Biocompatibility of Mesoporous Silica Nanoparticles - ACS Publications

Review pubs.acs.org/crt

Biocompatibility of Mesoporous Silica Nanoparticles Tewodros Asefa*,†,‡ and Zhimin Tao*,§ †

Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, New Jersey 08854, United States ‡ Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, 98 Brett Road, Piscataway, New Jersey 08854, United States § Department of Physics, Tsinghua University, Beijing 100084, China ABSTRACT: In this review, recent reports on the biocompatibility of mesoporous silica nanoparticles (MSNs) are reviewed, with special emphasis being paid to the correlations between MSNs’ structural and compositional features and their biological effects on various cells and tissues. First, the different synthetic routes used to produce the most common types of MSNs and the various methods employed to functionalize their surfaces are discussed. This is, however, done only briefly because of the focus of the review being the biocompatibility of the materials. Similarly, the biological applications of MSNs in areas such as drug and gene delivery, biocatalysis, bioimaging, and biosensing are briefly introduced. Many examples have also been mentioned about the biological applications of MSNs while discussing the materials’ biocompatibility. The cytotoxicity of different types of MSNs and the effects of their various structural characteristics on their biological activities, which are the focus of this review, are then described in detail. In addition, synthetic strategies developed to reduce or eliminate any possible negative biological effects associated with MSNs or to improve their biocompatibility, as necessary, are illustrated. At the same time, recent reports on the interactions between MSNs and various in vivo or in vitro biological systems, plus our opinions and remarks on what the future may hold for this field, are included.

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CONTENTS

1. Introduction 2. Synthesis and Functionalization of MSNs 2.1. Synthetic Mechanism of MSNs and their General Properties 2.2. Functionalization Strategies of MSNs 2.2.1. Stepwise Synthesis (or Post-Synthetic Grafting Method) 2.2.2. One-Pot Synthesis (or Co-Condensation Synthetic Method) 2.2.3. General Properties of Surface-Functionalized MSNs 3. Biological Applications of MSNs 4. Biocompatibility of MSNs 4.1. Overview 4.2. Effect of Particle Size 4.3. Effect of Particle Morphology 4.4. Effect of Mesoporosity and Pore Sizes 4.5. Effect of Surface Property 4.6. Effect of Cell Type 4.7. Effect of Methodology of Toxicity Assessment 5. Conclusions and Perspectives Author Information Corresponding Author

© 2012 American Chemical Society

Funding Notes Abbreviations References

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1. INTRODUCTION Because of their unique physical and chemical properties as well as potential biomedical applications, nanomaterials have long become the subject of intense research worldwide. Over the past two decades, synthetic routes to numerous nanosized particles that possess different chemical compositions and physical characteristics and that can induce a diverse range of biological effects have been developed.1−9 Among many nanosized materials, a class of nanomaterials named mesoporous silica nanoparticles (MSNs) stands out. MSNs have tunable nanoscale sizes, different shapes ranging from spheres to rods, uniform cylindrical mesopores, high surface areas, and easily functionalizable surfaces. Owing to these interesting structural features, the potential applications of MSNs as effective delivery vehicles for pharmaceuticals and bioactive molecules (e.g., nucleotides) to desired intracellular sites or as

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Received: April 16, 2012 Published: July 23, 2012 2265

dx.doi.org/10.1021/tx300166u | Chem. Res. Toxicol. 2012, 25, 2265−2284

Chemical Research in Toxicology

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Figure 1. Proposed LCT synthetic mechanism leading to MCM-41.10,11 The synthetic mechanism can go through two possible pathways as stated in the text. Reprinted from ref 11. Copyright 1992 American Chemical Society.

ibility of these nanomaterials still a subject of intense debate.35−50 Understanding the compatibility of MSNs within a biological context requires knowledge of not only the physicochemical characteristics of the nanomaterials but also the specific conditions in which the bio-nano interactions take place.51−53 On the one hand, MSNs come with different particle dimensions, shapes, pore sizes, surface topography, crystallinity, and mesoporosity as well as functional groups, while at the same time many more new ones are being reported. In addition, slight variations in the existing synthetic procedures and postsynthetic modifications of MSNs can result in an array of MSNs with different structural and surface properties. On the other hand, the complexity of biological conditions under which the nanoparticles are applied can also lead to a range of unpredictable biological responses to MSNs. Many of the biocompatibility issues associated with MSNs, or nanomaterials in general, are mainly to do with whether and how the nanoscale materials interfere with a variety of biological processes. At a cellular level, the potential cytotoxicities of MSNs could result from intracellular injuries caused by the interaction of MSNs with the biological systems through various complex mechanisms, including membrane peroxidation, glutathione depletion, mitochondrial dysfunction, and/or DNA damage. How specifically these processes are impacted by MSNs or how MSNs interact with biological systems varies depending on the type of cell or MSN in question. Moreover, the possible toxicity of a given MSN after the nanoparticle enters the animal’s or the human body could manifest itself in the form of impairments in lung, brain, skin, blood circulation, immune system, etc. Thus, putting together all of these variables involving the materials as well as the biological systems, a complex set of biocompatibility scenarios for these nanosized particles (i.e., MSNs) can be expected. Nevertheless, despite these complexities, because of the promising applications of MSNs in biological and medical fields, it is still vital to obtain sufficient information about their possible biocompatibility and the factors that affect their biocompatibility in order for these materials to be further advanced for clinical use. In this review, we first introduce the synthetic routes used to produce MSNs and the surface functionalization methods employed to modify their surfaces and compositions. We then briefly illustrate the applications of MSNs for drug and biomolecular delivery, bioimaging, and biosensing. Next, by categorizing the most important physiochemical properties of

