosensors used as a drug delivery material, cell attachment/detachment matrix, and hemostatic agent [77–80]. “Grafting to” and “grafting from” are two typical methods to attach poly(NIPAM) onto substrate by chemical covalent interaction. “Grafting to” method means that end-functionalized polymer chains are attached directly to an appropriate substrate. While in “grafting from” approach, an initiator is modified on the surface of aimed materials. Then, the initiator-modified materials can initiated the polymerization of monomers through living/controlled polymerization, such as the surface initiated atom transfer radical polymerization (ATRP).
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Fig. 2. SEM (A) and TEM (B) of MSN after calcination; TEM (C) of vesicles of lipid mixture; TEM (D) of lipid-MSN-HB. The samples of C and D were stained with 1% phosphotungstic acid. Reprinted with permission from ref. 19 (Copyright 2010 Royal Society of Chemistry).
For instance, You et al. grafted poly(NIPAM) to the surface of the preformed, thiol-functionalized MSN nanoparticles by the “grafting-to” method [81]. At the same time, we reported MSN@poly(NIPAM) core– shell structured nanomaterials by ATRP and the “grafting-from” method [15]. The synthetic process and typical sample are showed in Fig. 6. The synthesized composite nanomaterial has both a mesoporous silica core and a thermosensitive poly(NIPAM) shell. The ATRP technique can incorporate more condensed polymers onto the substrate surfaces compared to the “grafting-to” method. Polymer layers on the particle surface are more uniform with controllable thickness, while the inner channels remained. The measured LCST of MSN@poly(NIPAM) material is about 32 °C, which is consistent with pure poly(NIPAM) in water. 3. Potential applications and outlooks As mentioned above, such functional molecular/MSN nanocomposites provide new type of intelligent materials with core–shell structures. The soft organic layer endows MSNs with a novel functionality, while polyporus inorganic core makes nanocomposite more stable and also adsorbs more drugs when being used as drug carriers. Therefore, these organic–inorganic nanomaterials can be looked as novel nanotanks or nanocarriers in biomedical field. The application of such nanocomposites
might be a favorable resolution to some novel challenges in controlled drug delivery in intelligent therapeutic system, molecular recognition, bioimaging and so on.
3.1. In photodynamic therapy Photodynamic therapy (PDT) is an effective and selective means of suppressing diseased tissues without altering the surrounding healthy tissue. It is based on the systemic or topical administration of a photosensitive drug which is also known as a photosensitizer (PS) [82,83]. Light activated PS can generate reactive oxygen species which can irreversibly damage the treated tissues [84]. However, most PS molecules are hydrophobic and can aggregate easily in aqueous media, where PS aggregation will result in a decrease of their quantum yield and damage to healthy cells [85]. MSNs may be ideal PS carriers for PDT because they are hydrophilic and mono-dispersed. Nano-sized particles can also penetrate deep into tissues and be taken up efficiently by cancer cells. Especially for MSNs, they possess ordered porous structures and molecular oxygen can diffuse through the pores and interact with the PS loaded into the MSN carriers. The photogenerated reactive oxygen can diffuse out of the particle to generate the cytotoxic effect.
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Fig. 3. CLSM of lipid-MSN-HB particles in solution. The corresponding images of the HB (red) (A); NBD-PC in lipid mixture (green) (B); the overlapped image (C) and the pseudo-bright field image (D). Reprinted with permission from ref. 19 (Copyright 2010 Royal Society of Chemistry).
To take one example, lipid coated MSNs have been used as photosensitive drug (HB) carriers for photodynamic experiment in vitro [19]. With the lipid modification, the carriers can be internalized more easily by MCF-7 cells (human breast carcinoma cells) through an endocytic mechanism (Fig. 7). These intracellular HB-loaded lipid– MSNs show biocompatibility under darkness and high cytotoxicity after irradiation which provides the possibility of controlled delivery and release of drugs. The results indicate MSN based materials can be used as good photosensitizer carriers in photodynamic therapy. There will be further opportunity to use these materials for photodetection.
MSNs were found to be either adsorbed or embedded into Hela cell membranes (Fig. 8 A–D). When another cell line (ESF cells) was selected, we found that MSN@protein also presented to be cell membrane friendly (Fig. 8 E–H). As is well known, cell membranes consist of lipid bilayers and membrane proteins. In this work, the system of MSN@protein might be more favorable to the structure of the cell membrane and internalized by the cell membrane. These unique features make this nanocomposite a good material as cell marker for cell imaging.
3.2. In cell imaging
A facile functionalized surface of MSNs combined with the ATRP method provides a new strategy to construct stimuli-responsive polymer/MSN nanocomposites for controlled release. Recently, Yu and Sun et al. prepared pH sensitive poly(N,N-dimethylaminoethyl methacrylate) coated MSNs through the ATRP method for pH-responsive controlled release [86,87]. Another instance, poly(NIPAM) coated MSNs reported by us can also be used in thermosensitive controlled release system [15]. In detail, a fluorescent molecule, FITC, was used as model guest molecule to test the encapsulation ability of the MSN@ Poly(NIPAM) particles. FITC can be entrapped by the stretched polymer chains at 20 °C and be locked inside MSN at 40 °C. After that, FITC will
As we introduced before, nano-sized particles were particularly significant when they were used as cell markers to realize the maximization of cellular uptake. In our several work about biomolecules modified MSNs, most of composite MSNs presented good cell biocompatibility [15,19,20,61]. They could be readily attached onto the cell surface or internalized into the cells according to the uptake mechanism. For example, GA crosslinked protein (hemoglobin/GOD) coated MSNs present autofluorescent properties which make them become good carrier candidate to be used in biological tracing. Furthermore, this protein coated
3.3. In controlled release
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Fig. 4. TEM images of protein coated MSNs by different assembly layer numbers. (A) MSN; (B) MSN@(hemoglobin/GOD); (C) MSN@(hemoglobin/GOD)2; (D) MSN@(hemoglobinx/GOD)3. Reprinted with permission from ref. 20 (Copyright 2011 Royal Society of Chemistry).
