The application of mesoporous silica nanoparticle family in cancer theranostics

Accepted Manuscript Title: The application of mesoporous silica nanoparticle family in cancer theranostics Author: Yin Feng, Nishtha Panwar, Danny Jian Hang Tng, Swee Chuan Tjin, Kuan Wang, Ken-Tye Yong PII: DOI: Reference:

S0010-8545(16)30108-4 http://dx.doi.org/doi: 10.1016/j.ccr.2016.04.019 CCR 112248

To appear in:

Coordination Chemistry Reviews

Received date: Revised date: Accepted date:

14-3-2016 29-4-2016 30-4-2016

Please cite this article as: Yin Feng, Nishtha Panwar, Danny Jian Hang Tng, Swee Chuan Tjin, Kuan Wang, Ken-Tye Yong, The application of mesoporous silica nanoparticle family in cancer theranostics, Coordination Chemistry Reviews (2016), http://dx.doi.org/doi: 10.1016/j.ccr.2016.04.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The Application of Mesoporous Silica Nanoparticle Family in Cancer Theranostics Yin Fenga,c#, Nishtha Panwara#, Danny Jian Hang Tnga, Swee Chuan Tjina, Kuan Wangb,d, and Ken-Tye Yonga a

School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore, 639798 b Nanomedicine Program and Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan c Laboratory of Chemical Genomics, School of Chemical Biology & Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China d College of Biomedical Engineering, Taipei Medical University, Taiepi 110, Taiwan # These authors contributed equally to this work

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Contents 1. Introduction 2. Structure and Properties of Mesoporous Silica Nanoparticles 2.1. M41S type MSNs family 2.2. SBA-15 2.3. ORMOSIL nanoparticles 2.4. Hollow type MSNs 2.4.1. Hard-template MSNs 2.4.2. Soft-template MSNs 2.4.3. Self-Assembled MSNs 3. Silica Nanoparticles in Cancer Therapy 3.1. Early Cancer Detection and Diagnosis 3.1.1. Silica Nanoparticles as Imaging Contrast Agents 3.1.2. Mesoporous Nano Silica Chips 3.1.3. Fluorescent Silica Nanoparticles for Optical Imaging 3.2. MSNs-based drug delivery systems for cancer therapy 3.2.1. Passive delivery system 3.2.2. Active delivery system 3.2.3. Controlled-release drug delivery systems 3.2.3.1. pH-triggered drug release system 3.2.3.2. Temperature-triggered drug release system 3.2.3.3. Redox potential-triggered drug release system 3.2.3.4. Enzyme-triggered drug release system 3.2.3.5. Light-triggered drug release system 3.2.3.6. Other stimuli-triggered drug release system 3.3. Co-delivery of gene and drugs assisted by MSNs-based drug delivery system for combatting cancer multidrug resistance 4. Multifunctional delivery platform based on MSNs 5. Conclusion and Future perspectives References

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Review Article: Highlights  The properties and structures of different types of MSNs are discussed.  Various configurations of MSNs-based systems for cancer diagnosis and therapy are highlighted.  Role of MSNs-based drug delivery systems for combatting the cancer multidrug resistance is outlined.  A comprehensive timeline of the evolution of MSNs for biological applications is presented.

Abstract Cancer is among the most serious diseases characterized by uncontrollable cell growth and spread of abnormal cells. Cancerous cells form tumors that negatively impact the functions of the body, inducing serious malfunctioning leading to fatalities in most cases. Up to now, the effective diagnosis and treatments of cancer have remained a big challenge. Nanotechnology is an emerging field encompassing science, engineering and medicine, which has attracted great attention for cancer therapy in recent years. Among the numerous nanomaterials, Mesoporous Silica Nanomaterials (MSNs) have attracted great attention and are being considered as promising biomedical materials for the development of cancer therapies because of their size tunability, surface functionality, optically transparent properties, low toxicity and high drug loading efficiency. In this review, we first outline the properties and structure of different configurations of MSNs and their subsequent application in the field of cancer theranostics. Thereafter, the potential of MSNs as multifunctional delivery platforms for therapeutic agents and their significance in next generation cancer therapy is discussed.

Keywords: mesoporous silica nanoparticles, MCM-41, SBA-15, ORMOSIL, drug delivery, stimuli 1. Introduction

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Cancer is characterized by abnormal and uncontrollable cell growth. With malignant phenotypic behavior such as metastasis and invasion, cancer adversely affects different parts of the body and is considered among the most chronic diseases all over the world [1, 2]. In recent years, worldwide incidence and mortality rates of cancer have been rising sharply. Based on the 2004 World Health Organization (WHO) statistics, cancer stands as the primary factor of death in developed countries. In developing countries, it is the leading cause of fatality second only to cardiovascular disease. The number of worldwide deaths due to cancer is increasing at an alarming rate, from 10 million (13% of all deaths) in 2000 to 12 million in 2020 [3, 4]. Based on the different stages of cancer, patient age and health status, cancer treatments need to be customized and combined with several other therapies. Contemporary cancer treatment modalities such as surgery, chemotherapy, radiotherapy and photodynamic therapy (PDT) are able to prolong patient’s lives to some extent. Although physical methods like surgery are effective for patients with non-metastatic cancer, other systemic therapies are required in cases where the cancer has entered metastasis and spread throughout the body. Radiotherapy is the common alternative for surgery which utilizes high-energy rays to cause damage to cancer cells, followed by apoptosis. Nevertheless, radiotherapy can pose serious side-effects including the risk of secondary malignancy in the irradiated area and severe damage of normal and healthy tissues. Another method, chemotherapy, involves the use of one or more chemotherapeutic agents to destroy cancer cells. Most chemotherapeutic agents lack cell specificity, resulting in damage of normal cells with irreversible systemic side-effects. Besides the non-specificity, the development of multi-drug resistance (MDR) by cancer cells is a critical limitation for the low therapeutic index of chemotherapy [5]. More selective methods such as PDT, which is relatively new, use photosensitizing agents to kill cancer cells upon light activation. Photosensitizing agents are

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effective only if they have been activated by a specific type of light precisely directed at the cancer cells, thus making PDT more selective and less toxic than chemotherapy [6]. The limiting factor of this method however, is the low efficiency of light penetration for deeply located tumors within the body and the development of MDR towards the PDT agents in the treated cancer cells [7]. As a consequence, tumor recurrence, metastasis, resistance to chemotherapy and side effects caused by radiotherapy and chemotherapy remain the major bottlenecks in cancer therapy. Well-designed diagnostic, therapeutic and prognostic strategies are urgently needed for the effective treatment of cancer. Nanomedicine is an emerging field, integrating nanotechnology and biomedicine, which offers promising therapeutic potential for various diseases including cardiovascular disease, diabetes, tissue engineering and cancer theranostics. The rapid development of new nanomaterials has provided great opportunities to overcome chemotherapeutic side-effects while promising the diagnosis of cancer at preliminary stage. The first Food and Drug Administration (FDA)approved nano-drug, Doxil, is a typical example, where Doxorubicin (DOX) is encapsulated in liposomes for prolonged circulation time and bioavailability of DOX, and diminished sideeffects to heart muscles and other normal tissues [8]. In 2011, the first silica-based tumor diagnostic nanoparticles- Cornell dots (C-dots) were approved by FDA for stage I human clinical trial. C-dots are dye-entrapped silica nanoparticles with ultra-small size (<10 nm), which can be utilized as diagnostic tools to assist surgeons in identifying tumors [9]. Subsequently, tremendous efforts have been devoted towards functionalized nanoparticles for cancer theranostics. Christopher Loo et al. have pioneered this field through the engineering of immunetargeted nanoshells to detect and destroy breast carcinoma cells, by demonstrating bioimaging coupled with cancer therapy [10]. Another group has utilized anti-epidermal growth factor

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receptor (EGFR)-gold nanorods (AuNRs) to treat malignant oral epithelial cells, developing AuNRs as reagents for cancer cell diagnostics and selective photothermal therapy [11]. Similarly, MSNs bear enormous potential as functionalized nanomaterials. The earliest examples include the synthesis of folic acid (FA) modified-MSNs for targeted delivery of the hydrophobic anticancer drug camptothecin (CPT) [12, 13]. These studies have shown significant in vitro and in vivo tumor suppression effects by mesoporous silica nanoplexes, and achieved imaging and cancer therapy concurrently [12, 14]. With further progress in nanomaterial research, nanomedicine is envisaged to hold a strong stake in cancer diagnosis and therapy. Silicon dioxide (SiO2), also known as silica, is among the most abundant naturally available minerals on earth and a crucial component for human health, especially for skin, bones, hair and nails. Classified by the FDA as “Generally Recognized As safe” (GRAS), SiO2 is widely used in food additives, cosmetics and pharmacy. Due to the biosafety and easy synthesis of silica, silicabased nanomaterials occupy a prominent status in biomedical research. In recent years, MSNs have attracted increasing attention for optical imaging, magnetic resonance imaging (MRI), PDT and drug delivery [15-20]. Since the proposal of MCM-41 type MSNs as nanocarriers for delivering therapeutics in 2001 [21], a variety of MSNs such as MCM48 [22], SBA-15 [23, 24], TUD-1 [25], HMM-33 [24] and FSM-16 [26] have been engineered and applied as drug delivery systems extensively. As drug delivery vehicles, MSNs offer several advantages: (i) large internal surface area and pore volume enabling MSNs an effective drug delivery vehicle for a range of therapeutic agents, (ii) tunable particle size (50 -300 nm) permitting facile endocytosis across living animal and plant cells with minimal cytotoxic effects, (iii) a tunable porous structure with controllable narrow pore size distribution, allowing the loading of different therapeutic agents with highly precise drug release kinetics, (iv) a highly hydrophobic and rigid matrix structure