host materials for bioimaging, biocatalytic, and biosensing agents have been widely recognized.5−9 According to the International Union of Pure and Applied Chemistry (IUPAC), a mesoporous material is defined as a porous material with pore diameters between 2 and 50 nm. The first major publications on mesoporous materials were reported in the early 1990s by two independent groups, namely, Kresge et al.10,11 and Yanagisawa et al.12 In particular, the reports by Kresge and co-workers10,11 on the family of highly ordered mesoporous silica materials called M41S, which consisted of MCM-41, MCM-48, and MCM-50, had quickly paved the way to diverse types of ordered MSNs, on whose biocompatibility this review focuses. It is important to note here that both the words nanomaterials and micromaterials have often been used for MSNs in the literature. This is despite the fact that the traditional definition of nanomaterials limits the use of the word nanomaterials only to those materials possessing sizes in the range of 1−100 nm at least in one dimension. This traditional definition also emphasizes that nanomaterials should possess properties that are not extrapolations from a larger size and that are typically but not exclusively exhibited by materials in small size ranges. In our opinion, which is possibly shared by many others, the definition based solely on size is somewhat loose and not very relevant for MSNs because the properties of MSNs with sizes 100 nm. For example, MSNs with sizes of 105 nm or more often show properties similar to those with 90 nm or less (or as those defined as nanomaterials in a traditional sense). Furthermore, most of MSNs have sizes >100 nm in diameter (typically 60−1000 nm) and exhibit similar structure (e.g., size-, and shape-)-dependent properties as do other traditionally defined nanomaterials. So, our use of the word nanoparticles for MSNs in the review is not to imply that the sizes of MSNs are strictly 30 > 110 > 170 nm (Figure 4a).62 These results clearly indicate that endocytosis of MSNs is virtually a complicated process, determined by many more factors than just the particle size of the MSNs. In a recent report, the impact of particle sizes of MSNs on cell adhesion and migration was investigate by incubating 100 μg/mL of spherical MSNs with 80 and 500 nm in diameter with human dermal fibroblast cells.63 Owing to their similar surface charges as seen from their ξ-potential values, both types of MSNs were found to be internalized by the cells via similar mechanisms, which involved macropinocytosis, the clathrin-mediated pathway, and to a lesser extent, the caveolae-mediated process; however, the larger particles were found to be ingested more quickly and accumulated more in lysosomes than their smaller counterparts.63 Furthermore, when the dosage of the MSNs was increased from 5 to 200 μg/mL, the 80-nm size spherical MSNs inhibited cell growth and proliferation more than did the 500nm MSNs, although the dose-dependent effect was exhibited by both types of MSNs. These biological effects were results of the severe collapse of the mitochondrial membrane potential, decrease in dehydrogenase activity, and damage to the membrane integrity. Conversely, the 500-nm size MSNs reduced mRNA levels of major adhesion proteins (i.e., fibronectin, laminin, and focal adhesion kinase) less than did their 80-nm size counterparts.63 However, both types of particles seriously weakened cell migrations, possibly due to the presence of strong interactions between these nanoscaled particles and nucleotides.63 To evaluate the effect of size of MSNs on hemolysis, the Haynes group synthesized different batches of MCM-41 type 2271



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Figure 5. (a) Biodistribution of MSNs in a mouse model with xenograft tumor:46 (i) the percentage of silicon in urine and feces of mice several hours after injection of the MSNs through the tail vein of mouse compared to the total amount of material injected into the mouse; (ii) nanogram of silicon found per milligram of murine tissue collected several different times after injection of the MSNs.46 Reprinted with permission from ref 46. Copyright 2010 John Wiley and Sons. (b) Antitumor effects of MSNs loaded with CPT. Reprinted with permission from ref 46. Copyright 2010 John Wiley and Sons. (c) Scheme showing accumulation of MSNs in cancer tissues by to EPR effect.76 Reprinted with permission from ref 76. Copyright 2009 John Wiley and Sons.

the reticuloendothelial system (RES), mononulcear phagocytes are originally derived from bone marrow and widely migrated into a variety of organs through blood circulation, including brain, lung, heart, lymph nodes, liver, spleen, and subcutaneous tissues. Depending on their dimensions, particles, whether intercepted by RES or not, flow through the bloodstream and can reach the aforementioned tissues. While some of the

In addition to RBCs, MSNs' interaction with white blood cells (WBCs) has also been studied. As part of the body’s immune defense system, WBCs (sometimes called phagocytes) are known to engulf foreign particles > ∼100 nm in diameter upon encountering them.64 In some cases, active phagocytes would agglomerate into giant phagocytic cells in order to engulf large or (sub)micrometer sized foreign particles. Being part of 2272



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than the smaller ones.74 Conversely, MSNs with smaller sizes circulated more in the blood for longer times without being trapped in kidney.74 In another study, spherical MSNs with a diameter of ∼100−130 nm were injected into mice at doses ranging from 0.25 to 4 mg/mouse (i.e., ∼10 to 200 mg/kg mouse body weight) once per day and continuously for 10 days.46 A higher concentration of liver transaminase was detected in the mice treated with particles at a concentration of 2 mg/mouse or higher.46 Administration of up to 1 mg of MSNs/mouse either by i.v. injection twice during a two-week period or by i.p. injection twice per week for a period of 9 weeks caused no abnormality in mouse activity or organ histology.46 These results suggested that the MSNs did not induce acute and chronic toxicity. Elemental analysis of the mouse urine and feces 1 day after the injection of MSNs showed the presence of about 26.3% silicon in the mouse urine but a negligible amount in the feces (Figure 5a). However, 2 to 4 days after injection of the mouse with MSNs, the concentration of silicon (or silica) in feces kept increasing but still remained significantly lower than that in urine. By day 4, the total excreted silicon (or silica) in the urine and feces reached >94% of the amount originally injected into the mouse (Figure 5a).46 The biodistribution of MSNs was further investigated by administering fluorescently labeled MSNs in a mouse model transplanted with human breast cancer cells that later developed subcutaneous tumors.46 When the fluorescently labeled MSNs were administered by i.v. injection, the particles were found to preferentially bind to the tumor sites, possibly because of the size match between the porous cavity formed in the growing tumors and the nanoparticles circulating in the blood.46 Furthermore, when the MSNs were loaded with the anticancer drug camptothecin (CPT) and injected into the mouse by i.p., they not only ended up more in the tumor but also suppressed the tumor almost completely after 2 months. In contrast, the CPT alone reduced the tumor down to only ∼14% of its original size, while the MSNs alone did not exhibit any antitumor activity (Figure 5b).46 These results are clearly due to the fact that the tumor blood vessels built around newly grown cells are aberrant in their shapes from those around normal vessels (Figure 5c).75 In other words, the tumor blood vessels are leaky, thereby making the CPT-loaded MSNs enter the cancer cells effectively.75 Furthermore, the result once again indicates that pristine MSNs could be very promising drug carriers for nanomaterial-promoted cancer therapy by taking advantage of the enhanced permeability and retention (EPR) effect in solid tumors. This characteristic cancer angiogenesis particularly offers an opportunity for certain sizes of particles or macromolecules ( spleen > lung > kidney/heart. Furthermore, the accumulation of MSNs in each organ continuously increased in the first 5 days postinjection but significantly decreased at 1 month.74 Moreover, higher concentration of the larger particles was detected in urine samples 30 min after administration, indicating that larger particles were easily captured by RES 2273



