Mesoporous silica nanoparticles in drug delivery and biomedical applications Ying Wang MPharm, Qinfu Zhao PhD, Ning Han PhD, Ling Bai MPharm, Jia Li MPharm, Jia Liu MPharm, Erxi Che MPharm, Liang Hu MPharm, Qiang Zhang PhD, Tongying Jiang PhD, Siling Wang PhD PII: DOI: Reference:

S1549-9634(14)00546-2 doi: 10.1016/j.nano.2014.09.014 NANO 1010

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Received date: Revised date: Accepted date:

20 February 2014 22 September 2014 22 September 2014

Please cite this article as: Wang Ying, Zhao Qinfu, Han Ning, Bai Ling, Li Jia, Liu Jia, Che Erxi, Hu Liang, Zhang Qiang, Jiang Tongying, Wang Siling, Mesoporous silica nanoparticles in drug delivery and biomedical applications, Nanomedicine: Nanotechnology, Biology, and Medicine (2014), doi: 10.1016/j.nano.2014.09.014

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ACCEPTED MANUSCRIPT Mesoporous silica nanoparticles in drug delivery and biomedical applications

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Ying Wang, MPharma, Qinfu Zhao, PhDa, Ning Han, PhDa, Ling Bai, MPharma, Jia Li, MPharma, Jia Liu, MPharma, Erxi Che, MPharma, Liang Hu, MPharma, Qiang

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Zhang, PhDa, Tongying Jiang, PhDa, Siling Wang, PhDa*

a. Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua

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Road, Shenhe District, Shenyang, Liaoning Province 110016, PR China

Corresponding author: Tel./Fax: +86 24 23986348

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E-mail: [email protected]

Word count for abstract: 94

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Complete manuscript word count: 6667

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Number of figures: 11 Number of tables: 1

Number of references: 103

This work was supported by the National Basic Research Program of China (973 Program) (No. 2015CB932100) and the National Natural Science Foundation of China (No. 81473165). We declare no competing financial interests.

ACCEPTED MANUSCRIPT Abstract In the past decade, mesoporous silica nanoparticles (MSNs) with a large surface

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area and pore volume have attracted considerable attention for their application in drug delivery and biomedicine. In this review, we highlight the recent advances in

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silica-assisted drug delivery systems, including (1) MSN-based immediate/sustained drug delivery systems and (2) MSN-based controlled/targeted drug delivery systems.

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In addition, we summarize the biomedical applications of MSNs, including (1) MSN-based biotherapeutic agent delivery; (2) MSN-assisted bioimaging applications; and (3) MSNs as bioactive materials for tissue regeneration.

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1. Introduction

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biomedical applications

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Keywords: biocompatibility, mesoporous silica nanoparticles, drug delivery systems,

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With the development of nanotechnology, materials developed at the nanoscale level have attracted increasing attention in such fields as drug delivery, diagnostic and medical imaging and engineering [1-6]. Among all of the available nanomaterials, numerous studies have investigated porous silica nanoparticles (NPs) due to their unique properties, such as a large specific surface area and pore volume, controllable particle size and good biocompatibility [7, 8]. Compared with other porous silica nanocarriers, mesoporous silica nanoparticles (MSNs) with a pore size ranging from 2 nm to 50 nm are excellent candidates for drug delivery and biomedical applications [9-11]. Since the M41S family of ordered mesoporous silica was first reported in the 1

ACCEPTED MANUSCRIPT early 1990s [12], the number of studies on MSNs has increased rapidly. In general, MSNs are synthesized via a template-directed method [13] in the presence of a

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supramolecular assembled surfactant that acts as a structure-directing template. Common MSNs, including 2D hexagonal MCM-41 (Mobile Crystalline Material) and

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3D cubic SBA-15 (Santa Barbara Amorphous), exhibit a range of pore sizes (2 to 10 nm) [14]. MSN-based applications in biomedical fields have become quite promising

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since their first report in 2001 [15, 16]. The advantages of MSNs are the following: (1) a large surface area and pore volume [17] provides great potential for drug adsorption and loading within the pore channels, (2) excellent mesoporous structure and an

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adjustable pore size [18] enable better control of drug loading and release kinetics, (3)

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an easily modified surface for controlled and targeted drug delivery enhances the drug therapeutic efficacy and reduces toxicity [19, 20], (4) the in vivo biosafety evaluations

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of cytotoxicity [7, 21, 22], biodegradation [23-25], biodistribution and excretion [26,

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27], have yielded satisfactory results, (5) combinations with magnetic and/or luminescent compounds allow simultaneous drug delivery and bioimaging [14, 28, 29], and (6) those with excellent surface properties and porosity have proved to be attractive candidates as bioactive materials for bone regeneration [30]. Due to these unique merits, the number of studies on mesoporous silica materials has increased dramatically. Here, we highlight the recent research developments on mesoporous silica immediate drug delivery systems (IDDSs), sustained drug delivery systems (SDDSs), stimuli-responsive controlled drug delivery systems (CDDSs), and targeted drug delivery systems (TDDSs). In addition, we summarize the current biomedical 2

ACCEPTED MANUSCRIPT applications of MSNs, including MSN-based biotherapeutic agent delivery (such as protein, peptides, and genes), MSN-assisted bioimaging (magnetic resonance (MR)

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imaging and fluorescent imaging) and MSNs as bioactive materials for tissue

2. Drug Delivery Systems Based on MSNs

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regeneration.

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2.1 MSN-based immediate drug delivery systems

Many hydrophobic drugs have limited applications due to poor water solubility that results in poor absorption in the gastrointestinal tract after oral dosing [31].