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facilitating MSNs to remain uniformly dispersed in water and resist changes due to pH, heat, mechanical stress and hydrolysis-induced breakdown, (v) the internal and external surface can be selectively functionalized, enabling MSNs to offer targeted delivery and controlled release, (vi) a uniquely porous structure preventing premature release of its loaded components even when its pores are not fully capped [19, 20, 27, 28, 29]. MSNs exhibit incredible advantages over other drug delivery nanocarriers and provide promising opportunities for simultaneous cancer diagnosis and therapy. In this review, we focus on the current advances of MSNs as drug delivery systems for cancer theranostics. The structure and properties of different types of MSNs such as nanoparticulate MSNs, hollow/rattle MSNs and organically modified silica (ORMOSIL) nanoparticles are first discussed. Next, the recent research and progress of MSNs based-cancer therapies is highlighted. The applications of MSNs in cancer detection, diagnosis and drug delivery are summarized in this content. Furthermore, we pay special attention on discussing these MSNs-based drug delivery platforms in cancer therapies, which highlight their clinical applications for cancer theranostics, especially in the early diagnosis of pancreatic cancer and other malignant tumors. 2. Structure and Properties of Mesoporous Silica Nanoparticles MSNs are defined as a type of nanomaterial between the microporous and macroporous materials, with the pore diameter spanning from 2 nm to 50 nm [30]. MSNs can be classified as: M41S type MSNs family, organically modified silica (ORMOSIL) nanoparticles and hollow type MSNs (Figure 1). In 1992, MSNs were first synthesized through sol–gel technique and the pore diameter was adjustable from 2 to 10 nm [31]. This kind of MSNs, also known as Mobile Crystalline Material-41 (MCM-41), has since been extensively used in biomedical research. In 2001, the application of MCM-41 nanoparticles in drug delivery was first described by Vallet-

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Regi and co-workers, opening up a novel platform for the use of MSNs in biomedical engineering [21]. Thereafter, a range of MSNs have been proposed, developed and utilized for drug delivery. Presently, the most commonly studied MSN structures for drug delivery applications are based on MCM-41 and/or Santa Barbara Amorphous-15 (SBA-15). 2.1.M41S type MSNs family The discovery of a family of nano-structured MSNs called M41S, laid the foundation for the application of MSNs in drug delivery systems [32, 33]. MCM-41 is the basic model of mesoporous silica nanocarriers, with a hexagonal porous structure and is most widely studied material in the M41S family and has wide applications in biomedicine (Figure 2A and 3A). With large surface area, high thermal stability and narrow pore size distribution, MCM-41 is a substantial improvement over other mesoporous nanomaterials [34, 35]. Today, synthesis of MCM-41 is highly controllable, and the Stöber method (commonly called the sol-gel technique) is employed to synthesize monodispersed silica nanoparticles [36, 37]. The physical structures of these nanoparticles are highly controllable, with different structures being synthesized using different solution compositions, concentrations [38] and temperature [39]. Variations in the synthesis parameters alter the material structure at different levels, such as material size (Figure 3D) [40], surface area (up to 700 m2/g) and pore size (1.6 nm to 10 nm) (Figure 3B) [28, 34]. These structural tunabilities supplement M41S family with many beneficial properties for biomedical applications. Modifications in the shape of these MCM-41 nanoparticles considerably affect the drug delivery characteristics [21]. For instance, spherical and tubular shaped MCM-41 were commonly used as drug delivery systems due to their high surface area and narrow pore diameters (Figure 3C) [41]. Another property of MCM-41 nanoparticle is their ability to conjugate with metal ions and form complexes such as Al-MCM-41 [42], Mn-MCM-41

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[43], and Fe-MCM41 [44]. These metal ion-MCM-41 complexes enhance the performance of MCM-41 silica nanoparticles for MRI-based imaging capabilities in addition to its ability to act as efficient drug delivery systems [16, 17, 45]. When arranged into three dimensional structures to form the cubic MCM-48 and laminar MCM50, M41S MSNs exhibit even more unique properties which can be exploited for cancer treatment. MCM-48 features a three-dimensional pore system with a cubic structure, modeled as a gyroid minimal surface [46] (Figure 2B). MCM-48 possesses a higher surface area (upto 1600 m2/g), more than double of MCM-41. Additionally, it has an interwoven and branched pore structure providing it enhanced thermal stability [47]. The unique penetrating bicontinuous channel in MCM-48 allows rapid molecular transport and promotes easy molecular accessibility, which are vital in some drug delivery systems [48]. On the other hand, lamellar phase-MCM-50 consists of silicate or porous aluminosilicate layers separated by surfactant layers (Figure 2C). This phase is obtained by sheets or bilayers of surfactant molecules with hydrophilic head groups pointing towards the silicate at the interface [49]. MCM-50 nanoparticles are used as catalysts and sorbents in the fabrication processes of other mesoporous solids such as silica galleries, which have diverse biomedical applications [50].

2.2.SBA-15 Santa Barbara Amorphous (SBA-15) is another type of MSNs which is extensively explored as a drug delivery system. These MSNs are synthesized using polymer templates such as amphiphilic triblock copolymers, which have mesostructural ordering properties [51]. The structure of SBA15 mainly depends on the pH-levels during the synthesis process [52]. Likewise MCM-41, the temperature during synthesis varies its physical characteristics such as its pore size [53]. SBA-15

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nanoparticles share many similar characteristics with MCM-41 such as a well-ordered hexagonal mesoporous structure and one-dimensional parallel channels [54, 55]. However, compared to MCM-41, it has several structural differences. The thicker pore walls (3.1 - 6.4 nm) of SBA-15 provide a higher hydrothermal and mechanical stability. The wide pore sizes (5 nm to 30 nm), large particle diameter (>200 nm) and high internal surface area (400-900 m2/g) make SBA-15 a promising material for numerous applications, such as adsorption and separation [56, 57], advanced optics [58, 59] and catalysis [60]. In addition, SBA-15 has a rougher surface morphology with larger pore diameters of 5-30 nm, compared to MCM-41 which has relatively smoother pore wall surfaces and smaller pore diameters of 2-10 nm [55]. Modified or functionalized-SBA-15 nanoparticles are used as efficient catalysts [61], as tools for adsorption and separation [62] and for bulk drug delivery applications [63]. Recent research has concentrated on the synthesis of smaller SBA-15 type MSNs with particle size below 200 nm, which can further improve its drug delivery capabilities [64]. 2.3.ORMOSIL nanoparticles As discussed above, ordered mesoporous silica nanoparticles (M41S family, SBA-15, etc.) encompass a large scope for applications in various fields. A major reason for this is the interesting set of properties they offer, such as tunable diameter, pore size, large surface area, ordered mesoporous structure and good biocompatibility. However, their large particle size (>200 nm), structural and colloidal instabilities still need to be addressed for wider applications. ORMOSIL nanoparticles (Figure 4) have emerged as an exciting hybrid materials having attracted great interest in biochemical field in the past decade. With the combination of tetraethyl orthosilicate (TEOS)/ vinyltriethoxysilane (VTES) as inorganic silica precursors, (3-aminopropyl) triethoxysilane

(APTES)/

mercaptopropyltrimethoxysilane

(MPTMS)/

diethylenetriamine

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(DETA) as organic silica precursors and weak alkali solutions as catalysts, ORMOSIL nanoparticles can be easily obtained as an oil-in-water microemulsion at room temperature. Without the reliance on surfactants and corrosive solvents, the synthetic formation process of ORMOSIL is simple, feasible and time-saving. ORMOSIL nanoparticles bear various advantages for biomedical applications: (i) they can be incorporated with a host of surface functionalities (hydroxyl/amino/thiol/carboxyl groups depending on the organic precursors), supplementing them with additional functions such as fluorescence and bio-targeting capabilities [65]; (ii) the presence of hydrophobic and hydrophilic groups on the precursors help their selfassembly both as normal and reverse micelles to satisfy the polarity of cargos [66]; (iii) their particle size (from 10 to 100 nm) can be easily tailored by varying the concentrations of surfactant and precursors for different applications [67, 68]; (iv) biodegradation of ORMOSIL nanoparticles can be skillfully realized by the decomposition of their Si-C bond; (v) their inert and transparent properties make them suitable for doping with diverse fluorophores for optical imaging; (vi) their robustness and storage stability enhance the shelf-life and convenience for long-term research. 2.4.Hollow type MSNs The large porosity of MCM-41 and SBA-15 type MSNs have demonstrated excellent reagent loading capabilities. Optimization has resulted in MSNs with higher loading efficiency for a broader spectrum of biomedical applications. One of the obvious improvement is the emergence of hollow type MSNs, which possess a hollow core-mesoporous shell structure. These hollow type MSNs have been extensively explored in many research fields, including catalysis [69], acoustic [70], absorption [71] and bioimaging [72]. Due to their super-high drug loading capacity (> 1 g drug per 1 g silica) and ease of surface functionalization, hollow type MSNs are now