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submitochondrial particles, while neither particle was seen to induce oxidative stress (i.e., the glutathione levels in both MCM-41- and SBA-15-treated cells were not different from that in untreated cells).40 Contrary to our results, Heikkilä et al. reported that MCM-41 and SBA-15 microparticles (particle sizes ranging from 1 to 160 μm) caused elevated apoptosis in human colon carcinoma cells due to mitochondrial dysfunction, which was promoted by the large ROS generated due to the presence of the particles.47 The difference between the two results might be attributed to the differences in particle dimensions of the MSNs employed in the two studies, i.e., the MSNs used in our case were ∼300−650 nm in diameter and those investigated by Heikkilä et al. were 1 to 160 μm in dimension. In another study, we investigated the biological responses of various extracted murine tissues, including lung, liver, kidney, spleen, and pancreas, during exposures to 200 μg/mL calcined SBA-15 and MCM-41 particles for hours.48 The results showed that all the murine tissues did not exhibit any changes in their microscopic structures and bioenergetics after exposure to both types of MSNs.48 Further detailed in vitro studies with the lung tissue from a mouse model showed that both particles were widely distributed in pneumocytes, macrophages, endothelial cells, fibroblasts, and interstitium, regardless of the particles’ shapes and sizes.80 These results clearly suggested that calcined MSNs have very promising biocompatibility properties with mammalian tissues when administered at reasonable dosages. Similar examinations on the biocompatibility of MSNs, both in vitro and in vivo, using MCM-41 (100−150 nm), SBA-15 (600−800 nm), and mesocellular foam (MCF, 4000−5000 nm) type MSNs at concentrations ranging from 100 to 500 μg/ mL were conducted by the Kohane group.39 These materials exhibited in vitro toxicity at an incubation period of 4 days to a variety of cells, independent of the type of the MSN particles and their morphologies, in the following decreasing order: human mesothelial cells > mouse myoblasts > mouse peritoneal macrophases.39 More importantly, based on their in vivo data, Kohane and co-workers suggested that the type of administration of the MSN particles strongly influenced how toxic the nanoparticles would be. Whereas the administration of 30 mg MSNs/mouse intraperitoneally and intravenously were proven to be toxic, and even lethal in vivo, their subcutaneous injection was harmless.39 This, in turn, suggested that the death of the animal model treated with the MSNs might be due to pulmonary embolism or thrombosis. Conversely, the result implied that biodistribution of nanomaterials and their subsequent toxicity, if any, could be altered by adopting different routes of administration for them.76 In other words, different administration methods of nanoparticles into biological systems could be used to help accumulation of the nanoparticles at different desirable sites or targets. Kohane and co-workers further pointed out that the interactions between local tissues and MSNs could cause significant systemic toxicity while the MSNs circulate in the body.39 Hence, the fate of MSNs in the bloodstream deserves more research in order to unravel the possible mechanism of their possible toxicity in vivo and, more importantly, to understand why some MSNs induce toxicity while others do not. Tang and co-workers also investigated the biodistribution and excretion of different MSNs in mice, after injecting the tail veins of mice with two rod-like fluorescein-conjugated MSNs possessing different aspect ratios of 1.5 and 5 (or MSNs with

(80 to 150 nm in diameter) and rod shaped (400−1000 nm length × 80−150 nm width) MSNs, possessing the same types of fluorescent tags, similar surface charges (ξ-potential = −1.50 to −1.90 mv), similar surface areas (951.7 to 991.2 m2/g), and similar pore diameters (∼2.7 nm). The two different types of particles exhibited different transport properties to reach the cytoplasm of the cells.78 Although all these different types of particles got engulfed by the CHO cells more than by the normal human fibroblast cells, the spherical ones were internalized faster by both types of cells than the rod-shaped nanoparticles, possibly due to the lower tendency of the former to form aggregates.78 This result clearly demonstrates the effect of shapes of MSNs on their biological activities. It is worth noting that neither type of particle assaulted the nuclei of the cells.78 As cellular membranes are the first physical barrier in cells for nanoparticles to penetrate, MSNs with different morphologies were expected to have different degrees of interactions with cell membranes, and thus trigger different cascades of intracellular events after entering the cells. This hypothesis was tested and confirmed by several experimental works. For example, Huang et al. showed different cellular uptakes and subsequent cellular responses for MSNs with three different shapes.79 Although MSNs with different aspect ratios, namely, 1:1, 2:1, and 4:1, with dimensions ranging from 100 × 100 nm to 100 × 450 nm, and possessing surface areas ranging from 791 to 1169 m2/g were all found to be efficiently ingested by A375 human melanoma cells via encapsulation within endosomes, those with higher aspect ratios entered the cells more rapidly than those with lower aspect ratios.79 Furthermore, the MSNs with higher aspect ratios disrupted the cytoskeleton (F-actin disorganization) of the cells and induced more cytotoxicity (in a dosedependent manner). Compared to untreated cells, the MSNtreated cells also expressed less melanoma adhesion proteins.79 In particular, the particles with higher aspect ratios resulted in much less protein expression, although they did not affect the levels of mRNA concentrations,79 suggesting that some damages to protein translation or post-translational modification were dependent on the shapes of the MSNs.79 It is worth noting that this result is slightly different from those reported in refs 60 and 78; however, the latter were obtained with different cell lines and nanomaterials. Thus, general conclusions about the effect of shapes of nanomaterials on biological systems cannot be easily made or at least require the consideration of several other parameters including cell type and material composition. Our group also investigated the biological effects of two types of MSNs possessing sizes in the same range but having different shapes, i.e., spherical/oval shaped MCM-41 and irregular shaped SBA-15, on suspended and adherent human cancer cells.43 The results indicated that the MSNs had dose- and time-dependent cytotoxicity in the first 0−2 days when administered at concentrations in the range of 50−200 μg/ mL MSNs, with the MCM-41 MSNs exhibiting milder toxicity to both types of cells than the SBA-15 MSNs.43 Furthermore, the results indicated that both types of MSNs were efficiently engulfed by the cells after 1 h of incubation time.43 Moreover, our study on the impact of SBA-15 and MCM-41 MSNs on the bioenergetics of the cancerous cells showed that only the irregular shaped SBA-15 MSNs inhibited cellular respiration and ATP formation of the cells (in a dose-dependent manner).40 However, both types of MSNs were found to impair oxygen consumptions of isolated mitochondria and 2274