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Recently, Zhang et al. [22] discovered that mesoporous silica improves the dissolution

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rate and bioavailability of hydrophobic telmisartan (TEL) after oral administration. The results demonstrated that the dissolution rate of TEL loaded within MSNs was

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dramatically improved compared with crude TEL powders; the relative bioavailability

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of TEL-loaded MSNs was 154.4% ± 28.4% compared with that of the marketed product Micardis (Figure 1, E and F). The mechanisms responsible for the improved oral absorption were explored via permeability experiments in the human colon carcinoma (Caco-2) cell line. The permeability results suggested that MSNs significantly enhanced the permeability of TEL and decreased the rate of drug efflux, thereby improving oral absorption (Figure 1, A-D). Because the dissolution process is the rate-limiting step for the absorption of hydrophobic drugs, we have detailed the following factors that influence the drug loading efficiency and dissolution rate. 3

ACCEPTED MANUSCRIPT (1) Drug loading methods Drug loading methods based on MSNs mainly involve physical adsorption and

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solvent evaporation [32, 33]. Wang et al. reported the physical adsorption method [34] in which MSNs soak in a drug-containing solution until an equilibrium is reached, and

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most drugs penetrate deeply into the pore channels of the carrier. Solvent evaporation is another drug loading method that involves a combination of physical adsorption

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and subsequent rapid solvent evaporation [35]. The drug dissolution rate is faster with the solvent evaporation method than with the physical absorption method because the drug loaded or absorbed on the surface of MSNs undergoes immediate dissolution,

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whereas it takes much longer for the drug inside the pores to diffuse into the release

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medium. (2) Pore morphology

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Pore morphology is thought to greatly influence drug loading and release. The

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results reported by Wang et al. indicated that cilostazol had a faster dissolution rate from MCM-48 with interconnected pore structures than from MCM-41 with unconnected pore networks [34]. Hu et al. reported similar findings in which SBA-16 with a 3D cage-like cubic mesoporous structure exhibited a faster dissolution rate than MCM-41 with a 2D hexagonal arrangement because the interconnected pore structure reduced the diffusion hindrance and facilitated drug diffusion into the dissolution medium [18]. (3) Pore size Increasing the pore size also improves the drug dissolution rate because of 4

ACCEPTED MANUSCRIPT decreased diffusion hindrance. Zhang et al. [36] used spherical MSNs of three different pore sizes as carriers for loading TEL. The in vitro dissolution testing

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demonstrated that the dissolution of TEL accelerated with increasing pore size (Figure 2). A similar result was reported by Jia et al. [37]. The dissolution rates of paclitaxel

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from MSNs with different pore sizes were compared, and the results suggested that the paclitaxel dissolution rate was improved by enlarging the pore size from 3.03 to

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9.68 nm.

In summary, MSNs have unique features compared with other types of carriers as IDDSs. The large surface area and high pore volume enable the encapsulation of

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drugs with a high payload. The mesoporous channels keep drugs in the amorphous or

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noncrystalline state within the pores, which facilitates drug dissolution. Moreover, the

and release.

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marked chemical stability and inert behavior allow for better control of drug loading

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2.2 MSN-based sustained drug delivery systems As mentioned above, IDDSs cannot provide long-term drug release, and frequent administration is necessary. Therefore, a dosage form offering sustained release will have a significant advantage because it will be able to maintain a steady blood concentration for a prolonged period of time. Here, several MSNs used for sustained drug delivery have been categorized into two groups: unmodified and modified silica materials. Sustained drug release using unmodified silica can be achieved by regulating the pore structure, pore diameter and particle size of the carrier. With modified silica materials conjugated to organosilanes [38], the interaction between the 5

ACCEPTED MANUSCRIPT drug molecules and the functional groups delays drug dissolution from MSNs and enables a more sustained release.

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2.2.1 Pure MSN-based sustained drug delivery systems

Conventional mesoporous silica materials, such as MCM-type materials and

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SBA-type materials, are utilized as SDDSs by controlling their physical and textural properties. As discussed for the IDDSs, pore morphology and size significantly

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influence the release rate. To achieve a sustained release pattern, MSNs with unconnected pore morphology and a relatively small pore size should be utilized. In addition, Qu et al. demonstrated that drug release behavior was related to particle size,

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which was proportional to the pore channel length [39]. A comparison of various

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MCM-41 carriers with the same spherical shape revealed that, the drug release rate can be slowed down by extending the pore channel length. Moreover, the drug content

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also influences the release rate. Carriazo et al. discovered that the drug release rate

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decreased with increasing drug content since the solvents had difficulty penetrating into the pore channels, thus preventing drug from leaching out of the carriers [40]. Compared with conventional mesoporous silica, hollow mesoporous silica (HMS) materials have a higher storage capacity due to the special hollow core structure. Furthermore, drugs can diffuse into the cavities through accessible pore channels on the shell. To control drug release, Chen et al. [41] prepared double shelled HMS spheres (HMSs@mSiO2) with an average pore size for the two shells of 2.4 nm and 3.8 nm. The drug storage capacity of HMS with double mesoporous shells was greater than that of the conventional MCM-41. Moreover, drug release was much slower from 6

ACCEPTED MANUSCRIPT HMSs@mSiO2 than from single shelled HMSs because of the longer diffusion

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2.2.2 Modified silica-based sustained drug delivery systems

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distance and the delay caused by the smaller pores on the second shell.

The modification of MSNs with appropriate functional groups (such as -NH2, -Cl,

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-SH, or -CN [42]) or polymers directly affects the drug release rate by increasing the drug diffusion resistance. Therefore, the surface modification on MSNs is an

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important strategy for achieving sustained drug release. He et al. [43] covalently grafted rhodamine B (RhB) on MSNs to create a SDDS. The zeta potential results revealed that the MSNs were negatively charged, whereas the MSN-RhB particles

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were positively charged; the RhB covalently grafted on the MSNs was hypothesized

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to attract the negatively charged model drug, salvianolic acid B (SAB), into the mesoporous channels via electrostatic interactions. Because of this electrostatic

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interaction, SAB molecules or ions were released from the SAB@MSN-RhB in a

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sustained manner, which improved the long-term efficacy. Sun et al. [19] developed polymer chitosan-functionalized MSNs as oral SDDSs. The results showed that the factors leading to sustained drug release were the powerful dispersing effect of the MSNs and the blocking effect due to the swelling of chitosan. The ability of chitosan to swell in an acidic environment and shrink in a relatively alkaline environment enables the regulation of drug release in a simulated gastrointestinal fluid. As SDDSs, MSNs differ from other drug delivery carriers because of their unique pore network that facilitates drug dispersion and simultaneously regulates the drug release rate. In addition, their easily functionalized surface enables numerous 7

ACCEPTED MANUSCRIPT strategies for achieving sustained drug release via interactions between drug

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2.3 MSN-based controlled/targeted drug delivery systems

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molecules and functional groups that decrease the drug diffusion rate.