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being considered as an ideal drug delivery system for chemotherapy, to overcome the chemotherapeutic failure by inefficient intracellular accumulation [69, 73, 74]. Hollow type MSNs are further classified according to the fabrication methods used in their synthesis: hardtemplate MSNs, soft-template MSNs and self-template MSNs. 2.4.1. Hard-template MSNs Hard-template MSNs are the most commonly utilized MSNs due to their simple and effective fabrication methods. These MSNs have structures with tunable thickness, mesoporosity, and functionality. To fabricate these structures, non-silica nanomaterials such as polymer beads, metal or metal oxide nanoparticles and semiconductor nanoparticles are usually used as templates for assembly of the silica-shell structure. Major steps involved in the hard-template method are: core template synthesis, surface activation of the polymer latexes, silification to form shell layer and selective removal of templates (Figure 5) [75]. The structural properties of the synthesized MSNs are primarily governed by the templates used. Excellent size tunability has been achieved using polymer core templates [76]. A unique characteristic of hollow type MSNs is that the direction of the pores can be precisely controlled, and mesopores perpendicular to the core surface can be fabricated using dual-latex surfactant templates [77]. The direction of the porosity greatly influences the MSNs release characteristics, providing a promising advantage over solid core MSNs [78]. Shell thickness, which determines the diffusion length of loaded reagents, can be controlled using precursors such as a basic ethanol/water mixture that affect the core properties [79]. Furthermore, the use of stimuli-responsive agents (or capping agents) can block the pore entrances of drug-encapsulated MSNs, regulating drug release while concurrently avoiding aggregation or fusion. To date, a series of capping agents such as metal nanoparticles

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[80], polymers [81] and proteins [82] have been produced for the controlled release of MSNsbased drug delivery systems, which promote further applications of MSNs in biomedicine. A significant advantage of the hollow type MSNs is their ability to leverage upon other functional nanoparticles. Using other nanoparticles as templates for MSN shell fabrication, the strengths of both the nanostructures can be combined for diverse applications. Additionally, using polymer cores based on other functional nanoparticles solves many of the limitations faced in traditional synthesis routes, such as the aggregation or fusion of hollow type MSNs, nonuniform hollow type MSNs with rough shell texture, and a mixture of solid MSNs in the final product [83]. Metal, metal-oxide and semiconductor nanoparticles form the core templates for the fabrication of these hollow type MSNs. Mesoporous silica-coated gold nanorods (Au@SiO2) is a classic example for cancer theranostics, possessing a high drug payload and photothermal effect [84]. With mesoporous silica as shells, the silica-shelled single quantum dot (QD) micelles is used as fluorescent cell tracers without cytotoxicity [85]. Using functional nanoparticles as templates generate uniform pore channels in hollow, yolk-shell structured mesoporous spheres that exhibit high catalytic activity [86]. Although hard-template MSNs have been extensively utilized in the synthesis of hollow type MSNs, the major weakness of hard-template MSNs is the tedious multistep and time-consuming procedures in fabrication. These factors call for further refinement in synthesis procedures for the widespread use of hard-template MSNs. 2.4.2. Soft-template MSNs Soft-template MSNs are fabricated using soft materials such as “micelles”, microemulsion droplets or vesicular structures formed by surfactants as soft templates. A variety of amphiphilic surfactants have been studied and utilized to build the structure of hollow type MSNs. Compared with hard-template MSNs, soft-template MSNs involve simpler fabrication processes and are

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facile under mild reaction conditions [87]. Another attractive feature of soft-template MSNs is their ability to encapsulate other reagents during its synthesis process [88]. This potentially allows soft-template MSNs to have a wide range of multifunctional capabilities, making them more versatile than hard-template MSNs. Mou et al. have used a simple ternary water-in-oil microemulsion system to produce rattle-type gold nanocatalysts embedded within hollow type MSNs (Au@HSNs), creating poison-resistant nanoparticles with high levels of catalytic activity [88]. More recently, magnetic hollow type MSNs have been fabricated by a co-surfactantindependent water-in-oil microemulsion system. This resulted in MSNs with magnetic properties and minimal residual surfactant impurities after synthesis, which enabled them to be used for wide applications including MRI [89]. One of the limitations of soft-template MSNs is their poor size controllability due to the large amount of surfactants used in their synthesis processes [90]. In order to overcome this limitation, the chemical processes during synthesis must be closely controlled. Currently, by adjusting the hydrolysis and condensation kinetics of precursors and surfactants, it is possible to fabricate small hollow type MSNs of approximately 20 nm in size [91]. More recent work using emulsion/micelle dual-templates have not only increased the sizes of soft-template MSNs to 80 - 220 nm, but also improved the control on other parameters like shell thickness and pore size [88]. 2.4.3. Self-Assembled MSNs Unlike hard-template MSNs and soft-template MSNs, self-assembled MSNs are formed without the use of an additional template structure. These MSNs make use of their own structure as a “self-template” to create the hollow structure. The self-template method is a simple, self-driven and low-cost process, with no additional requirement of templates or protective surfactants [28, 92]. In general, there are two major steps involved in fabrication: the synthesis of template

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nanomaterials and the formation of hollow structures from these templates. The field of selfassembled MSNs is wide and there are various synthesis methods available to produce hollow MSNs of different structural properties. The size of these MSNs is particularly determined by the dissolution and regrowth of their corresponding preformed solid spheres (Figure 6A). Tang et al. had first proposed the preparation of hollow type MSNs by alkaline treatment of the cationic polyelectrolyte pre-coated mesoporous silica spheres (Figure 6B) [93]. In this approach, polydimethyldiallylammonium chloride (PDDA) was coated over the mesoporous silica spheres, followed by treatment of these PDDA coated-mesoporous silica spheres in ammonia solution. Hollow MSNs were obtained by the interaction of the anionic silicate oligomers and the polyelectrolyte shell. Through the selection of suitable etching reagents and starting core structures, monodispersed hollow MSNs of 70 nm to several micrometers can be synthesized [94]. Based on these works, other functional nanomaterials have also been incorporated into the hollow core [95]. Precise control of the surface area and pore size of self-assembled MSNs has been achieved through selective etching technique based on structural differences. This has produced hollow type MSNs-based structures with variable morphologies and particle/pore sizes (Figure 6C-D) [96]. In this synthetic process, selective etching of the silica core (as homogeneous template) was performed while the mesoporous shell was unmodified. In addition, the interior volume and pore structure of these self-assembled MSNs could be tuned based on the reaction conditions during the core transformation, such as etching time, concentration of silica and acidity of the environment [97]. Polymer protective layers used during core transformation allow the hollow structures to be formed with even higher morphological fidelity (Figure 5A) [98]. Core transformation using this route is known as surface-protected etching. 3. Silica Nanoparticles in Cancer Therapy

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Over the past few decades, there has been extensive research towards safe and effective treatment for cancer. The emergence of nanomaterials offers high expectation for better detection and treatment of cancer. Silica-based nanoparticles play a significant role in cancer therapy due to their distinct advantages such as tunable and uniform pore size, high surface area and interior pore volume, nontoxicity and biocompatibility. The mesoporous structure of silica nanoparticles does not only make them active drug delivery systems for cancer treatments, but also allow their doping with diverse materials (such as fluorophores, nanoparticles etc.) for various applications in cancer therapy (Table 1).

3.1.Early Cancer Detection and Diagnosis In order to provide timely and more effective cancer treatment, early detection and diagnosis of cancer is essential for minimizing mortality. Currently, tissue biopsy, where tissues are removed from the patient to look for cancerous cells, is the most widely used method of diagnosis. With imaging guidance from ultrasound (US), X-ray, computed tomography or MRI, suspected cancerous tissues can be detected through biopsy. However, low sensitivity and poor selectivity of the traditionally used contrast agents in these types of imaging modalities limit early diagnosis. During the last decade, various nanomaterials like gold nanoparticles [99], quantum dots (QDs) [100] and silica nanoparticles [101] have been employed for early detection of cancer and diagnosis. Although gold nanoparticles have achieved encouraging development in cancer therapy, optical imaging using gold nanoparticles possess limited clinical future because of the weaker optical signal of gold nanoparticles compared with certain fluorescent dyes or QDs [102]. Perhaps QDs are being considered as multifunctional nanoparticles in biomedical applications including in situ optical imaging and drug delivery, more efforts have nonetheless to be devoted to toxicity effects before their extensive application in clinical diagnosis and therapy [103]. On