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The first example deals with MSNs' catalytic activity toward the oxidation of epinephrine. As mentioned above, both MCM41 and SBA-15 MSNs were shown to catalyze the oxidation of epinephrine; however, the two materials exhibited different catalytic activities toward this reaction, which was attributed mainly to their differences in mesostructures.82 In another example, whereas SBA-15 type MSNs impaired cellular respiration and mitochondrial electron transport chain in cells when incubated in vitro with Jurkat cells, MCM-41 type MSNs at the same dosage barely resulted in any noticeable effect.40 In addition, the MCM-41 type MSNs generally produced milder toxicity than SBA-15 type MSNs.43 Although these two types of MSNs have similar hexagonally ordered mesostructures, they have some subtle differences. For instance, in contrast to MCM-41, SBA-15 has a unique porosity as it it possesses pore interconnectivity between the ordered cylinderical channel pores; this in turn, contributes to a substantial part of total surface area and leads to a different catalytic activity from MCM-41.82 Thus, some different biological effects exhibited by these two types of MSNs can be explained based on these structural differences. The relatively larger surface area and smaller pore diameter of MCM-41 can make these nanoparticles thermodynamically more favorable for cellular ingestion, according to the proposed model regarding nanoparticle endocytosis shown in Figure 4b.65 Our previous study also indicated that the MCM-41 and SBA-15 underwent cellular internalization through slightly different endocytotic mechanisms, as shown in Figure 6a.43 In an additional example, MSNs having different mesostructures (short-range worm-like or long-order hexagonal structure) were reported to rupture RBCs differently.64 This finding further confirms that a defined mesoporous structure, including pore size, pore volume, degree of mesostructure order, and integrity, can determine how MSNs interact with biological systems. Thus, like the particle size, morphology, and surface area, the type of mesostructure in MSNs should not be neglected as far as the biological responses of MSNs are concerned. The difference in mesoporosity of the MSN materials can also result in different drug adsorption capacities and drug release profiles, which in turn leads to different pharmacokinetics when the drugs are delivered by different MSNs as drug carriers. All these results point out the significance of the mesoporous structures of MSNs to their biocompatibility as well as bioactivity in various biological systems. 4.5. Effect of Surface Property. Given the interests in the potential biological applications of MSNs, synthetic methods that make MSNs as biocompatible as possible are necessary. In this regard, surface modification of MSNs is very important as it may result in enhanced cellular uptake and endosomal escape, and reduced collisions with some undesired organelles during cellular trafficking of the nanoparticles. This can be exemplified by the work performed by Lin and co-workers, in which a variety of surface-functionalized MSNs were found to be internalized differently into human cancerous HeLa cells expressing folate receptors.84 The different surface-modified MSNs were synthesized by functionalizing MCM-41 with 3aminopropyl (AP), guanidinopropyl (GP), 3-[N-(2guanidinoethyl)guanidine]propyl (GEGP), and N-folate-3aminopropyl (FAP) groups, All these organic-functionalized MSNs exhibited lower surface areas and pore volumes compared with their parent MCM-41 in the order of parent MCM-41 > AP-MCM-41 > GP-MCM-41> GEGP-MCM-41 >

similar diameters but different lengths of ∼185 and ∼720 nm, respectively).81 Two hours up to one day after injection, stronger fluorescent signals were detected in the RES of the murine liver, lung, and spleen, while weaker signals were observed in the murine kidney. Whereas the MSNs were found in both urine and feces samples, they were detected in lower amounts in the kidney than in the liver, suggesting that the MSNs could be more rapidly excreted via renal uptake than hepatic digestion,81 a result which was consistent with that reported in ref 46. The fluorescent signals of these MSNs started to fade away seven days after administration as a result of degradation or excretion of the particles. Meanwhile, no fluorescent signals were observed in limb lymph nodes and the brain, suggesting the inability of MSNs to cross some physical barriers such as the blood−brain barrier.81 Furthermore, 2−24 h after administration, the concentration of the MSNs with the larger aspect ratios remained almost unchanged in the bloodstream, whereas the concentration of the MSNs with the smaller aspect ratios dramatically decreased,81 indicating that the higher aspect ratio rod-shaped MSNs circulated for longer periods in the bloodstream than the shorter ones. Hence, it can be concluded that the shape (or aspect ratio) of MSNs can also influence the MSNs’ circulation times in the bloodstream and possibly their interactions with biological systems as well. 4.4. Effect of Mesoporosity and Pore Sizes. The mesoporous structures and the so-caused high surface areas are characteristic features of MSNs that can also impact the materials’ biological activities. This is well demonstrated by some comparative biological studies involving MSNs, and as reference materials, some dense silica nanoparticles that possess chemical composition similar to that of MSNs but have much less surface area than MSNs were used. For instance, whereas MSNs catalyzed the oxidation of epinephrine, a biogenic hormone, in phosphorated buffer solution due to the possible generation of oxygen radicals inside the mesoporous channels of MSNs, the dense silica nanoparticles did not catalyze this oxidation reaction.82 Furthermore, when incubated with cancerous cells, the solid silica spheres exerted a more acute and permanent injury to cells and induced more cell death than MSNs. This indicates that the dense silica nanospheres were more severely cytotoxic, or conversely, the MSNs were more biocompatible. This is presumably due to the presence of mesoporous structure in the latter or the absence of mesoporosity in the former, although other subtle structural differences between the two types of materials might have also led to the differences in their biological effects.43 Two independent groups also showed that MSNs exhibited less hemolytic activity compared with their dense silica counterparts possessing sizes similar to those of the MSNs but no mesoporous structures in them.64,83 The authors attributed this difference in the hemolytic effect exhibited by these two materials to the differences in their surface silanol density and overall cell-contactable surface areas. MSNs with fewer silanol groups on their cell-contactable surfaces were considered to trigger the hemolysis of RBCs less than their nonporous silica counterparts containing higher density of cellcontactable surface silanol groups.64 As introduced above, MSNs can be synthesized with different mesostructures such as hexagonal, cubic, worm-hole, etc. Besides sizes and shapes, the type of mesostructures existing in MSNs was also found to affect the MSNs’ biological activities as illustrated with the following examples. 2275



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determining the degree of endocytosis of the MSNs. The mechanisms for the endocytosis of the particles, except for GEGP-functionalized MSNs, were also included in the study, and they were reported to be different from material to material.84 The authors concluded that the parent MCM-41 MSNs were ingested through a clathrin-mediated pathway, whereas the AP- and GP-grafted MCM-41 MSNs were internalized into cells via a caveolae-dependent mechanism, and the FAP-MCM-41 MSNs were engulfed by both clathrinand foliate receptor-mediated endocytosis, followed by more confinement of positively charged particles inside endosomes.84 These results clearly showed the effects of surface properties, such as surface electrochemistry, of MSNs on their cellular uptake and subsequent endosomal escape. Notably, a particular surface chemistry, surface charge, or surface functional group may help one of the steps in the given biological process, but may also inhibit the others in the process. Therefore, all the steps in a given biological process, e.g., cellular uptake, need to be taken into account when rationally designing and synthesizing nanomaterials for biological applications. Beginning with cellular uptake of the foreign nanomaterials, the consequent cellular events can depend on the various physical and chemical factors of the nanomaterials, such as their sizes, surface charges, surface ligands, etc. Hence, evaluations on the combined effects of all the material variables in a series of the biological processes, which consist of membrane trespassing, vesicular coating, endosome development, and lysosome degradation, would be necessary for one to determine how exactly the cellular trafficking of MSNs occurs. We emphasize here that the surface chemistry of MSNs, including their surface charges and ligand affinity, needs to also be assessed when the biological responses of MSNs are investigated or the potential biomedical applications of MSNs are evaluated. For example, we previously found that quaternary amine-functionalized MSNs (positively charged) tended to stick onto negatively charged cell membranes of the cells, rather than penetrating the cells and entering the cytoplasm.43 Generally, the uptake of nanosized particles into cells can be a complicated process, typified by phagocytosis or different receptor-mediated endocytosis (shown in Figure 6b). In eukaryotic cells, regardless of each specific pathway, endocytosis usually takes place when the plasma membrane (inner cellular membrane comprising glycolipids and integral proteins and covering the cytoskeleton) forms vesicles to wrap the particles and generate early endosomes. This process is reversible as endosome-transported particles can be repatriated out of the cells, and endosomes are recycled to compensate the plasma membrane. Alternatively, early endosomes mature into latestage endosomes with increasing acidity due to the elevated amount of acid hydrolases.85 The matured endosomes then fuse with lysosomes, which are mainly produced by the Golgi apparatus and endoplasmic reticulum.85 The lysosomes, which are full of hydrolytic enzymes, then help the degradation of the particles. The nondegradable particles are eventually exocytosed.85 Thus, in order to become effective in therapeutic or diagnostic applications, MSNs need to be well designed so that they are capable of crossing cell membranes, fleeing endosome capture, dodging unwanted intracellular binding processes, and finally reaching their cellular targets and releasing their payloads of drugs, biomolecules, and bioactive agents, as necessary. Among these processes, the endosomal escape of MSNs constitutes a crucial step or checkpoint, which

Figure 6. (a) TEM images showing the endocytosis of MCM-41 and SBA-15 MSNs by Jurkat cells.43 Reprinted from ref 43. Copyright 2009 American Chemical Society. (b) Endocytotic pathway of MSNs in mammalian cells.