release again slowly from MSN@Poly(NIPAM) at 20 °C, but not at 40 °C (Fig. 9). The polymer chains on the surface of MSN will be stretched below LCST which let guest molecule go into pores freely. However, the chains will collapse and block guest molecule from being release
when raising the temperature above LCST. This process is reversible which proves that the systems can be used in DDSs. What's more, the composite materials can be internalized into MCF-7 cells easily which make it a promising material for application in drug carriers.
Fig. 5. (A) Real-time monitoring of glucose catalysis at different concentrations within MSN@protein to analyze glucose consumption with respect to time using fluorescence spectrofluorometer. (B) Fluorescence intensity increased with the concentration of glucose at 585 nm after 10 min for glucose consumption. Reprinted with permission from ref. 20 (Copyright 2011 Royal Society of Chemistry).
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Fig. 6. A schematic illustration of the surface functionalization for MSN and the fabrication of MSN@Poly(NIPAM) and TEM of MSN@Poly(NIPAM) through the “graft-from” method. Reprinted with permission from ref. 15 (Copyright 2008 Royal Society of Chemistry).
Fig. 7. CLSM of MCF-7 cells and lipid-MSN-HB suspensions after 12 h of co-culturing. The corresponding images show the NBD-PC in lipid mixture (green) (A) and the HB (red) (B) taken up by the cells; the overlapped image (C) and the pseudo-bright field image (D). (E) MCF-7 cells after incubation in lipid-MSN in the presence of the FM 4-64. The main image shows the area of xy section, while the white and yellow lines indicate the area of the xz and yz sections, respectively. The image shows the lipid-MSN particles surrounded by cell membrane (red). Reprinted with permission from ref. 19 (Copyright 2010 Royal Society of Chemistry).
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Fig. 8. CLSM images of Hela cells (A–D) and ESF cells (E–H) stained with FM 4-64 and MSN@protein suspensions after 12 h co-culturing. The corresponding images of the MSN@protein nanoparticles (green) (A, E); FM 4-64 labeled cell membranes (red) (B, F); the overlapped image (C, G) and the pseudo-bright field image (D, H). (in Fig. E–G, blue fluorescence come from cell nucleus marker, Hoechst 33342). Reprinted with permission from ref. 20 (Copyright 2011 Royal Society of Chemistry).
3.4. In selective recognition Recent attention has been devoted to the design of nanodevices with highly efficient ligands that selectively recognize tumor-associated or tumor-specific antigens [88]. Nucleic acid ligand (aptamer) is a novel class of targeting molecules for therapeutic and diagnostic applications [89]. For example, 15-mer thrombin binding aptamer (TBA) is such targeting molecules which can inhibit the enzymatic function of thrombin [90–92]. The significant neoplastic biological effect of thrombin involves clotting-dependent mechanism and protease-activated receptor-1 (PAR-1) related signaling; this leads to several tumor functions, specifically proliferation and angiogenesis [93–96]. In our work, we used TBA-tethered lipid-coated MSN composite as an extra and intracellular anticancer nanocarrier [63]. The composites comprise MSN as support core, mixed lipid layers with the incorporation of thrombin
binding aptamer and anticancer drug docetaxel as well as a hydrophilic PEG shell. Two approaches are involved in the whole tumor cell proliferation suppression process. Firstly, TBAA15 recombined on the bioconjugate selectively recognizes thrombin and inhibits its proteolytic capacity in the extracellular surroundings which results in interference of signal transduction pathways activated by the interaction of thrombin and PAR-1. Furthermore, incorporated docetaxel releases into cytoplasm where it triggered higher cellular cytotoxicity. The hybrid system constructed will be extended for combined anticancer treatment related to PAR-1 overexpressed. Conclusions We have provided a brief overview of the immobilization of the functional molecules on the MSNs' surfaces by layer-by-layer methods, covalent linkage or electrostatic interaction. The composite nanomaterials create various possibilities where both sophisticated functions of the molecules shells and mechanical stability of MSN cores are fulfilled. It is clear from the introduced work that functional molecules coated MSNs are stable and biocompatible. Moreover, such nanocomposites display much potential applications in biomedical field, such as photodynamic therapy, cell imaging, selective recognition and so on. These strategies might be universal methods for constructing hybrid organic–inorganic nanomaterials which can be widely applied in biomedical field. Acknowledgments The authors acknowledge the financial support of this work by the National Basic Research Program of China (973 Program 2009CB930100, 2013CB932800) and the National Nature Science Foundation of China (21003027, 21273055). References
Fig. 9. FITC release from MSN@Poly(NIPAM) at different cycle times which was recorded by UV absorbance at 480 nm. The fresh water was replaced and FITC loaded MSN@ Poly(NIPAM) particles were redispersed at 20 °C (▲) or 40 °C (■) for 15 min. The inset image illustrates the whole process. Reprinted with permission from ref. 15 (Copyright 2008 Royal Society of Chemistry).
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