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the other hand, silica nanoparticles are biocompatible, non-toxic, biodegradable, and have a high loading capability for different agents, which thus renders silica-based treatments potentially more effective and safer for diagnostic approaches. 3.1.1. Silica Nanoparticles as Imaging Contrast Agents Imaging technologies such as US and MRI have been largely used for cancer diagnosis as they are low-cost, possess low radioactivity and real-time monitoring properties [101]. However, the commonly used contrast agents for US or MRI are small molecules including gadolinium chelates and calcium and other metal ions [104], which cannot provide high contrast images for early cancer diagnosis, due to the low specificity and inherent noise present during imaging [101]. Due to their robustness, high drug loading capacity, multiple-functionalization and facile biodegradation in the body in a timely fashion, silica nanoparticles are used as US- and MRIcontrast agents with specific targeting and low toxicity, and show promising results for cancer diagnosis (Figure 7A-C). In 2010, perfluorocarbon gas-filled hollow porous silica microshells were developed to be injected directly into tissues. These nanoparticles could remain in the tissues for several days without toxic effects and were easily imaged by US imaging in human breast tissue in all three dimensions. The long residence time of these agents within the body promoted novel applications, such as Doppler imaging using US and contrast specific imaging for longer duration [105]. In subsequent studies, hollow type silica as well as silica-boron nanoparticles were systemically applied in tumor bearing mice for US imaging [106]. For example, Daewon and co-workers engineered MSNs with Herceptin (a special recognizer for epidermal growth factor receptor 2), which can selectively target certain types of breast cancers. These functionalized MSNs could confer sufficient mean pixel intensity to generate higher quality US images [107]. MRI can access deep tissues and provide valuable high spatial

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resolution information without radioactivity; however, it requires highly sensitive contrast agents for practical application [108]. Further studies have investigated the possibility of using silica nanoparticles as a novel type of MRI agent [109]. With longer nuclear relaxation time and ease of surface modification with biologically compatible ligands, silica nanoparticles can be used as hyperpolarized, targetable MRI agents. The in vivo sensitivity of silica nanoparticles and other MRI nanoparticle probes have also been compared. The hyperpolarized silica nanoparticles were injected into the prostate tumor bearing mice and could be easily imaged with high sensitivity at low magnetic field, subsequently demonstrating other potential applications such as real-time MRI [110]. Furthermore, Kazuya et al. have developed a novel MRI contrast agent composed of a core micelle containing liquid perfluorocarbon inside a robust silica shell [111]. This type of silica nanoparticle-based agent has high sensitivity, sufficient in vivo stability, modifiability of the surface, and biocompatibility, which can propel promising future applications in early cancer detection and diagnosis. 3.1.2. Mesoporous Nano Silica Chips Proteomic analysis by mass spectrometry and chromatography has greatly revolutionized the early diagnosis of cancer, which can distinguish the differences between normal cells and cancer cells at molecular level [112-114]. However, there are several limitations to be addressed [115, 116], such as the signal interference from high concentration proteins to low concentration proteins, distinct spectra features of one sample from different types of mass spectrometry or chromatography and poor selectivity to identify changes in protein concentration between the normal and abnormal states. Moreover, the traditional samples have complicated preparation steps and the well-fractionated quality of samples is difficult to obtain. These difficulties cause detection to become time-consuming and inaccurate [117, 118]. Mesoporous silica-based chips

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(Figure 8A-B) with specific pore size and inert properties, act as a filter in protein mass spectrometry for the identifying cancer biomarkers in early detection and diagnosis [119-122]. Nanoporous silica chips allow the separation of low molecular weight proteins in serum from the higher weight proteins [122]. This in effect concentrates the low molecular weight proteins and enhances the signals. With the aid of mass spectrometry and biostatistical analysis, unique protein signatures pertaining to various stages of cancer development can be identified. In addition, surface engineering by the attachment of metal ions [120] or other functional groups [123] enhance the selectivity and sensitivity of mesoporous silica chips, which can selectively concentrate the low molecular weight proteins and identify proteomic biomarkers in various cancers. The use of functionalized mesoporous silica chips therefore provides a promising platform for the analysis of post-translational modifications in the human proteome and the potential diagnosis of early symptoms of cancer and other diseases, which may significantly enhance the possibility and accuracy of early cancer detection and therapy. 3.1.3. Fluorescent Silica Nanoparticles for Optical Imaging Fluorescent optical imaging has attracted great attention and has become essential in imagingbased therapy for preclinical investigations, especially for early cancer detection and diagnosis. Fluorescent dyes, fluorescent and bioluminescent proteins are traditional fluorescent probes that have been applied in optical imaging, but the rapid degradation, inadequate photostability and unpredictable toxicity limit their further application [124, 125]. The combination of silica nanoparticles and fluorescent materials overcomes these limitations and offers more effective, safer and affordable approaches for early cancer detection and diagnosis. There are two main types of fluorescent silica nanoparticles: (i) dye-doped silica nanoparticles and (ii) combination of QDs with silica nanoparticles.

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Dye-doped silica nanoparticles, which are prepared by incorporating fluorescent organic dye into silica nanospheres, have been extensively utilized for optical imaging. Among numerous fluorescent nanoparticles, dye-doped silica nanoparticles stand out and exhibit several advantages: (i) they contain high amounts of dye units within a silica matrix and exhibit a much stronger intense fluorescence signal compared with normal organic fluorophores [125] (e.g. small organic dyes, fluorescent proteins, metal-ligand complexes); (ii) the silica matrix serves as a robust shell and prevent the fluorophores from quenching and degradation; (iii) they can be surface-functionalized for a range of applications such as targeted imaging or evasion of capture by the reticuloendothelial system [126-128]. The organic dye-doped silica nanoparticles are commonly synthesized by two methods - the Stöber method [127] and the microemulsion method [126]. The simple microemulsion method is usually preferred as the conjugated celltarget moieties (e.g. antibodies [129], peptides [130], aptamers [131]) have higher cancer selectivity. With these specific cell-target moieties conjugated onto the surface, organic dyedoped silica nanoparticles can target specific cancer cells more efficiently and selectively. This allows the in situ diagnosis and treatment monitoring during clinical therapy. In our studies, we synthesized dye-entrapped and FA-conjugated ORMOSIL nanoparticles for in vivo cancer targeting and imaging (Figure 9A-B) [132]. With low-toxicity, biocompatibility and robust properties, our optical nanoprobes exhibit promising potential in clinical applications for cancer theranostics, especially in the early diagnosis of pancreatic cancer and other malignant tumors. Fluorescent semiconductor nanocrystals, or QDs, have been widely utilized in biomedical research over the past decade. In contrast to the conventional organic dye, QDs possess many superior optical characteristics, such as size-tunable wavelength absorption and emission, broad excitation wavelength, narrow emission bandwidth and long fluorescent lifetime [125, 133].

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However, the potential toxicity of QDs [128, 129] during in vivo applications [134, 135] and instability of nanocrystals in buffers [136], are some limitations which need to be addressed before one can utilize them for translational medicine research. To overcome these limitations, QDs can be embedded into silica-based nanoshells or alternatively, incorporated into silica nanostructures, forming silica-QDs hybrid nanoparticles. In one study by Tomas et al., QDdoped silica nanoparticles were surface functionalized with neutravidin to target T-lymphocytes [137]. In this research, the fluorescent nanoassembly was subjected to receptor-mediated endocytosis by Jurkat T-lymphocyte cells and was partially released to lysosomes, which was an excellent representation for construction of specific intracellular nanoprobes and transporters. Another group has embedded silica nanoparticles with a large number of hydrophobic QDs and obtained QD-embedded silica nanoprobes with high quantum yield [138]. These nanoprobes exhibited high fluorescent activity and are useful for tumor imaging in vivo (Figure 10). Unlike the silicon QDs, their particle surface have been modified with hydrogen, halogen or oxide terminated surface, thereby resulting in different photoluminescence properties [139]. The intracellular internalization of QDs alkyl-functionalized silica nanoplex has been investigated recently and a higher cellular uptake rate of the nanoplex is observed in the malignant cells as compared with normal cells [140]. These nanoparticles point to a feasible direction towards selective tumor imaging. According to these studies, QDs-embedded silica nanoparticles envision an attractive roadmap for cancer cell imaging and detection in vitro. However, more research for silica nanoparticles needs to be implemented in vivo for the further application in cancer detection and diagnosis. 3.2.MSNs-based drug delivery systems for cancer therapy

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During the last decade, silica nanoparticles, particularly MSNs have been widely used as drug delivery nanocarriers [48, 78, 141, 142]. With tunable particle size, large surface area and interior volume, uniform mesoporous structure and multi-functional surface, MSNs are a preferred drug delivery system over other drug carriers. First, the tunable pore diameter endows the MSNs with the ability to deliver drugs of diverse sizes for different kinds of cancer [19]. Secondly, the encapsulation of drugs inside MSNs effectively prevents the premature release and degradation, as well as reduces the toxicity impact to the healthy tissues during the transport process in vivo [28]. Thirdly, the water solubility of hydrophobic drugs can be improved by encapsulating them with water-dispersible silica nanoshells [12]. Meanwhile, the sustained release effect caused by MSNs’ mesoporous structure enhances the medication persistency [143]. Moreover, the multi-functional surface of MSNs can be tailored to serve as case-specific drug delivery systems for different cancer treatments [141]. These advantages outline MSNs as an excellent and flexible drug delivery platform for cancer therapy [144-146]. In general, MSNsbased drug delivery systems are classified into three types : passive delivery system, active delivery system and controlled-release drug delivery system.