FAP-MCM-41.84 Interestingly, the degree of particle uptake by the HeLa cells increased in the same trend as their surface charges, i.e., in the order of parent MCM-41 < AP-MCM-41 < GP-MCM-41 < 0 mv < GEGP-MCM-41 < FAP-MCM-41.84 Furthermore, among these materials, the FAP-modified MSNs were internalized the most by the folate receptor-expressing cells, indicating that chemical affinity of functionalized particles to cells could play greater roles than physical properties in 2276



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particle sizes.74,81,92,93 In addition, PEGylation prolongs the blood circulation of MSNs by reducing RES organ uptake, bypassing renal and hepatobiliary clearance, and thus enhancing permeability and retention of the nanoparticles. Hence, the surface modification of MSNs using PEG or other similar functional groups not only improves the biocompatibility of MSNs but also promotes the ability of the MSNs to deliver drugs or biomolecules to desired sites in a sustained manner. The surface property of nanomaterials can also affect how nanoparticles undergo exocytosis as much as it affects their endocytosis and intracellular trafficking. However, unlike endocytosis, which is more often looked into while studying the biocompatibility of nanomaterials, exocytosis of nondegraded nanoparticles such as MSNs has been largely overlooked. Generally, exocytosis is a cellular process by which cells remove the unwanted material or wastes from the plasma membrane typically via the following processes: (1) intracellular vesicles containing the material to be disposed are transported to the vicinity of the inner plasma membrane; (2) once the intracellular vessicles are in contact with the plasma membranes, the vesicles are further fused and integrated onto cell membranes; and (3) with vesicular membranes turning themselves inside-out, the waste to be disposed is wrapped and released out of the cell.85 Exocytosis of nanomaterials is an inevitable step that determines the final fate of the nanoparticles used as drug delivery vehicles or hosts for bioactive reagents. Furthermore, it determines how compatible the nanoparticles are with the biological systems. Therefore, exocytosis also requires attention during investigations of the overall effects of nanoparticles on biological systems. Not surprisingly, like endocytotic processes, exocytosis can be influenced by many physiochemical variables associated with the nanomaterials such as size, shape, surface property, etc. and the biochemical or biological factors of host cell/tissue, such as cell/tissue origins, protein expression, and ion strength of the intracellular environment. Although only a few studies have been conducted on exocytosis, there have been some reports on exocytosis of MSNs in different cell lines. For example, Slowing et al. studied the exocytosis of MSNs by normal (HUVEC) and cancerous (HeLa) cells and reported two interesting findings.94 First, the MSNs were found to be ingested by the cells and reached a constant intracellular amount within 2 h, indicating the attainment of a balance between the rates of endocytosis and exocytosis of the particles (or equilibrium) in 2 h of incubation time. Second, the exocytosis as well as transcytosis (a process in which materials are transported from one to another cell) of MSNs were found to be much more efficient in healthy HUVEC cells than in malignant HeLa cells.94 Despite these exocytosis studies, systematic in vitro studies on the effect of size of MSNs on their exocytosis are, however, limited. On the contrary, such studies have been conducted for many other materials including silica spheres (60−600 nm in diameter), gold nanoparticles (10 nm in size), or metal quantum dots (4 nm radius), which generally showed higher exocytotic efficiency for the smaller nanoparticles.95−97 Nevertheless, the effect of surface charge on secretion of the nanoparticles in murine models was well investigated for MSNs; the results showed that the surface charge on MSNs plays an important role in regulating the cellular excretion of MSNs.98,99 Specifically, the study showed that MSNs, especially those with sizes of >100 nm, were primarily retained by the liver and to a lesser extent by the spleen after being

can be significantly improved by properly functionalizing the surfaces of the MSNs. For example, polycations such as poly(Llysine), poly(ethyleneimine) (PEI), polyamidoamine dendrimers, natural chitosan, and endosome-disrupting peptide were successfully used to coat the outer surfaces of MSNs in order to improve cellular uptake and endosomal release of MSNs.86 Furthermore, as branched PEI contains a high density of amine groups, it can act as a proton sponge and thus serve as an excellent proton buffering medium during endosomal encapsulation, where pH values abruptly decline (from 7.4 to ∼5.5 in the late endosome).87 Therefore, due to this high endosomolytic activity, PEI-coated MSNs exhibit an improved ability to deliver nucleotides and drugs to intracellular or even nuclear sites.88 Similarly, chitosans, poly(alkyl acrylic acids), and anionic amphiphilic peptides (derived from viral fusion peptides), which get protonated in response to pH decrease, can also be tethered on MSNs’ surfaces via covalent bonding or electrostatic interaction. This functionalization can help MSNs accumulate with high charge density when acidity increases during endosome development, destabilizing the endosomes’ membranes.86 In addition, surface modification of MSNs with functional groups such as photosensitive porphyrins was shown to result in a novel MSN-based delivery system for photoinduced endosomal escape in living cells.89 After entering cells via endocytosis and binding to endosomal membranes, these photosensitized MSNs were excited by light and further quenched by a triplet oxygen moiety to produce a singlet oxygen species.89 The singlet oxygen species then damaged the endosomal membranes, thus releasing the cargo-loaded MSNs. Tuning the surface chemistry of MSNs is imperative also for potential in vivo administration or future clinical uses of MSNs as drug delivery vehicles or bioimaging agents. Surface modification can help MSNs camouflage, and this process assists the MSNs to avoid massive opsonization and be rapidly eliminated by RES of the body’s immune system. Polyethylene glycol (PEG) has been widely utilized for these purposes as it has the ability to improve the surface hydrophilicity of MSNs, reduce their protein adsorption, and shield them from nonspecific bindings.90 Compared to their naked counterpart, PEGylated nanoparticles (including PEGylated MSNs) were already shown to have much higher dispersion and stability (resistance to degradation by aging) in biological media and much lower adsorption of serum proteins and less uptake by macrophages.90−92 PEGylated MSNs with enhanced biocompatibility could be produced more easily by applying PEGs with molecular weights >10 kDa on the nanoparticles and by ensuring homogeneous surface coverage and optimal PEG density on the nanoparticles’ surfaces rather than by increasing their chain lengths.90−92 In addition, properly performed PEGylation could also help the mesoporous structures of MSNs remain intact.21,64,65,92 Furthermore, PEGylated MSNs were shown to exhibit significantly lower effects toward hemolysis of human RBCs, possibly because the PEG groups shielded the silanols on the surfaces of MSNs and prevented the latter from possibly generating ROS that could damage cell membranes. This was confirmed both with in vivo and ex vivo mouse model studies in which the distributions of PEGylated MSNs in various tissues following i.v. injection were probed. PEGylated nanoparticles also exhibited longer retention times in the bloodstream and lower accumulation in the spleen, liver, kidney, and urinary bladder, compared to their parent MSNs or their carboxylated or hydroxylated counterparts with similar 2277