2.3.1 MSN-based stimuli-responsive CDDSs

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MSN-based CDDSs have been developed by applying controls such as ‘gatekeepers’ over the pore entrance. The drugs cannot leak out from silica carriers

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unless the drug-loading system is exposed to external stimuli, such as pH, redox potential, temperature, photoirradiation, or enzymes, which trigger the removal of the gatekeepers. Researchers have investigated various types of gatekeepers that are

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summarized in Figure 3. In Type I gatekeepers, solid NPs (CdS, ZnO, and Fe3O4 NPs)

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are attached to the pore openings via covalent bonds that can be removed by external stimuli. The liner molecule (Type II) and multilayer (Type IV) gatekeepers are either

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covalently bound or adsorbed onto the surface of MSNs to achieve controlled drug

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release. The macrocyclic molecule (Type III) gatekeepers (e.g., cyclodextrins, crown ethers, and cucurbit[6]uril) are attached to the pore outlets of MSNs through covalent/non-covalent interactions that can be cleaved/weakened by certain stimuli, thereby clearing the pore entrances. Among the various stimuli-responsive drug delivery systems, pH-responsive CDDSs have been widely investigated due to the pH gradients that are present in different tissues and subcellular compartments [44]. Compared with normal tissues, tumor and inflammatory tissues are more acidic, thereby providing a potential internal trigger to control drug release. In this section, a macrocyclic β-cyclodextrin 8

ACCEPTED MANUSCRIPT (β-CD)-capped MSN-based drug delivery system (Type III) that offers autonomously modulated drug release will be discussed [45]. In this system, β-CD serves as a

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gatekeeper that encircles the stalks previously attached to the surface of the carriers via non-covalent binding to block the pore outlets. The binding between β-CD and the

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stalk is weakened under mildly acidic conditions, leading to the sliding of β-CD and subsequent drug release from the MSNs (Figure 4, A). The drug release profiles

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follow a typical pH-responsive release pattern because the drugs are tightly blocked in neutral environment but can easily diffuse out in response to acidic stimuli (Figure 4, B). The doxorubicin (DOX)-loaded nanoparticles were efficiently take up into KB-31

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cells within endosomal acidification environments, and DOX (red fluorescence)

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molecules were released into the nucleus and subsequently stimulated apoptosis by 60 h and nuclear fragmentation by 80 h, as expected based on the pharmacological

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effects of DOX (Figure 4, C). The results suggested that these pH-sensitive,

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nanovalve-capped MSNs can be triggered by lysosomal acidification and can serve as drug delivery carriers into cells. Redox potential has become another popular stimulus for triggering drug release due to the significant difference in the glutathione (GSH) concentration between the extracellular (2 μM) and intracellular (10 mM) environments. Moreover, the intracellular GSH levels in most tumor cells are at least 4-fold higher than those in normal cells [46]. Lin et al. developed a redox-responsive CDDS involving MSNs in which CdS NPs were used as gatekeepers (Type I) [47]. They found that the disulfide linkages between the MSNs and the CdS NPs could be cleaved by GSH, leading to 9

ACCEPTED MANUSCRIPT the subsequent rapid release of the mesopore-entrapped drugs. A cross-linked polymeric network anchored to the pore entrance has also been used as a

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redox-responsive cap [48]. In this system, poly(N-acryloxysuccinimide) (PNAS) was grafted onto the surface of MCM-41, and the outlets of PNAS-grafted MCM-41 were

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blocked by the addition of cystamine, which binds the PNAS chains together via covalent capturing of the N-oxysuccinimide groups to form disulfide linkages (Type

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II). The cross-linked, network-capped MCM-41 CDDS exhibited a good capping efficiency, and the mesopore-entrapped dye molecules were rapidly released after the addition of disulfide-reducing agents. Moreover, the release rate was proportional to

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the concentration of the reducing agents.

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Among the various stimuli used for CDDSs, temperature has also emerged as a promising trigger. A temperature-sensitive polymer, poly(N-isopropylacrylamide)

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(PNIPA), has been used as a gatekeeper (Type II) due to the hydrophilic-hydrophobic

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transition of PNIPA at the ‘lower critical solution temperature’ (LCST) of approximately 32 °C [49]. Based on the pore size of MSNs, PNIPA can be modified onto the interior surface of MSNs with a larger pore size or onto the exterior surface of MSNs with a small pore size. Zhang et al. reported a silica-PNIPA drug delivery system in which PNIPA was polymerized inside MSNs [50]. The effect of temperature on drug release was such that below the LCST, the drugs were locked within the mesostructure due to the swelling of the PNIPA, which led to the closure of the pore outlets. The formation of a hydrogen bond between PNIPA and the drug (ibuprofen in this case) can further prevent drug release. When the temperature increased, the 10

ACCEPTED MANUSCRIPT PNIPA chains became hydrophobic and shrunk within the mesopores, thereby opening the pores and disrupting the hydrogen bonds.

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Compared with other stimuli, light-triggered CDDSs have the significant advantage of rapidly responding to light at ‘specific or specified’ location and time,

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which does not change the physiological environment. For example, He et al. described a light-operated system in which thymine derivatives were grafted on the

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pore outlet of MSNs (Type II) [51]. When the thymine-modified MSNs were exposed to 365 nm UV light irradiation, cyclobutane dimmers formed in the pore entrances, which blocked the pores. With 240 nm UV light irradiation, the cleavage of the

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cyclobutane dimer allowed for the release of the mesopore-entrapped drugs.