3.2.1. Passive drug delivery system Briefly, passive drug delivery strategies are based on the enhanced permeability and retention (EPR) effect - a consequence of pathophysiological characteristics of diseased tissues for better drug accumulation in pathological sites [147]. With leaky vasculature in the blood vessels’ epithelial layers and inefficient lymphatic drainage system, tumor tissues offer superior conditions for passive drug delivery (Figure 11A). However, the size of most commonly used anticancer drugs is not large enough for passive delivery. To overcome this limitation, a number

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of nanovehicles including liposomes [148], micelles [149], dendrimers [150] and nanoparticles [151], have been designed to facilitate the delivery of these anticancer drugs into the tumor site with improved pharmacokinetics. Among these numerous nanovehicles, MSNs with unique structural features are largely applied for passive drug delivery. In 2001, the application of MCM41 type-MSNs as drug delivery systems was first reported, opening a new era in MSNsbased drug delivery [21]. To date, researchers have made numerous efforts to explore the application of MSNs-based drug delivery systems for cancer theranostics. One significant achievement is the loading of hydrophobic anticancer drugs into MSNs to transport them into human cancer cells. With MSNs-based nanoshells, the poor water solubility of hydrophobic anticancer drugs has been overcome, achieving highly efficient cellular uptake [12]. These findings suggest the prominent role of MSNs as an effective vehicle in overcoming the insolubility issue of many anticancer drugs. Consequently, MSNs with size of ~100 nm were utilized as a drug delivery vehicle to deliver anticancer drug into a nude mice bearing human breast cancer (MCF-7) xenograft [13]. These nanoplexes were taken up and accumulated in tumors by EPR effect, following which the growth of tumors was significantly suppressed. To determine the appropirate size of MSNs for EPR effect, Andre et al. calculated the EPR effect for different sizes of MSNs-based passive drug delivery systems [152]. They demonstrated a drastic improvement in the EPR effect by the size reduction of MSNs down to ∼50 nm and by modification of the particle surface with a PEI-PEG copolymer. Notably, EPR effect does not exist in all kinds of tumors, which may limit the application of passive delivery systems [153]. Furthermore, even in a single tumor, the permeability of vessels may not be the same. Therefore, MSNs-based passive delivery approaches should be modified prior to application in particular cases of cancer therapy.

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3.2.2. Active drug delivery system An effective way order to overcome the limitation of passive delivery approaches is to conjugate affinity ligands on the surface of nanocarriers (Figure 11B). These affinity ligands include antibodies [154, 168-171], peptides [155, 172-180], proteins [156, 164, 184], aptamers [157, 185, 186], small molecules [13, 14, 160, 162-167] and saccharides [18, 144, 187, 188] (Table 1). Due to the expression of receptors on the target cells, nanocarriers can accurately recognize and bind to these cells by ligand-receptor interaction, as well as transfer drugs to the target cells actively and efficiently. Depending on the type of receptor expressed on the target cells, diverse kinds of multi-functional MSNs-based drug delivery vehicles have been developed. FA, a vital nutrient required by all living cells, has high affinity for the folate receptor (FR). Because the expression of the FR is selectively upregulated in certain malignant tumor cells, such as ovarian, lung, kidney, breast, endometrial, brain and colon cancer cells, FA is considered as a common target molecule for these cancer cells. Lin et al. first studied the cellular uptake properties of surface functionalized-MSNs and reported the active role of FA groups functionalized on MSNs in facilitating receptor-mediated endocytosis for increased uptake by tumor cells [158]. Further studies have confirmed that the internalization of FA functionalized-MSNs in HeLa cells (human epithelial cells) was 5-6 times higher as compared with normal cells [159], which suggest FA functionalized-MSNs as a useful drug delivery system for FR-positive cancer cells due to increased toxicity of FA-conjugated MSNs prodrug in FR-positive cancer cells [160]. In addition, the biomolecular targeting agents like peptides and proteins are conjugated to the MSNs surface for other treatment effects. For instance, TAT peptide-conjugated MSNs were used as a nucleartargeted drug delivery system, which exhibited significant enhancement in the anticancer activity by nuclear internalization [155]. Transferrin is another candidate for conjugation on the surface

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of MSNs to enhance the recognition of brain glioma cells [156]. Transferrin increases the transport efficiency of nanoplex across the blood-brain barrier, thus enhancing the chemotherapeutic efficiency for treating brain glioma. Till date, the MSNs-based active drug delivery systems are being extensively utilized in in vitro and in vivo cancer-related research. However, for the clinical application, much more effort needs to be devoted to address the limitations, including harmful degradation byproducts, and the high cost of preparing the nanoformulation [161]. 3.2.3. Controlled-release drug delivery systems Using numerous kinds of ligands with different cell affinities conjugated onto the MSNs surface, MSNs-based drug delivery systems are capable of accurately recognizing and targeting diverse types of tumors. Furthermore, the most distinguishing feature of MSNs-based drug delivery systems is the “zero premature controlled-release” property. Briefly, drug delivery systems are able to deliver drugs with precisely controlled release to the target cells and do not prematurely release their loaded drugs en route. This property is one of the necessary prerequisite in the evaluation of a system’s degree of therapeutic enhancement and decrease in the cytotoxicity of its loaded chemotherapeutic drugs [189]. This is a major challenge for other available drug delivery systems. Recently however, several multifunctional MSNs-based drug delivery schemes have achieved “zero premature controlled-release” [190-192], by blocking the pore entrances of drug-encapsulated MSNs with stimuli responsive agents, also named as “caps”. Once triggered by external or internal stimuli unique to the desired location, these caps disassemble from the pore entrances and the loaded drugs are released to the targeted sites (Figure 11C). Lin and coworkers first introduced the “zero premature controlled-release” concept in the control release system of MSNs [190]. They utilized MCM41 type-MSNs as drug nanocarriers capped with

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inorganic CdS nanoparticles to efficiently deliver drugs into neuroglial cells with “zero premature controlled-release”. Up to now, a variety of materials have been applied as capping moieties, such as CdS [190], Au [193], Fe3O4 nanoparticles [194], rotaxanes [163], dendrimers [195], polymers [196] and proteins [192]. However, there are some potential risks of toxicity or biocompatibility from the capping chemical or metal agents [184, 197]. Subsequent developments in these caps propose the use of biomolecules, especially proteins, to reduce toxicity. Lin and coworkers designed a glucose-responsive drug delivery system by functionalizing the external surface of MSNs with gluconic acid-modified insulin 8 (G-Ins 8) protein [191]. The release of G-Ins 8 and cyclic adenosine monophosphate (cAMP) inside MSNs-based nanocarriers was triggered by the controlled introduction of glucose. Another example is the biomolecule-based enzyme-responsive cap system constructed from biomolecules and MSNs composites [192]. In this research, tetrameric protein avidin was utilized as a cap and could be disintegrated by enzymatic hydrolysis. As highlighted in the previous section, the application of caps in drug delivery can efficiently prevent premature release and enhances the therapeutic effect of a drug. Based on the different types of caps, many reports have emerged highlighting the development of the stimuli-responsive triggers, including pH, temperature, redox potential, light, enzyme, etc. These triggers have been successfully applied in vitro and in vivo, and promote the application of MSNs-based drug delivery systems in cancer theranostics. 3.2.3.1.pH-triggered drug release system Due to acute hypoxia, disorganized vasculature and elevated interstitial pressure in the internal environment of tumors, the lactate produced through glycolysis in tumor cells cannot be exhausted rapidly enough, resulting in low pH in tumor tissues (pH<7) [198]. Therefore, pH triggering is the most common and feasible approach for application in a controlled drug release

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system. pH-responsive linkers have been conjugated to MSNs-based nanocarriers for controlled drug release in tumor cells. Latorre and co-workers explained the earliest example of a pHtriggered release system [199]. In their studies, they utilized MCM-41 type MSNs as drug carriers and conjugated the surface with amino groups. Employing this pH-controlled and anioncontrolled drug delivery system, successful release of squaraine dye aided by MSNs nanocarriers was obtained. Subsequently, other groups proposed pH-triggered MSNs drug delivery systems decorated with different functional groups. For example, Che et al. have developed a novel pHtriggered MSNs-based drug delivery system which uses coordinate bonding of the functional groups present on the pore surface with metal ions and drugs [200]. The “host-metal-guest” architecture exhibited attractive stability and rapid pH-responsivity, and provided a new direction for pH-triggered release system in cancer therapy. Lee and co-workers fabricated calcium phosphate (CaP) capped-MSNs as drug delivery system, which could release drugs under pH control [201]. As a novel pore blocker and nontoxic inorganic biomineral, CaP plays an instrumental role in the natural bone regeneration process (Figure 12A). Compared with traditional MSNs, the capped-MSNs can effectively prevent the premature release of DOX and administer DOX under pH influence (Figure 12B). The in vivo anti-tumor studies strongly support capped MSNs-based nanocarriers as promising specific intracellular drug carriers for cancer therapy (Figure 12C). 3.2.3.2.Temperature-triggered drug release system Unlike the normal tissues which are generally stable in the body, the tumor tissues are highly active with continuous cell replication. Therefore, the temperature in tumor-bearing tissues is higher as compared with most normal tissues [202]. This temperature difference is used as an internal trigger for instigating controllable drug release. In addition, near infrared (NIR) light or