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exocytosis of these materials in the two cell lines.43 Therefore, in addition to the cellular metabolism, the growth pattern of cells (i.e., whether being adherent or suspended cells) can also determine how the cellular responses during exposure to MSNs would be. By using hydroxyapatite nanoparticles with similar particle size, shape, and crystallinity but different surface charges, Chen et al. recently showed that these differently charged nanoparticles underwent different cellular uptake in murine osteoblasts in vitro.101 The particles with positively charged surfaces were accumulated inside the cells more than their negatively charged or neutral counterparts. This result is consistent with a previous report on the uptake of differently charged MSNs by HeLa cells.84 Interestingly, the cells even with highest particle uptake (i.e., those containing positively charged hydroxyapatite nanoparticles) had cell membrane integrity, viability, and normal replication, with no difference compared to those containing less particles inside (i.e., the cells treated with negatively charged or neutral particles).101 This result was unprecedented, considering the fact that substantial residues of nanomaterials remained inside the cells or tissues and that such materials are traditionally expected to result in severe injury or toxicity based on previous findings on the internalizations of hydroxyapatite nanoparticles in other cells (e.g., fibroblasts or hepatic cancer cells) and their cytotoxicity and cell growth inhibitory effect.101 Nonetheless, this again corroborated the results that surface charge on MSNs could play a major role on cell uptake of the materials and cell viability. Moreover, the results showed that the degree of cell uptake of the functionalized MSNs and their subsequent biological effects and biocompatibility could vary with the cell type used in the study. In the work by Chung et al., a similar observation on the effect of surface charges of MSNs on the biological activities of these materials on human stem cells was also reported.102 Specifically, the authors adjusted the density of positive charges on the surfaces of MSNs by varying the relative density of their surface amine groups and then monitored how well the resulting particles were engulfed by the cells. Whereas the MSNs were effectively internalized by human mesenchymal stem cells regardless of the nanoparticles’ surface charges, the more positively charged MSNs were found to be more ingested in mouse embryonic cells, without affecting cell viability, division, and differentiation.102 These results clearly indicate that different endocytotic mechanisms must be at play during internalization of a given MSN in different cell types. Therefore, the biocompatibility of a given nanomaterial or MSN can vary depending on what type of cell the materials are with. For this reason, one may have to also expect that the nanomaterials administrated as nanomedicines could interact differently with different targets, such as tumors with different origins, sizes, growth rates, biochemical markers, etc. As a result, different therapeutic or diagnostic effects can be anticipated by a given nanomaterial-based drug delivery vehicle at different parts of the body. 4.7. Effect of Methodology of Toxicity Assessment. Generally, the assessment employed in a given study could affect the experimental outcomes and the final conclusions one could make with regard to the possible biocompatibility of a certain nanomaterial on the given cell line. Thus, it is essential to discuss the toxicity assays that different researchers have frequently used, which might also be responsible for some contradictory results reported (or possibly will be reported in

administered via tail-vein injection in nude mice and Sprague− Dawley rats.98 Compared with their negatively charged counterparts, the positively charged MSNs at physiological pHs were more adsorbed by serum proteins, thus being more rapidly transported via hepatobilic secretion into the gastrointestinal tract and finally eliminated with feces.98 When hepatic metabolism of MSNs was further investigated at subcellular resolution in real-time, the results revealed that the negatively charged MSNs had a tendency to remain in blood vessels or be sequestered by Kupffer cells that constitute the walls of liver sinusoids.99 As a result, negatively charged MSNs could be a potential threat to hepatic health. On the contrary, after efficiently endocytosized by hepatocytes, the positively charged MSNs were effectively eliminated via hepatobiliary pathway.99 These results not only showed the effect of surface charges of MSNs on cellular uptake and host clearance of the nanoparticles but also depicted charge-dependent surface recognition and disposal routes by different cells/tissues. 4.6. Effect of Cell Type. As mentioned in some of the examples in the above section, the biocompatibility of MSNs could be strongly dependent on the type of cell- or tissue/organ in question. Thus, to properly evaluate the biocompatibility of engineered nanoparticles such as MSNs, an appropriate biological system needs to be wisely chosen, and the evaluation should be accompanied by accurate, reliable, and expedient assessment methods. Generally, compared to the more complicated in vivo models, in vitro studies present much quicker, relatively clearer, and more specific results for screening the biocompatibility of a new material while also minimizing animal sacrifices. Different in vivo and in vitro studies on the interactions between various nanomaterials (e.g., carbon nanotubes and quantum dots) and diverse biological entities (animal/plant cells, microorganisms, tumor, or transgenetic models, etc.) have been widely documented in the literature.1−6 However, here we mainly focus on those studies adopting in vitro systems because most of the reports in this area have dealt with such systems. Furthermore, the discussion here will mainly illustrate the possible interference of the cell types on toxicity assessments. It is demonstrated with some examples above that the potential cytotoxicity (or biocompatibility) of nanomaterials is dependent on the cell type in question. As an additional example, it is worth mentioning the work by Chang et al. on the effect of dense nanosized silica particles on a variety of adherent cancerous (lung, gastric, and colon epithelial cancers) and normal (skin and lung fibroblast) cells.100 The authors found that the cytotoxicity of the silica nanoparticles was strongly correlated with the types of the cells employed in the study, where different cell types exhibited different metabolic activities and doubling times with the nanoparticles. Overall, the nanosized silica particles induced lower cytotoxicity, and thereby shorter cell doubling times, on the cells having higher metabolic activity.100 Our research with MCM-41, SBA-15, and solid spherical nanosized silica particles, with and without surface amine groups, also indicated that the particles exhibited different toxicities on different cell lines.43 In contrast to Jurkat (human T-cell lymphoma) cells grown in suspension media, adherent SK-N-SH cells derived from human neuroblastoma showed more drug resistance and less recovery when treated with all the three types of nanomaterials, i.e., MCM-41, SBA-15, and solid spherical nanosized silica particles, whether they were modified with amine groups or not. This difference appeared to be mainly due to the possible differences in endocytosis and 2278