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In addition, endogenous enzymes in living organisms have been used as stimuli to trigger drug release. Bernardos et al. loaded dyes as model drugs into the mesopores

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and modified different saccharides on the surface of MSNs (Type II) [52]. This

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‘intelligent’ controlled delivery system exhibited limited release until the addition of β-D-galactosidase, which can hydrolyze the glycosidic bond between β-D-galactose and β-D-glucose and induce the opening of the pore outlets. Compared with other types of stimuli-responsive nanoparticles for CDDSs, MSNs have unique advantages. First, the distinctive mesoporous structures make MSNs perfectly suitable for designing various types of gatekeepers to control drug release via regulated opening and closing of the pore outlets. Second, during the drug release process, only the grafting moieties break away from the carrier under certain stimuli; the MSNs will not disintegrate. 11

ACCEPTED MANUSCRIPT 2.3.2 MSN-based targeted drug delivery systems MSNs have also emerged as appealing candidates for targeted drug delivery

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systems. MSNs with a particle size in the nanoscale range can accumulate in tumor tissues via the enhanced permeation and retention (EPR) effect [53, 54]. In addition,

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specific drug delivery can be achieved via active targeting by decorating MSNs with targeting ligands such as folate (FA) or EGF [55, 56]. Peptides, antibodies and

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magnetic materials can also be conjugated onto MSNs, thereby acting as homing devices [57-61]. In the targeting process, the particle size and surface modification of MSNs critically influence particle pharmacokinetics and bio-distribution [62, 63].

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These parameters should be considered when aiming for a high targeting efficiency

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because they will directly affect the particle stability, circulation time, tumor accumulation, cellular uptake and therapeutic efficacy.

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2.3.3 MSN-based controlled and targeted drug delivery systems

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Recently, many researchers have focused on MSN-based multifunctional drug delivery systems [64-66], which can deliver antitumor drugs in a targeted fashion and release them on demand to increase their cellular uptake without any premature release prior to reaching the target site (Figure 5, A). Table 1 summarizes several MSN-based controlled and targeted drug delivery carriers. The preparation of MSNs possessing both a targeting ability and a stimuli-responsive ability involved two approaches. One involves designing a multifunctional targeting molecule as both a targeting and a capping agent to achieve MSN-based controlled and targeted drug delivery (Figure 5, B). For example, superparamagnetic iron oxide (Fe3O4) NPs have 12

ACCEPTED MANUSCRIPT been utilized as both gatekeepers for CDDSs and magnetic materials for TDDSs. Fe3O4-capped MSNs have been prepared by linking Fe3O4 NPs to the surface of

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MSNs via different sensitive chemical bonds (such as cleavable disulfide bonds and acid-labile boronate ester bonds [67, 68]) that block the pores and effectively inhibit

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drug diffusion. When the Fe3O4 NPs were released from the MSNs in response to certain stimuli, the pore outlets were re-exposed, and the mesopore-entrapped drugs

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were released at the target site. Chen et al. [69] reported a hyaluronic acid (HA)-conjugated MSN that can be utilized as a targeted and enzyme-responsive drug delivery system. In this system, polysaccharide HA acted as both a targeting ligand

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that specifically binds to CD44 receptors overexpressed on cancerous cells and as a

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smart valve to achieve controlled drug release. The CLSM results revealed that, compared with HA-free MSNs, HA-containing MSNs were internalized more easily

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by the cancerous cells due to the highly specific interaction between HA and the

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CD44 receptors. Additionally, drug release was instantly triggered upon HA degradation by hyaluronidase-1, which is only present in the tumor microenvironment. A similar finding was reported by Guo et al. [70], wherein FA served as both a targeting and a capping agent. FA anchored to the pore outlets of MSNs by disulfide bonds eliminated premature drug leakage in circulation and enabled drug release within the cytoplasm where the concentration of GSH was high enough to cleave the disulfide bonds. Additionally, FA-MSNs were efficiently internalized into HeLa cells via FA receptor-mediated endocytosis. The second approach involves designing a stimuli-responsive gatekeeper that is 13

ACCEPTED MANUSCRIPT further modified with a target moiety to achieve multifunctional drug delivery (Figure 5, C). Zhang et al. [71] reported another novel type of multifunctional

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redox-responsive CDDS in which β-CD was directly attached to the surface of the MSNs through disulfide bonding for glutathione-induced intracellular drug release.

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Then, an Arg-Gly-Asp (RGD) motif and a Pro-Leu-Gly-Val-Arg (PLGVR) peptide were decorated on the surface of the β-CD-modified MSNs via a host-guest

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interaction. Poly(aspartic acid) (PASP) was also introduced onto the nanoparticles to protect the RGD targeting ligand (Figure 6, A). The advantages of this smart MSN are the following: (1) the polyanion PASP prolongs the systemic circulation time in blood

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and improves the EPR effect, (2) the matrix metalloproteinase (MMP) substrate

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peptide PLGVR can be hydrolyzed after the drug carrier arrives at the tumor, resulting in the removal of PASP and the exposure of the RGD motif, (3) the RGD tumor

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targeting ligand enhances the tumor cell uptake of the MSNs, and (4) the drug

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encapsulated in the MSNs can be released rapidly into the tumor cells after GSH-mediated the cleavage of the disulfide linkers. The tumor-triggered targeting ability of this drug delivery system was demonstrated by the PASP release profile and CLSM. The smart MSNs rapidly released PASP after an incubation with MMP for 4 h, and no PASP was released after co-incubation with MMP and MMP inhibitors (Figure 6, B). In the presence of MMP, the polyanion PASP detached from the MSNs, and the exposure of the RGD motif enabled the MSNs to be specifically taken up by the cancerous cells (Figure 6, C and D). Thus, this system was highly sensitive to MMPs and exhibited tumor-triggered targeting ability. Drug release from the β-CD-capped 14

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significant as a CDDS.

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3. Biomedical Applications

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3.1 MSN-based biotherapeutic agent delivery

Proteins, enzymes and peptides are being used as promising therapeutic agents in

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various medical fields including cancer therapy, vaccines and regenerative medicine. However, protein delivery is limited by particular intrinsic properties: (1) rapid degradation by proteases or peptidases present in body fluids; (2) denaturation due to

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the fragile structure; and (3) membrane impermeability [72, 73]. MSNs with tunable

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pore sizes have emerged as potential carriers for biotherapeutic agents. Moreover, MSNs can transport these biotherapeutic agents into the cytosol via endocytosis of the

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carriers and then escape from the endosomal compartment [74]. For example, Lin et

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al. [75] loaded cytochrome c inside MSNs and investigated the in vitro uptake of the cytochrome c-containing MSNs. The results demonstrated improved cellular uptake of these particles by HeLa cells. Moreover, the cytochrome c released from the MSNs retained high activity in catalyzing their substrates. To preserve the activity of the encapsulated

proteins,

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[76]

designed

multilayered

polyelectrolyte-modified MSNs in which the activity of the encapsulated catalase was retained even after exposure to enzyme-degrading substances; the multilayers protected the catalase from degradation. For vaccines, MSNs can function as drug delivery carriers and as adjuvants that increase the immune response. Carvalho et al. 15

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SBA-15 was compared with that of aluminum hydroxide salts and Incomplete Freund’s adjuvant. The results demonstrated that SBA-15 induced the production of

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both IgG1 and IgG2a isotypes. Moreover, this protein delivery system increased the immune response of mice that were poor responders.