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magnetic materials which induce the hyperthermal effect can also be utilized as external triggers for controlled drug release. The most commonly employed thermal-sensitive polymer, poly (N-isopropylacrylamide) (PNIPAAM), is been functionalized onto MSNs for modulating controlled drug release [203205]. These polymers present swollen hydrated states below a low critical solution temperature (LCST) and are able to cover the cavities of MSNs to prevent the drug release. As the temperature exceeds LCST, the polymers undergo a reversible phase transition to a shrunken hydrophobic state in aqueous medium, thereby causing pore opening and drug release [206]. Early studies conducted by Lopez group, have demonstrated switchable behavior (from hydrophilic to hydrophobic state) of PNIPAAM on the surface of silica materials upon an increase in temperature and suggest that the hybrid material composed of silica and PNIPAAM could act as a controlled-release system under temperature transition [207-209]. Consequently, more studies have been devoted to the application of PNIPAAM-functionalized silica nanoparticles in drug delivery. Voelcker et al. grafted PNIPAAM to the surface of porous silicon materials by surface-initiated atom transfer radical polymerization technique [210]. The composite material exhibited high drug loading ability and excellent drug controlled-release property. Zhao et al. described a novel NIR-stimulus controlled drug release system, which comprised of cores composed of gold nanocages, shells composed of mesoporous silica and PNIPAAM as the thermal-sensitive gatekeepers [205]. Under NIR light, Au-nanocage cores convert NIR light to heat following which the PNIPAAM gatekeepers change from the hydrophilic to hydrophobic state, resulting in pore opening and drug release (Figure 13A). With the synergistic photothermal therapy effect and efficient drug release by NIR light, these MSNs-

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based nanocarriers exhibit cancer cell killing ability (Figure 13B) and can be further applied for biomedical research. Although PNIPAAM as thermal-sensitive gatekeepers have been utilized for MSNs-based controlled drug release systems, the LCST of pure PNIPAAM is 32°C and may be not very useful for clinical applications (normal body temperature ranges between 36.1 and 37.2°C). A series of PNIPAAM analogues have been developed for applications at higher temperature. For example, Poly (ethyleneoxide-block-N-vinylcaprolactam) can be used as a gatekeeper on MSNs surface that maintain the pore opening temperature at ~36°C [211]. Zwitterionic sulfobetaine copolymer which is conjugated onto the MSNs enhances the LCST to 50°C [212]. Recent studies on other temperature-responsive caps, such as DNA oligomers or peptides, have made notable progress in drug release. Thomas group conjugated biotin-labeled DNA strands with the external surface of MSNs and modulated the pore opening temperature by the length of DNA strands (Figure 13C) [213]. Kros and co-workers utilized coiled-coil peptide motifs as a temperatureresponsive cap to control the drug release inside MSNs (Figure 13D) [214]. These gatekeepers are nontoxic and biodegradable, which place them as worthy candidates for clinical cancer therapy. 3.2.3.3.Redox potential-triggered drug release system In general, the level of antioxidant species in intracellular space is considerably higher than in extracellular space, which is the reason for high redox potential difference [215]. The basic principle of redox potential-triggered drug release system is to utilize the high redox potential difference to destroy the disulfide bond between caps and carriers. Compared to the normal cells, the redox potential difference is more pronounced in tumor cells. Therefore, the disulfide linkage is more susceptible to breakage in cancer cells, amounting to higher drug concentration at tumor

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locations [216]. Wang et al. assembled a multilayer on the surface of drug encapsulated-MSNs by disulphide bond [157]. No obvious release of loaded drug molecules was detected in cells even after 24 hours of incubation. In contrast, dithiothreitol (DTT) treatment led to deconstruction of the disulphide bond and the consequent release of drug molecules. With the cancer cell-specific DNA aptamer sgc8, the MSNs-based nanocarriers possess high cell recognition and cause pronounced cancer cell death. Besides the polymers, Lin and co-workers conjugated the inorganic Fe3O4 nanoparticles as caps to the surface of MSNs and achieved drug release by cell-produced antioxidants [217]. These magnetic-MSNs-based nanocarriers possess “zero release” prior reaching the target site and release the cargos after internalization by the cells. 3.2.3.4.Enzyme-triggered drug release system The development of enzyme-triggered controlled-drug release systems is viewed as effective strategy to cancer therapy, owing to the excellent biocompatibilities, rapid and specific biological activities of enzymes. The first MSN based-enzyme-triggered release system was shown by Zink and co-workers [218]. They used α-cyclodextrin ring as a snap-top, which consisted of polyethylene glycol chemically bonded to MSNs through an enzymatically cleavable bond. Following enzyme-mediated hydrolysis, the snap-top system could successfully release the encapsulated cargo molecules. In recent studies, the enzyme-triggered release system based on MSNs has been widely applied for drug delivery in cancer theranostics research. There exist FDA-approved peptide capping agents, such as protamine, which have been complexed with MSNs to produce a non-toxic and biocompatible drug delivery system [82]. When the protamine-capped MSNs carriers encounter the proteolytic enzyme trypsin, the caps disintegrate and the encapsulated drug is released (Figure 14A,B). The nanocarrier itself exhibits non-toxicity

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and can enhance the toxicity of hydrophobic anti-cancer drugs for cancer cell treatment (Figure 14C,D). The next phase of development of these enzyme-triggered release systems would be to increase their specificity to the cellular level [219]. Biopolymer capping agents such as chondroitin sulfate, are multifunctional capping agents, which provide MSN drug retention, cell targeting and bio-responsive drug release. Chondroitin sulfate capping agents specifically target cancer cells-CD44 biomarkers over-expressed on the cell membrane, releasing the encapsulated drugs after being triggered by enzymes such as lysosomal hyaluronidase which are abundant within cancer cells. 3.2.3.5.Light-triggered drug release system Leveraging on the various advancements in PDT, light irradiation as a means of triggered release is effective for site-specific drug release. The speed and range of drug release is controlled via the exposure of the MSNs to light with specific wavelengths and duration. The first lighttriggered release system based on MSNs was reported by Tanaka group [220]. In their approach, coumarin ligands, which are UV-light sensitive, were used as capping agents at the surface of MSNs, effectively regulating porosity of the MSNs in response to UV light (λ=250 nm). Since then, various photochemical responsive linkers for example, azobenzene, thymine, o-nitrobenzyl ester, aluminium phthalocyaninedisulfonate and graphene oxide (GO) were discovered to be effective light-triggered release capping reagents for MSNs. These different capping reagents respond to different wavelengths of light, allowing MSNs to be used for a wider variety of applications. For instance, aluminium phthalocyaninedisulfonate, a red-light sensitive photosensitizer (~800 nm), was used for light-triggered release in deeper tissues, as the penetration of higher light wavelengths is more effective through tissues [164]. When used with other target ligands, such as FA and epidermal growth factor (EGF), these MSN drug

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nanocarriers can specifically recognize and target cancer cells. These multifunctional MSNs are the next wave of development. Other approaches used photochemical responsive linker GO and cancer cell recognizer-AS1411 (Figure 15A) to create a light-triggered system which exhibited manageable drug release and accurate cancer cell recognition [221]. NIR light was utilized as the exogenous stimuli to trigger drug release (Figure 15B,C). Based on the lower toxicity and photothermal effect of NIR light, the light-triggered drug delivery system showed synergistic dual-mode chemotherapy and photothermal therapy to cancer cells (Figure 15D). Two-Photon Excitation (TPE) in the NIR region is a noteworthy alternative which was used as a trigger for MSNs-based drug delivery system [222]. This system possesses zero premature release of anticancer drugs and is efficient for TPE triggered-drug release in cancer cells upon irradiation from a focused laser beam. 3.2.3.6.Other stimuli-triggered drug release system Besides enzyme- and light-triggered drug release systems, other types of stimuli for the controlled release of drug reagents from MSNs are developed which aim to increase the specificity of such treatments. Lin et al. demonstrated a stimuli-triggered release system, namely, a glucose-responsive drug release system, described in section 3.2.3 [191]. In this system, G-Ins 8 encapsulate cAMP molecules into the MSN mesopores. The nanocarriers are endocytosed by cells and release the drugs upon the introduction of glucose. A biomolecule-sensitive controlled release system was described by Wang group [223]. Depending on the high affinity-based aptamer-target interaction, cargos can be accurately released at the specific site without premature release. Following with the investigations on single stimuli-triggered drug release systems, there is rapid ongoing effort to create the next generation of therapeutic MSNs with multiple stimuli-triggered

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drug release systems. These systems achieve complex drug release profiles either independently or synergistically, regulating drug release under different specific conditions for better targeting of the therapeutic agents. Yang and co-workers utilized the copolymer of 2-(2-methoxyethoxy) ethyl methacrylate and oligo (ethylene glycol) methacrylate cross-linked by disulfide bonds to coat hollow type MSNs [224]. The presence of glutathione or temperature change can trigger the drug release, causing the death of glutathione overexpressing-cancer cells [225]. Cui et al. developed a pH- and temperature-based dual-controllable drug release system [226]. In such a system, MSNs act as the drug carriers, wherein the dual stimuli responsive-gating shell is composed of the copolymer-lipid layer. With greater drug loading capacity and dual stimuliresponsive releasing ability, this system exhibits high anticancer ability and can be further used for clinical applications. In recent years, stimuli-responsive drug delivery systems are gaining advancing position in biomedical research. However, most studies are focused on in vitro studies and there are fewer reports on in vivo applications. Although cell-based assays can provide some information, cultured cells still cannot mimic the complicated physiological environment and complex interactions in the cell-tissue habitat. To achieve the goals of clinical cancer therapy, more efforts should be devoted to in vivo studies based on these stimuli-responsive drug delivery systems. 3.3.Co-delivery of gene and drugs assisted by MSNs-based drug delivery system for combatting cancer multidrug resistance Cancer MDR is the process wherein cancer cells become simultaneously resistant to a variety of drugs with dissimilar structure, molecular target and mechanism of action [227]. The development of MDR to chemotherapy has become a major limitation to cancer therapy. Cancer MDR can be classified into two types: pump resistance and non-pump resistance. Pump resistance is related to ATP-binding cassette transporters, which function as drug efflux pumps,