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of the cytotoxicity of nanosized porous materials, including MSNs.41,43,103,106,107 High-throughput screening of the toxicity of nanomaterials using multiwall cell plates for adherent cells has been widely investigated in recent years. However, care needs to be exercised while using this method, particularly when differentiating the cytotoxicity inherently caused by the nanoparticles from that caused by the possible physical blockage of oxygen or nutrients to the cell surfaces by the nanoparticles.39,43 There is one solution to this potential problem, which is achieved by putting the particles into the multiwall cell plates first, followed by adding the cells into the solution, and then determining the biological responses of the cells to the particles.39 The toxicity data obtained with this approach can then be compared with those obtained by performing the experiment in a reverse order, that is, growing the cells in the wells first, followed by adding the particles, and then measuring the biological effects of the particles. However, concerns about whether the initial coverage of MSNs on the bottom of cell plates would influence the cell seeding should be also taken into consideration, as a significant amount of MSNs might occupy the cell growth area and affect cell attachment to the cell plates. In addition, sedimentation of insoluble particles by gravity from their suspension to land onto adherent cells and contact the cell surface remains a major issue in many cytotoxicity tests.108 This is because, without the particles virtually contacting the cells, their possible effect on the cells cannot be properly studied. This is especially more serious for MSNs with small sizes, low densities, and large surface areas per unit mass, and MSNs possessing surface charges. However, other forces (e.g., convection force) would help such nanomaterials contact the cells more than gravitational force does.109 In any case, the reported dosage and incubation time that cause cell mortality could be overestimated if the experiments are conducted without taking these issues into consideration. Moreover, realistic time periods required for the nanoparticles to diffuse and reach cells should be subtracted from the typically reported incubation times in most studies, which usually start right after the addition of the nanoparticle suspensions into cell media. Therefore, during days-long cell incubation, the real portion of particles to reach surface layers of the cells at the bottom of the wells, where adherent cells reside, could be very small, invalidating any possible non- or low-toxicity conclusion about certain nanoparticles, including MSNs of certain sizes with high surface areas and low density. Furthermore, to make matters worse, some small particles can form aggregates and rapidly sediment onto cell surfaces differently under different conditions.109 Thus, materials (e.g., MSNs) should be sonicated until a well-suspended solution is obtained, and then the cell plates after the addition of nanomaterials should be gently and briefly centrifuged to promote the incubation of cells with the dispersed nanoparticles. A possible source for the evident toxicity induced by the treatment of MSNs comes from the possible residual organic templating reagents in the MSNs. In addition, due to the different synthetic procedures and postsynthetic processes, subtle differences in the density of surface silanol groups in the MSNs can also sway the final conclusion on their biocompatibility with certain biological objects, such as RBCs. Thankfully, many kinds of very good characterization tools, ranging from solid state NMR spectroscopy and elemental analysis to thermogravimetric analysis and infrared spectrom-

the future) on the toxicity (or biocompatibility) of MSNs in different studies. For example, an important study conducted by Krug and coworkers in 2006 revealed many possible sources of artifacts in cell viability tests, which led to contradictory results about the toxicity (or biocompatibility) of nanosized materials.103 For instance, when human A549 lung epithelial cells were treated with single-walled carbon nanotubes (SWCNTs), the traditional MTT assay using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide indicated that SWCNTs caused severe cell injury, whereas other standard tests suggested otherwise.103 This underscores how the information reported about the biocompatibility of nanomaterials relies also on the methods employed to assess them. Thus, the underlying results from these tests may have to be taken with caution. This also applies to MSNs when their biocompatibility is under examination. Some possible sources of artifacts and errors during biocompatibility tests might be related to how the assays work or the inherent processes associated with them. MTT or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide is a yellow salt that can be reduced by mitochondrial dehydrogenase in living cells to produce a water-insoluble purple formazan salt intracellularly. After the cells are homogenized and the salt is solubilized (e.g., by dimethyl sulfoxide), the formazan product can be quantitated by spectrometrically monitoring the intensity of the absorption maxima at ∼570 nm, which in turn gives information about the number of living cells (or cell viability).104 This easy-to-use assay allows the rapid analysis of cell growth and proliferation, particularly in drug screening and cytotoxin evaluation; however, the possible cytotoxicity of MTT itself in different cell lines remains a question. Furthermore, during toxicity tests with MTT, the formazans sometimes form insoluble crystals, which could not be further removed by a solubilization reagent, inside porous nanomaterials such as carbon nanotubes.103 This might, in turn, cause significant decrease in the color or absorbance of the solutions and underestimate values of cell viability. MTT was also found to be an inefficient reagent for examination of the potential toxicity of mesoporous silicon microparticles because it oxidized the surface of silicon into silica,105 thus leading to overestimated viability compared to that expected for such materials. Although silica cannot be further oxidized and does not interact much with MTT, cellmetabolized formazan products were visualized on cellular surface after a half-hour treatment of the cells with MSNs, indicating the presence of enhanced formazan exocytosis due to MSNs, which in turn led to an overestimation of the MSNs’ cytotoxicity.106 Moreover, as surface functionalization did not alleviate this effect, the MTT assay was shown to be somewhat inappropriate for assessing the toxicity of MSNs.106 Later on, different tatrazolium salts that can produce watersoluble formazans, including XTT (2,3-bis-(2-methoxy-4-nitro5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymehoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), WST series 1−11 (water-soluble tetrazolium salts developed by Dojindo Laboratory, Japan), and INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5phenyl-2H-tetrazolium chloride) emerged as possible replacement reagents for MTT. These reagents require no solubilization and render much improved signals. Thus, these nontoxic tatrazolium compounds and their corresponding water-soluble formazans are more suitable and effective for more precise determination 2279