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Therapeutic genetic material, including plasmid DNA, siRNA or antisense oligonucleotides, encompasses another important class of biotherapeutic agents that requires careful loading and delivery. The delivery of therapeutic genetic material is

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difficult because intracellular degradation processes after cellular internalization

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dramatically decrease nuclear access. MSNs are promising carriers for gene delivery because they provide an insulated space for incorporated genetic material, thereby

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protecting it from nuclease degradation [78]. For example, Lin et al. [79] prepared a

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novel MSN-based gene transfection system (Figure 7, A). G2-PAMAM-capped MSNs (G2-MSNs) were used to form a complex with a plasmid (pEGFP-C1) that encoded enhanced green fluorescence protein. The G2-MSNs effectively protected pEGFP-C1 DNA from restriction endonucleases. Compared with the transfection efficiency of other commercial transfection reagents (PolyFect: 5% ± 2%, SuperFect: 10% ± 2%, and Metafectene: 16% ± 2%), the transfection efficiency of the G2-MSNs reached 35% ± 5%. The permeability studies demonstrated that many G2-MSNs-DNA complexes were endocytosed into the cells, indicating that G2-MSNs can be employed as transmembrane delivery carriers for gene therapy (Figure 7, B). In 16

ACCEPTED MANUSCRIPT addition, Gu et al. packaged siRNA into the mesopores of magnetic MSNs using a strongly dehydrated solution [80]. Unlike the conventional DNA or RNA loading

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process, almost all the siRNA could be loaded into the mesopores of magnetic MSNs; the main driving forces for DNA adsorption into magnetic MSNs were shielded

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intermolecular electrostatic forces, dehydration effects and intermolecular hydrogen bonds. Then, polyethyleneimine (PEI) was chosen to coat the surface of the

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siRNA-loaded MSNs to prevent premature siRNA release from the magnetic MSNs. The successful knockdown of exogenous EGFP and endogenous Bcl-2 confirmed the efficient gene transfection.

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3.2 MSN-assisted bioimaging

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3.2.1 MSN-assisted fluorescent imaging MSNs have also emerged as appealing vehicles for fluorescent agents. It has been

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reported that MSNs are optically transparent due to their nanoscale particle size [81].

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Consequently, MSNs would not disturb the emission of fluorescent agents. Because of the hydrophilic surface of MSNs, fluorescent agent-containing MSNs become well dispersed in aqueous solution. In this section, we mainly discuss MSNs containing quantum dots (QDs), organic dyes or rare earth elements. After being embedded into MSNs, QDs can be protected from destruction by the chemical stability of MSNs. Considering that QDs can be easily oxidized, leading to fluorescence quenching, Pan et al. developed PEGylated liposome-coated QDs/MSNs to prevent the oxidation of QDs and to further improve the dispersion stability [82]. The in vitro release of cadmium ions (a type of QD) indicated that the 17

ACCEPTED MANUSCRIPT liposome-modified MSNs effectively prevented the degradation of QDs. In numerous studies, MSNs have been modified with various fluorescent dyes for bioimaging

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applications. Tsou et al. designed a wide range pH sensor based on HMS by modification with two different pH-sensitive dyes, FITC and rhodamine B

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isothiocyanate (RITC), to measure the intracellular pH in the cytosol and endosome-lysosome regions [83]. The co-localization of green and red spots indicated

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the coexistence of these two dyes on MSNs. By estimating the fluorescence intensity ratios of five regions of interest (ROIs) using CLSM, the corresponding pH values were calculated for the ROIs using a pH calibration curve; the data indicated that

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these dye-doped MSNs could measure local cellular pH values.

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However, QDs and fluorescent dyes that belong to the down-conversion luminescent material class are restricted by their inability to penetrate into tissue.

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Therefore, an alternative material, near-infrared (NIR)-to-vis up-conversion (UC),

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was developed to circumvent this limitation. These materials often contain rare earth metals, such as lanthanide. UC materials exhibiting high chemical stability can increase the signal-to-noise ratio when an essentially dark UC background is used. Zhang et al. developed a hexagonal α-NaYF4/Yb3+, Er3+-doped HMS with magnetic Fe3O4 NPs (named MUC-F-NR) for bioimaging and targeted chemotherapy (Figure 8, A) [84]. Electron microscopy suggested that the prepared MUC-F-NR had an α-NaYF4/Yb3+, Er3+ shell and a Fe3O4 core in the center of a hollow cavity (Figure 8, B). Then, DOX was loaded inside the mesopores of MUC-F-NR to verify the potential of this novel carrier for drug delivery and bioimaging. Figure 8 C illustrates the 18

ACCEPTED MANUSCRIPT following: (1) MUC-F-NR exhibited green luminescence under 980 nm laser excitation, indicating that this novel carrier can be used for bioimaging; (2) red DOX

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fluorescence was observed in the cell nuclei, confirming that DOX was released from MUC-F-NR into the nucleus; and (3) DOX was toxic to the tumor cells, as evidenced

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by apoptotic bodies and cell death. To further confirm the in vivo application of this UC bioimaging technique and its magnetic targeting efficiency, researchers compared

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the accumulation of DOX-loaded MUC-F-NR in hepatocarcinoma (H22) murine xenograft tumors in the absence or presence of a magnetic field (MF). The mice were imaged using 980 nm laser excitation. A significant increase in luminescence intensity

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at the tumor site was observed in the presence of a MF, indicating that the

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MUC-F-NR was suitable for targeted delivery and bioimaging applications.