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for example, MDR-associated protein 1 (MRP1) and P-glycoprotein (Pgp) [228]. These efflux pumps can exploit the energy generated by ATP hydrolysis to pump the drugs and decrease the intracellular drug concentration. With these efflux pumps, intracellular drug uptake is reduced which thereby induces drug resistance. Non-pump resistance is mainly caused by the activation of cellular anti-apoptotic defence, such as the expression of anti-apoptosis protein Bcl-2 [229]. The application of drug delivery systems based on MSNs is a major advancement towards the suppression of MDR in cancer therapy. These nanocarriers have changed the routes of drugs entering into tumor cells to avoid binding by the efflux pumps, resulting in the accumulation of drugs in tumor cells [230, 231]. Furthermore, due to high loading capacity and efficient encapsulation of cargos, multiple drugs can be simultaneously delivered into cancer cells without premature release and degradation by intracellular enzymes [232]. Several groups have utilized MSNs-based delivery system to co-deliver genes and anti-cancer drugs, which effectively target both pump and non-pump resistance and significantly increase drug efficacy to cancer cells [233, 234]. Improvement of cytotoxicity in cancer cells promotes this strategy as a suitable approach for the therapeutic treatment of MDR-dominant cancers. For example, Zhao and co-workers improved the MSNs-based co-delivery systems with high cell recognition and pH-triggered controllable–release [235]. They utilized FA as the target ligand and PEI as the gatekeeper to functionalize the surface of MSNs (Figure 16A-D). PEI layers collapse due to the acidic intracellular environment of cancer cells, following which drugs and siRNA can be released naturally. Besides the co-delivery of siRNA and anti-cancer drugs, multiple combination delivery systems based on MSNs have been developed, such as therapeutic peptides with anticancer drugs [236], biomacromolecules with anti-cancer drugs [237] and hydrophilic molecules with hydrophobic drugs [238], etc. In contrast to single delivery systems, the co-delivery systems

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provide more effective therapeutic strategies to reverse MDR in cancer cells [239]. MSNs-based drug delivery systems offer high loading capability, numerous controlled-release approaches and outstanding biocompatibility, which are considered to become the best transporters for therapeutic drugs to cure cancers and other diseases in clinical trial. 4. Multifunctional delivery platform based on MSNs To date, nanotechnology has provided a varied range of technologies for developing new pathways for cancer detection, diagnosis and treatment. Owing to the unique properties, which include high loading capability and multi-functionalized surface properties, MSNs serve as nanocarriers which can simultaneously be conjugated with versatile molecules to bring forward multifunctional capabilities. The combination of targeted drug delivery and concurrent real-time in vivo monitoring is the most representative example of multifunctional delivery platform based on MSNs, which successfully implements the synchronization of tumor diagnosis and treatment. Yeh et al. utilized hollow type MSNs to co-deliver fluorescence and anti-cancer drugs [240]. This co-delivery system can simultaneously act as imaging probes for tumor optical imaging in vivo and perform drug release by the appropriate selection of pH-dependent molecules to kill cancer cells. To expand the application of MSNs in cancer therapy, combinations of MSNs with additional functional nanoparticles have been developed. This combination can integrate diverse diagnostic and therapeutic methods into one single system for better cancer theranostics. One of the noteworthy benefits of MSNs over other nanoagents is the capability to co-deliver hydrophobic and hydrophilic anti-cancer drugs [241]. The co-delivery system predominantly enhances tumor cell apoptosis and inhibition of growth, and simultaneously traces and investigates the process of endocytosis in cells. Meanwhile, the magnetic core supplements the MSNs with magnetic

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properties for MRI or magnetic field manipulation for extended biomedical applications. Combination of (AuNRs) and MSNs is another commonly used platform for cancer theranostics [84, 242, 243]. In the combined platform, AuNRs efficiently convert the NIR light to heat and induce photothermal effect. This multifunctional platform combines chemotherapy, photothermal therapy and multimodal imaging into a single system simultaneously, as well as provides a more comprehensive approach for cancer theranostics (Figure 17A-D). In addition, the combination of QDs and MSNs has been investigated by Brinker group [244]. In their system, targeting peptides on the surface of MSNs exhibit greater affinity for human hepatocellular carcinoma, multitherapeutic drugs and QDs that are encapsulated inside MSNs realize the synchronization of cancer diagnosis and treatment, and the fluid-supported lipid bilayer prevents premature release. This system sums up the multiple applications based on MSNs nanocarriers and promotes the development of their integration for cancer diagnosis and treatment. Table 3 is an elaborate timeline of the journey of MSNs since their synthesis and evolution pertaining to cancer theranostics and gene delivery applications.

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5. Conclusion and Future perspectives The advancement of MSNs-based nanocarriers is considered of great importance in drug delivery and provides a wide range of strategies for cancer theranostics. In this review, we discuss the structure and properties of various configurations of MSNs and their applications in cancer theranostics. Based on Stöber method, diverse synthesis conditions have been developed to successfully fabricate a series of MSNs, such as MCM-41, SBA-15, hollow type MSNs, ORMOSILs, etc. By regulating the reaction conditions (for e.g. the molar ratio of silica precursors and surfactants, temperature, pH value and addition of co-surfactants), the particle size, pore structure and morphology of MSNs can be facilely controlled. The particle diameter spans tens to hundreds of nanometers, which can passively target diverse cancerous tissues by EPR effect [13]. Furthermore, the most distinguishing feature of MSNs-based drug delivery systems is their “zero premature controlled release” property [308]. This occurs because MSNs can encapsulate drugs inside and immobilize other materials as porous capping reagents on the surface to block the pore entrances. These capping reagents are sensitive to a certain trigger and open the entrance by dissolving or phase transition. Triggers can be internal or external, for example, pH, light, redox potential, temperature and enzymes. Functionalizing the surface of MSNs with capping reagents and targeting ligands can fabricate a powerful drug delivery system with controlled-release and cell recognition properties, which exhibit incomparable advantages over other drug delivery systems for cancer theranostics. Despite encouraging progress and research in the biomedical applications of MSNs-based drug delivery systems, several major challenges still need to be addressed for achieving clinical success. The first issue is the in vivo toxicity caused by MSNs. Albeit silica is considered as GRAS and the C dots have secured FDA approval for stage I human clinical trial [308], many in

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vivo studies describe dose-dependent toxicity of MSNs [309], such as inflammatory response [310], liver injury [311], neurotoxic [312] and pregnancy complication [313]. In addition, the particle size, surface charge and surface functional groups also play significant roles in MSNs cytotoxicity [314]. Therefore, more careful and sufficient in vitro and in vivo studies require to be conducted to outline the proper dose and type of MSNs before clinical trials. Another critical issue is bridging the gap between the successful in vitro experiments to challenging in vivo applications. By conjugating with targeting ligands and multifunctional “caps”, various in vitro studies have demonstrated the capability of MSNs-based drug delivery systems to recognize the target cancer cells and release drugs controllably. However, the complicated physiological environment and highly dynamic and heterogeneous properties of tumors impede the active targeting and effective release in vivo. To overcome these barriers, MSNs-based drug delivery systems should be modified to have longer circulation time, ability to extravasate into tumors and multiple functional groups for cancer cell targeting. Furthermore, a single formulation of drug delivery system is impossible to effectively cure all patients with cancer. The development of multifunctional MSNs-based nanocarriers, such as the combination of AuNRs nanoparticles and MSNs, magnetic nanoparticles and MSNs, that can achieve the synergistic effect of photothermal therapy, chemotherapy and multiple imaging together, exhibit much better therapeutic effect than single therapy. In conclusion, the research on MSNs-based drug delivery holds promising evaluation for cancer theranostics. With the high loading capacity, good biocompatibility, tunable particle and pore size and multifunctional surface properties, MSNs are considered as an ideal drug delivery system. This system holds great promise for reversing MDR by co-delivery of multiple drugs and targeting the tumor site actively, and possesses the “zero premature release” until a certain