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understand these structure-related biological effects of MSNs, including their possible negative impacts on human health, some of the studies also revealed contradictory results about the biocompatibility (or cytotoxicity) of various MSNs, as we discussed in this review. The chemical modification, along with the original synthetic condition, can tailor the particle size, shape, or surface property of the MSNs. This, in turn, could influence the biological activities of the nanomaterials. This aspect about functionalized MSNs was also considered in this review. In particular, it is illustrated that the favorable size, shape, or surface property of organic-functionalized and nonfunctionalized MSNs alike that leads to insignificant interference in a given biological system can generally be attributed to those that cost the least interfacial energy at the nanobio boundary. Specifically, in contact with cell or intracellular organelles, MSNs of a particular size and shape with larger surface area can be thermodynamically favored in their cellular interactions, leading to more intracellular accumulation but less cell injury. Thus, proper surface modification of MSNs (e.g., making the MSNs with positively charged surfaces) can enhance their endocytosis as well as exocytosis, while also mollifying their intracellular trafficking. However, size may play a superior role over shape in causing toxicity in vivo, if any, as the body generally recognizes a certain dimension of foreign particles, allowing or disallowing their further penetration into different organs or sites in the body to take part in some biological processes. At the same time, different surface properties regulate the particle circulation in the bloodstream, whereas the different affinities to certain biological receptors due to functionalization could dictate the final fate or biocompatibility of the particles. In this review, we also highlighted how the dosage of MSNs affect the biological responses to MSNs, and why their dosage should be properly chosen when they are considered for biological applications to minimize or eliminate their unwanted side effects (e.g., toxicity). In addition, the different chemical functional groups that can be immobilized on the surfaces of MSNs via surface modification to improve the selectivity and the affinity of MSNs to bind to specific biological sites were discussed. In our opinion, mesoporosity is among the main structural features of MSNs that also strongly affects the biological activities of these materials, besides the size, shape, and dosage of MSNs. This structural character also substantially determines whether MSNs can serve important functions in biological/ medical applications, such as drug delivery vehicles. Compared to the solid nature of other silica nanospheres, the mesoporosity of MSNs is responsible for their lower rigidity, buffering their interactions with biological entities. The mesoporous structure is also considered to be the major reason for the lowered interfacial energy between these nanomaterials and biological surfaces. Such structure-related biological features of MSNs have also been discussed in this review. For a given MSN, it was clearly shown that different biological systems are expected to exhibit different responses. At this point, it is worth noting that the conclusions made about the biocompatibility of a given nanomaterial, including MSN, are strongly determined by the accuracy or reliability of the chosen biological assay. Moreover, when using traditional assays to probe the biological effects induced by nanoscaled materials, the possible interruption in data collection or analysis during the assessment remains a big concern, which may result

etry, are now widely available to allow a routine and reliable determination of the structural as well as compositional parameters associated with these nanomaterials. These help to easily rule out the possible toxicity due to the detrimental effects of residual organic templating agents in MSNs after the preparation of the MSNs. In toxicity assessments, colorimetry is one of the most commonly used methods to conveniently elucidate the biological effects of nanomaterials and to quickly determine their general cytotoxic profiles.110−113 Besides colorimetric methods, other conventional measurements that enable the detection of ROS generation or DNA damage, such as flow cytometry, high-performance liquid chromatography (HPLC) or immunostaining, have also been utilized to evaluate the cytotoxicity of MSNs and unravel the possible mechanism behind their cytotoxicity, if any.110−116 As there are no reports regarding the severe interference of nanomaterials in these analytical methods, these techniques could be reliably used to assess cellular injury induced by MSNs. Nonetheless, to fully probe and verify the biocompatibility of well-defined nanoscaled materials, including MSNs, it is still highly recommended that many complementary methods as well as more than one type of cell line should be employed.

5. CONCLUSIONS AND PERSPECTIVES In this review, we first introduced the various synthetic strategies used to make and functionalize different types of MSNs. We then discussed how the physiochemical properties of MSNs could be tuned, either by shape-/size-controlled synthesis or by functionalization of their surfaces with organic groups. Furthermore, we described how the surface functionalization and shape/size control of MSNs could, in turn, result in MSNs with versatile properties for various potential biological and medical applications. The review also highlighted many of MSNs’ unique structural features such as nanosize pores, mesostructures, high surface area, high pore volume, and easily modifiable surfaces and elaborated on how these structural features enabled MSNs to become a very promising class of candidate materials for drug delivery, host materials for bioimaging agents, and platform materials for biosensing/biocatalytic moieties. However, it was also shown that many different structural features of MSNs could lead to different biological activities for these materials. Thus, probing and summarizing the overall biological responses induced by these materials, which possess a range of structural features and compositions, could be challenging. Nevertheless, in light of their great potential applications in biomedical areas, many experts in the field seem to agree that the biological activities of these materials need to be fully assessed and evaluated in order to determine the proper physical or chemical traits that could make MSNs highly biocompatible. These efforts and some typical results reported in these areas over the last two decades have been discussed as the main subject in this review. A broad evaluation at the interface between the material’s properties and its biological surroundings is crucial to fully determine whether a given material is biocompatible. This task, however, is very complicated and challenging in the case of engineered nanomaterials such as MSNs because they possess a variety of physical properties and chemical compositions, which could lead to a diverse range of biological responses and trigger a cascade of different biological events. While intense studies were being conducted by various researchers worldwide to fully 2280



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propyl; HUVECs, human umbilical vein endothelial cells; LCT, liquid crystal templating; MSN, mesoporous silica nanoparticle; PEI, poly(ethyleneimine); PEG, polyethylene glycol; RBCs, red blood cells; RES, reticuloendothelial system; SWCNTs, singlewalled carbon nanotubes; TEM, transmission electron microscope; WBCs, white blood cells

in artifacts or lead to wrong conclusions. This issue has long been overlooked in many studies and may be a substantial reason (at least, partially) for many conflicting results reported for a given nanomaterial (e.g., MSN) by different research groups, who have employed different experimental methods for their studies. Thus, possible pitfalls in those traditional assays used to determine the biocompatibility of MSN were also analyzed in the review. The investigation of the overall biocompatibility of the nanomaterials, including MSNs, can be complicated by the fact that the nanomaterials produced with typical nanomaterial synthesis often have a range of physicochemical properties and some size distribution, among other things. Thus, in our opinion, it is highly important and critical that the biological research involving MSNs should start with finding or developing reliable synthetic routes to a set of nanoparticles with well-defined structures. This has to be followed by choosing reliable and appropriate biological systems to conduct the biological studies on biocompatibility. Sometimes, more than one type of reliable assay (based on different distinguished and complementary analytical methods) is also needed to properly determine the overall biocompatibility of the nanomaterials within a defined biological system. With an accumulated knowledge on the effects of a certain nanomaterial, such as MSN, under a range of biological conditions, a general biocompatibility of such a material can thus eventually emerge. For example, complete information about the exact cutoff size of MSNs that results in biocompatibility or causes cytotoxicity, information that is still missing in the literature but is extremely important to move the field forward, can be obtained. Another important immediate research in this area has to be determining the exact effect of surface silanol groups and surface morphology on the interaction of MSNs with biological systems. With the recent advent of more powerful atomic force microscopes (AFMs) and their successful applicability to soft materials, this type of study should now be more feasible to conduct. This type of study, in turn, could provide an enormous amount of information regarding MSN− bio interfaces, MSNs’ interactions with biological systems, and why some MSNs could be biocompatible while others could be toxic.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (T.A.); [email protected] (Z.T.). Funding

This work was supported by Tsinghua Research Fund 100401007, Chinese Postdoctoral Science Foundation Grant 20100480314 (awarded to Z.T.), and the US National Science Foundation (NSF) under Grant Numbers CAREER CHE1004218, NSF DMR-0968937, NSF NanoEHS-1134289, NSFACIF for 2010, and NSF Special Creativity grant in 2011 (awarded to T.A.). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS AP, 3-aminopropyl; CHO, Chinese hamster ovarian; CPT, camptothecin; DCs, dendritic cells; EPR, enhanced permeability and retention; FAP, N-folate-3-aminopropyl; GP, guanidinopropyl; GEGP, 3-[N-(2-guanidinoethyl)guanidine]2281



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