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3.2.2 MSN-assisted MR imaging Magnetic resonance imaging (MRI) is an effective biomedical tool and offers the

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ability to non-invasively obtain anatomic and metabolic/functional information with high spatial and temporal resolution [85]. MSNs containing inorganic NPs, such as gadolinium (Gd) chelates, Fe3O4, or manganese oxide, have been used for MRI. MSN-based MR contrast agents are more sensitive due to their enhanced relaxivity and large surface area, which has high payloads of active magnetic centers [86]. More importantly, the mesopores of MSNs provide the protons of water molecules easy access into the magnetic center, which significantly reduces the T1 and T2 decay relaxation times [87, 88]. In addition, MSNs with a hydrophilic surface prevent inorganic NPs from aggregating in vivo [89-91]. Furthermore, MSNs modified with 19

ACCEPTED MANUSCRIPT targeted ligands can be effectively directed to abnormal tissues for diagnostic purposes. The accumulation of these MSN-based MR contrast agents at the targeted

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site contributes to the increased imaging sensitivity.

Lin et al. [92] coated MCM-41 with a Gd-Si-DTTA complex. The resulting

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MSN-Gd had a high Gd-DTTA loading efficiency, and the particles exhibited large longitudinal (r1) and transverse (r2) relaxivities. The r1 and r2 values on a per

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millimolar Gd basis were greater than those for a previously reported solid silica NP coated with a multilayered Gd-DTPA derivative [93]. The increased MR relaxivity was attributed to the ready access of water molecules into the magnetic centers.

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MSN-Gd can be utilized as an intravascular MR contrast agent due to the T1-weighted

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enhancement that was clearly visible in the aorta of a DBA/1J mouse 15 min after injecting 2.1 µmol/kg body weight of MSN-Gd using a 9.4 T scanner (Figure 9, A and

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B). This dose was much lower than that used for currently available contrast agents

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(0.1-0.3 mmol/kg) [94]. Moreover, at a higher dose of 31 µmol/kg, MSN-Gd was an effective T2-weighted contrast agent for MR imaging of liver abnormalities (Figure 9, C and D). MSNs containing Fe3O4 NPs (Fe3O4@mSiO2) have been prepared by Kim et al. for MRI [29]. The effectiveness of Fe3O4@mSiO2 as a T2-weighted MR contrast agent was evaluated by injecting these particles into tumor-bearing nude mice. Two hours after injection, Fe3O4@mSiO2 accumulation in tumors was detected in the T2-weighted MR images. Even at 24 h post-injection, Fe3O4@mSiO2 remained at the tumor sites. Shi et al. [95] developed a manganese oxide-based hybrid MSN (HMSN) as a 20

ACCEPTED MANUSCRIPT contrast agent for pH-responsive MRI and drug delivery. The relaxation rate, r1, of the sample was almost twice that of commercial GdⅢ-based contrast agents and was the

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highest r1 value ever reported for a manganese oxide NPs-based MRI-T1 contrast agent. Moreover, due to the dissolution nature of manganese oxide under weak acidic

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conditions, the relaxation rate r1 was markedly increased. The morphology and principles of MRI-T1 enhancement in an acidic environment are presented in Figure

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10. The color of the HMSN solution changed gradually at pH 5.0 because of the dissolution of MnOx, whereas no such change occurred at pH 7.4 (Figure 10, G and H). The dissolution of the manganese ions in the acidic solution significantly

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increased the T1-weighted image. To test manganese oxide-based MSNs as an

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effective MRI contrast agent in vivo, a sample was injected into tumor-bearing mice. The peripheral tumor vasculature exhibited significant contrast enhancement during

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the initial stage, and there was a gradual enhancement of the T1 contrast in the tumor

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interior over the time course, which was attributed to manganese ions leaching out in the weakly acidic tumor microenvironment (Figure 10, I-M). 3.3 MSNs as bioactive materials for tissue regeneration Bioactive materials play a significant role in the field of tissue regeneration [96]. MSNs with excellent surface properties and porosity have shown to be attractive candidates as biomaterials for bone regeneration. The silanol groups on the surface of MSNs react with body fluids to generate active nano-sized carbonated apatite, which can bind to natural bone (Figure 11, A). This bioactive bond ensures the integration of implanted bone [30]. Furthermore, a recent report suggested that 50 nm silica NPs 21

ACCEPTED MANUSCRIPT effectively inhibited osteoclast differentiation and stimulated osteoblast differentiation,

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the use of MSNs in bone tissue regeneration will be discussed.

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thus increasing bone mineral density in vivo [97]. In this section, recent advances in

Vallet-Regí et al. synthesized three different types of mesoporous silica materials

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(SBA-15, MCM-48 and MCM-41) to evaluate their potential application in bone regeneration by soaking them in simulated body fluids. In vitro bioactivity studies

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suggested that an apatite-like layer was produced on the surface of SBA-15 and MCM-48 after 30 and 60 days, respectively, indicating that these mesoporous materials were valuable for bone regeneration engineering [98]. Although pure

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MCM-41 did not exhibit any bioactive behavior, phosphorus- or bioactive

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glass-doped MCM-41 induced the formation of an apatite layer [99, 100]. Therefore, the textural and structural properties of MSNs are important factors that influence

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bioactive behavior, and the kinetics of apatite formation can be regulated and

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improved by combining phosphorous with mesoporous silica or by adding small amounts of bioactive glass or other bioactive ingredients. In addition, MSNs can encapsulate osteogenic agents, which promote the formation of new bone in vivo; therefore, the ability of MSNs to act as bone regenerators and to carry osteogenic agents suggests the potential for designing MSNs with specific medical applications. For example, Balas et al. [101] used SBA-15 as a bioactive matrix to simultaneously induce bone-tissue regeneration and load L-Tryptophan (L-Trp), a hydrophobic amino acid that accelerates the bone healing and formation process. Due to the hydrophobic nature of L-Trp, bioactive SBA-15 was not capable of loading L-Trp inside its 22

ACCEPTED MANUSCRIPT mesopores. Therefore, SBA-15 was modified with quaternary amines to increase the surface hydrophobicity, resulting in increased L-Trp loading efficiency and sustained

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L-Trp release due to the electrostatic and hydrophobic interactions between the amino acid and the modified MSNs. Moreover, MSNs modified with different groups (such

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as thiol or amino groups) can recruit or bind to specific proteins (such as osteogenic peptides and growth factors) in vivo to accelerate tissue regeneration (Figure 11, B)

applications.

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4. Conclusions and Outlook

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[102], which opens up a new avenue for designing bioactive-silica for biomedical

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In this review, we highlighted the major research advances involving drug delivery and biomedical applications based on MSNs. With their versatile mesoporous

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structure and porosity, MSNs have significant advantages. One, the pore size and

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morphology of MSNs are adjustable, which makes them suitable for loading various cargos. Two, as drug delivery systems, surface-modified MSNs can release their cargo in a controlled manner. Three, multifunctional (magnetic and luminescent) MSNs offer the possibility of simultaneous bioimaging and drug delivery. Overall, the research advances in MSN delivery systems are exciting and hold great potential for future biomedical applications. However, there remain several significant challenges that need to be addressed and investigated to enable the development of practical applications in the treatment of human diseases. For example, methods for designing novel multifunctional MSNs 23

ACCEPTED MANUSCRIPT with efficient, regulated drug release that can also be monitored in real-time in target tissues via bioimaging must be developed. Although there have been some studies

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involving multifunctional MSNs, innovative methods for functionalization remain limited. More detailed knowledge about drug delivery systems and bioimaging is

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necessary to develop more comprehensive diagnostics and therapeutics in the field of nanomedicine.

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Although the toxicity at a cellular level has been investigated, we are still far from using MSNs for clinical diagnostics and treatment. First, the biodistribution and acute/chronic toxicity differ based on the MSN administration route. Second, recent

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reports have indicated that 110 nm MSNs can elicit an inflammatory response around

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the injection site after intramuscular or hypodermic injection [103]. This suggests that more comprehensive and detailed toxicity studies are imperative before MSNs are

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used in patients and must have a particular focus on the toxicity induced by different

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administration routes or MSN particle sizes. To create MSNs that are suitable for clinical applications without undesirable side effects, better and more extensive in vivo testing is necessary for this new and rapidly expanding field of nanomedicine.

24

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[93] Cormode DP, Skajaa T, van Schooneveld MM, Koole R, Jarzyna P, Lobatto ME, et al. Nanocrystal core high-density lipoproteins: a multimodality contrast agent platform. Nano letters. 2008;8:3715-23. [94] Seo WS, Lee JH, Sun X, Suzuki Y, Mann D, Liu Z, et al. FeCo/graphitic-shell nanocrystals as advanced magnetic-resonance-imaging and near-infrared agents. Nature materials. 2006;5:971-6. [95] Chen Y, Yin Q, Ji X, Zhang S, Chen H, Zheng Y, et al. Manganese oxide-based multifunctionalized mesoporous silica nanoparticles for pH-responsive MRI, ultrasonography and circumvention of MDR in cancer cells. Biomaterials. 2012;33:7126-37. [96] Castner DG, Ratner BD. Biomedical surface science: Foundations to frontiers. Surface Science. 2002;500:28-60. 35

ACCEPTED MANUSCRIPT [97] Beck Jr GR, Ha S-W, Camalier CE, Yamaguchi M, Li Y, Lee J-K, et al. Bioactive silica-based nanoparticles stimulate bone-forming osteoblasts, suppress bone-resorbing osteoclasts, and enhance

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bone mineral density in vivo. Nanomedicine: Nanotechnology, Biology and Medicine. 2012;8:793-803. [98] Izquierdo-Barba I, Ruiz-González L, Doadrio JC, González-Calbet JM, Vallet-Regí M. Tissue

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regeneration: a new property of mesoporous materials. Solid state sciences. 2005;7:983-9. [99] Vallet-Regí M, Izquierdo-Barba I, Rámila A, Pérez-Pariente J, Babonneau F, González-Calbet JM.

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Phosphorous-doped MCM-41 as bioactive material. Solid state sciences. 2005;7:233-7. [100] Horcajada P, Rámila A, Boulahya K, González-Calbet J, Vallet-Regí M. Bioactivity in ordered mesoporous materials. Solid state sciences. 2004;6:1295-300.

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materials to promote bone formation. Acta Biomaterialia. 2008;4:514-22. [102] Vallet-Regí M, Ruiz-González L, Izquierdo-Barba I, González-Calbet JM. Revisiting silica based

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ordered mesoporous materials: medical applications. Journal of Materials Chemistry. 2006;16:26-31.

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[103] Fu C, Liu T, Li L, Liu H, Chen D, Tang F. The absorption, distribution, excretion and toxicity of mesoporous silica nanoparticles in mice following different exposure routes. Biomaterials. 2013;34:2565-75.

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ACCEPTED MANUSCRIPT Figure Captions Figure 1. MSNs increase the oral bioavailability of poorly soluble drugs via an

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improved dissolution rate and enhanced drug permeability. (A-B) TEM photographs of the cellular uptake of MSNs. (C) Confocal Laser Scanning Microscopy (CLSM)

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images of Caco-2 cells after incubation for 0.25 h, 1 h or 2 h at 37 °C with fluorescein isothiocyanate (FITC)-MSN particles. The cell nucleus is blue. The plasma membrane

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is red, and the FITC-MSNs are green in the cells. (D) The mean cumulative transport (apical to basolateral permeating direction) versus time was graphed for 3.88 µmol/L TEL in a Caco-2 cell monolayer. CsA is an inhibitor of P-glycoprotein. (E)

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Dissolution profiles of TEL. (F) The mean plasma concentration versus time for TEL

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after an oral dose in beagle dogs. (Reproduced with permission from [22], copyright by the American Chemical Society).

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Figure 2. (A-C) TEM images of MSNs revealed that the respective mean pore sizes

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for samples A (MSNs-1), B (MSNs-2) and C (MSNs-3) were approximately 4, 9 and 15 nm. (D) Release profiles of TEL from TEL-MSNs and from pure crystalline TEL in enzyme-free simulated intestinal fluid (pH 6.8). (E) Structural formula of TEL. (Reproduced with permission from [36], copyright by Elsevier). Figure 3. Different gatekeepers on the pore outlets of MSNs for stimuli-responsive CDDSs. Figure 4. (A) Stalk synthesis, cargo loading, pore capping, and cap release under acidic conditions. (B) elease profiles of DOX from β-CD-MSNs. (C) KB-31 cancer cells effectively endocytosed DOX-loaded FITC-MSN at 3 h; apoptotic bodies 37

ACCEPTED MANUSCRIPT appeared by 60 h, followed by nuclear fragmentation after 80 h. (Reproduced with permission from [45], copyright by the American Chemical Society).

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Figure 5. (A) In vivo process of a MSN-based controlled and targeted drug delivery system. The two approaches to multifunctional MSN-based drug delivery systems are

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to design (B) a multifunctional targeting molecule that acts as both targeting and capping agent or (C) a stimuli-responsive gatekeeper that is further modified with a

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(A)

Schematic

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redox-responsive CDDS. (B) PASP release from unloaded MSNs under different

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conditions (■ in the presence of MMP- ; ▲ in the presence MMP-2 and an MMP

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inhibitor). (C) and (D) CLSM images of SCC-7 and HT-29 cells treated with DOX-loaded MSNs in the (A−C) absence or (D−F) presence of an MMP inhibitor. (A,

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D) Red fluorescence images, (B, E) confocal field images, and (C, F) overlap of the

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confocal fluorescence and brightfield images. (G) Flow cytometry analysis of SCC-7 and HT-29 cells treated with or without DOX-loaded MSNs in the absence (red) or presence (blue) of an MMP inhibitor or without treatment (blank). (Reproduced with permission from [71], copyright by the American Chemical Society). Figure 7. (A) Schematic illustration of a Texas Red-loaded, G2-PAMAM dendrimer-capped MSN complex containing enhanced fluorescence protein plasmid DNA. (B) TEM micrographs of G2-MSN-DNA complexes (black dots) endocytosed by CHO cells (a), HeLa cells (b), and astrocytes (c). Subcellular organelles, such as mitochondria and Golgi, are near MSNs in panels (b) and (c). Panel (d) shows a 38

ACCEPTED MANUSCRIPT logarithmic plot of HeLa cell growth with (O) or without ( △ ) G2-MSNs. (Reproduced with permission from [79], copyright by the American Chemical

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Society).

Figure 8. (A) Synthetic procedure for the drug-loaded Fe3O4@SiO2@α-NaYF/Yb, Er

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nanorattles (DOX-MUC-F-NR). (B) SEM (left) and TEM (right) images of MUC-F-NR. (C) Fluorescence microscopy images after incubation with DOX-loaded

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MUC-F-NR (a-f) or with the negative control (g). In the fluorescence microscopy images, the cell nuclei are blue (Hoechst 33258 dye), DOX produced red fluorescence after 480 nm excitation, and MUC-F-NR produced green luminescence after

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excitation by up-converted 980 nm light. Mice bearing H22 xenograft tumors were

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injected with DOX-MUC-F-NR (1 mg/kg) and subjected (+MF) or not subjected (-MF) to a magnetic field for 1 h. At 24 h post-injection, in vivo imaging of the mice

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was performed (h). (Reproduced with permission from [84], copyright by the

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American Chemical Society). Figure 9. (A) Pre-contrast and (B) post-contrast (2.1 µmol/kg dose) T1-weighted mouse MR image showing aorta signal enhancement. (C) Pre-contrast and (D) post-contrast (31 µmol/kg dose) mouse MR images showing liver signal loss due to T2-weighted enhancement. (Reproduced with permission from [92], copyright by the American Chemical Society). Figure 10. (A) Schematic illustration of the microstructure of HMSNs. (B) TEM image of HMSNs (inset: SEM image). (C-E) Element mapping of Si (C), O (D) and Mn (E) in HMSNs. (F) Color merged image of C, D and E. (G-H) T1-weighted 39

ACCEPTED MANUSCRIPT solution MR images of HMSNs under different pH environments (G: pH 5.0; H: pH 7.4) after co-incubation at 37 °C for 4 h. In vivo T1-weighted MRI of a tumor before (I)

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and after the intravenous administration of HMSNs (J: 5 min, K: 15 min, L: 30 min and M: 60 min; arrows indicate the tumor tissue). (Reproduced with permission from

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[95], copyright by Elsevier).

Figure 11. Schematic illustration of bone tissue regeneration based on MSNs.

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MSN-based bone tissue regeneration involves two approaches. (A) MSNs react with body fluids to generate active apatite for bone regeneration. (B) Modified-MSNs enable the attachment of specific proteins (osteogenic peptides and growth factors)

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that accelerate bone regeneration.

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ACCEPTED MANUSCRIPT Table 1. MSNs-based controlled and targeted drug delivery carriers Gatekeeper

Stimuli

Trigger

Polyelectrolyte layer

pH

acid

Targeting agent ErbB-2

Lipid bilayer

pH

acid

SP94 peptide

Doxorubicin

61

CaP

pH

acid

Doxorubicin

64

Fe3O4 NPs

pH

acid

Dexamethasone

67

β-Cyclodextrin

Redox

Doxorubicin

71

Collagen

Redox Redox

Folate

Redox

Fluorescein isothiocyanate Fluorescein isothiocyanate Doxorubicin

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Fe3O4 NPs

Hyaluronic acid

Enzyme

Reducing agent (DTT, GSH) Reducing agent (DTT, GSH) Reducing agent (DTT, GSH) Reducing agent (DTT, GSH) Hyaluronidase-1

Hyaluronic acid Magnetic Fe3O4 Arg-Gly-Asp (RGD) motif Lactobionic acid Magnetic Fe3O4 Folate

Doxorubicin

69

Rotaxane

Enzyme

Camptothecin

66

Ref.

TNF-α protein

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Model drug

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Porcine liver esterase

Hyaluronic acid Folate

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Mesoporous silica nanoparticles in drug delivery and biomedical applications.

In the past decade, mesoporous silica nanoparticles (MSNs) with a large surface area and pore volume have attracted considerable attention for their a...
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