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stimuli is triggered. Moreover, MSNs-based drug delivery systems can be conjugated with versatile molecules to bring forward multifunctional capabilities simultaneously, which offer a multifunctional delivery platform including drug delivery, optical imaging and controlled-release for treatment. With tremendous efforts to overcome the existing bottlenecks, it is worthwhile to envision a conclusive position of multifunctional MSNs-based drug delivery systems to catalyse the progress of clinical cancer theranostics. Acknowledgements This study was supported by the Ministry of Education, Singapore (Grants Tier 1 M4010360.040 RG29/10 and Tier 2 MOE2010-T2-2-010 (M4020020.040 ARC2/11)), NTU-NHG Innovation Collaboration Grant (No. M4061202.040), A*STAR Science and Engineering Research Council (No. M4070176.040), NTU-A*STAR Silicon Technologies, Centre of Excellence under the program grant No. 11235100003 and School of Electrical and Electronic Engineering at Nanyang Technological University. References [1] P. Anand, A.B. Kunnumakara, C. Sundaram, K.B. Harikumar, S.T. Tharakan, O.S. Lai, B. Sung, B.B. Aggarwal, Pharm. Res. 25 (2008) 2097-2116. [2] D. Trichopoulos, F.P. Li, D.J. Hunter, Sci. Am. 275 (1996) 80-87. [3] M.D. Mignogna Huang, S. Fedele, L. Lo Russo, Eur. J. Cancer Prev. 13 (2004) 139-142. [4] J.R. Heath, M.E. Davis, Annu. Rev. Med. 59 (2008) 251-265. [5] J.P. Gillet, M.M. Gottesman, Methods Mol. Biol. 596 (2010) 47-76. [6] J.W. Ryu, Y.S. Kim, Tuberc. Respir. Dis. 78 (2015) 36-40. [7] M. Triesscheijn, P. Baas, J.H. Schellens, F.A. Stewart, Oncologist 11 (2006) 1034-1044. [8] S. Tinkle, S.E. McNeil, S. Muhlebach, R. Bawa, G. Borchard, Y.C. Barenholz, L. Tamarkin, N. Desai, Ann. N. Y. Acad. Sci. 1313 (2014) 35-56. [9] J.M. Rosenholm, V. Mamaeva, C. Sahlgren, M. Linden, Nanomedicine (Lond) 7 (2012) 111-120.

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Figure 1. Schematic illustrations of three types of MSNs with the mean particle size range: a) M41S-type MSNs, b) organically modified silica (ORMOSIL) nanoparticles and c) hollow type-MSNs (HMSN).

Figure 2. Structural arrangements of M41S family members: A) MCM-41 nanoparticles: hexagonal phase, B) MCM-48 nanoparticles: cubic phase and C) MCM-50 nanoparticles: lamellar phase.

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Figure 3. The schematic illustration of MCM-41 preparation by surfactants and silica precursors and their characterization. (A) Synthesis of MCM-41; a) micelle formation, b) condensation, c) alignment, d) calcination. The surfactant self-assembles into micelle formation and then condensates into micellar rods in a hexagonal array, while the silicate precursors solidify the structure. (B) TEM images of MCM-41 materials with different pore sizes; a) 20 A°, b) 40 A°, c) 65 A° and d) 100 A° (Reproduced from ref. [34] with permission of American Chemical Society). (C) SEM (a, c) and TEM (b, d) images of two main shapes of MCM-41 materials: spherical and tube-shape silica particles (Reproduced from ref. [41] with permission of Elsevier Science B.V.) (D) SEM images of various spherical shapes of MCM-41 under different reaction conditions. (Reproduced from ref. [40] with permission of Elsevier Science B.V.)

Figure 4. TEM images of ORMOSIL nanoparticles of sizes A) 30 nm, B) 50 nm and C) 80 nm. (Reproduced from ref. [68] with permission of Royal Society of Chemistry)

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Figure 5. Synthesis of hard-template method for hollow type MSNs by using surfactant or polymer. (Reproduced from ref. [75] with permission of Royal Society of Chemistry)

Figure 6. The self-template method for synthesizing hollow type MSNs. (A) Spontaneous transformation of silica colloids from solid spheres to hollow structures; a) Fabrication process of hollow type MSNs, b) TEM images of SiO2 spheres, c-d) TEM images of hollow type MSNs in different etching conditions. (Reproduced from ref. [98] with permission of WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) (B) Fabrication process of hollow type MSNs with silica shell in the presence of ammonia solution. TEM images show the PDDA pre-coated mesoporous silica spheres during different instants of 1M ammonia solution treatment. (Reproduced from ref. [93] with permission of American Chemical Society) (C) The selective-etching procedure (left) for the fabrication of hollow type MSNs (left) with different etching conditions are exhibited in Route A (Na2CO3 solution) and Route B

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(ammonia solution). The local microscopic structure (right) shows the existence of long carbon chains of C18TMS that constitute the hydrophobic cores. (D) TEM images of hollow type MSNs in different etching conditions; a-b) sSiO2@mSiO2, c-d) in 0.6M Na2CO3 solution at 80°C for 0.5 h, e-f) in 0.12M and 0.24M ammonia solution at 150°C for 24 h, respectively. The inset of panel shows SEM image of broken hollow type spheres, g-h) The role of different initial precursor concentrations (45 nm and 450 nm) on the fabricated hollow type MSNs. (Reproduced from ref [96] with permission of American Chemical Society)

Figure 7. Silica nanoparticles as contrast agents for US and MRI. (A) SEM images of porous hollow silica nanoand micro-shells with different particle size: a) 100 nm, b) 500 nm and c) 2000 nm. (Reproduced from ref. [106] with permission of Elsevier Ltd.) (B) US imaging of gas-filled silica microshells in tumor-bearing mice; a) Interperitoneal IGROV-1 ovarian tumor in the dissected nu/nu mouse, b) Cadence contrast pulse sequencing (CPS) image and c) B-mode image of the particles through the tumor cross section 1 h post injection with silica nanoparticles, d) Overlay of CPS image and B-mode image (red arrow - tumor, green arrow - spinal column, blue arrow - bottom of the mouse). (C) In vivo accumulation of silica nanoparticles at tumor site; a) Structural schematic of functionalized silica nanoparticles, b) In vivo MRI of functionalized silica nanoparticles in tumor-bearing mice. Liver and tumor positions are represented as L and T, respectively. (Reproduced from ref. [111] with permission of WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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Figure 8. Production and assembly of mesoporous silica based chips for proteomic applications. (A) The chemical evolution in the coating solution during the production stages of a mesoporous silica film; a) Fresh solution, b) Formation of micelles, c) Spin-coating process leading to self-assembly, d) Magnified image of a pore post aging at high temperature. (B) SEM and TEM cross-sectional images of GX6 chip on a) bulk silicon wafer surface (upper) and b) mesoporous silica film-coated silicon wafer surface (lower). Scale bar is 500 nm. (Reproduced from ref. [119] with permission of WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Figure 9. The organically modified silica nanoparticles (ORMOSIL) for optical imaging in vivo. (A) Characterization of synthesized ORMOSIL nanoparticles; a) TEM image of the synthesized ORMOSIL nanoparticles, b) Absorption and c) Photoluminescence spectra of the ORMOSIL nanoparticles (ORM), dye-doped

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nanoparticles (ORMD) and DCM dye (DCM). Inset of c): The corresponding solutions under natural and UV light. d) Hydrodynamic size distribution of the dye-doped ORMOSIL nanoparticles, e) FTIR spectra of FA, ORMD and FAconjugated dye-doped nanoparticles (FA-ORMD). (B) In vivo luminescence imaging post tail vein injection; (a-d) Fluorescence images of Miapaca-2 tumor tissues (pointed by white arrows) in mice taken at specified times, where PBS served as control (right). (Reproduced from ref. [132] with permission of Royal Society of Chemistry)

Figure 10. The application of silica-QDs nanoparticles. (A) Cellular uptake in vivo test of QDs-embedded silica nanoparticles; a) Schematic illustration of QDs-embedded silica nanoparticles in HeLa cells, b) Fluorescence images of QDs-embedded silica nanoparticles in HeLa cells (silica QDs: Red, DAPI: blue), c) In vivo fluorescence images of QDs-embedded silica nanoparticles in cell-transplanted mouse. (Reproduced from ref. [138] with permission of WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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Figure 11. Schematic representation of passive and active delivery system. (A) Passive drug delivery system: drugloaded MSNs nanocarriers cannot extravasate through normal endothelium and only small molecules of free drug can traverse normal endothelium; with the large gaps between endothelial cells in tumor tissues, nanoparticles can extravasate and accumulate in tumor tissues creating high local drug concentrations. (B) Active drug delivery system: drug-loaded MSNs nanocarriers are modified with a specific ligand which is able to recognize certain binding sites on the tumor cell surface. As a result, these nanocarriers can attach to the cell surface and release the drug therein or can be internalized bringing the drug inside target cells. (C) Controlled-release process of MSNsbased drug delivery systems: The capping agents block pore entrances to block the premature release of drug molecules. After the specific stimuli triggers the disassembly of caps from MSNs, the encapsulated drugs can be released to the target location.

Figure 12. pH-tunable CaP-coating MSNs as drug delivery systems for cancer theranostics. (A) Drug release from CaP-coated MSNs under pH control; a) Synthetic route to urease-MSNs (UR-MSNs): 1) APTES, 2) removal of

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CTAB, 3) modification with UR. b) TEM image of UR-MSNs. c) Drug release by surface CaP mineralized-DOXloaded UR-MSNs by pH-triggering. HAp=hydroxyapatite. (B) Fluorescent images of MCF-7 cells treated with LysoTracker (Green color), free DOX (Red color), and DOX-MSNs-CaP; a) Free DOX after 1 h exposure, DOXMSNs-CaP after 1 h b) and 5 h c) exposure, DOX-MSNs-UR for 1 h d) and 5 h e) exposure. Scale bar: 20 mm. (C) a) The tumor volumes after treatment with saline (●), free DOX (■), DOX-MSNs-UR (▲) and DOX-MSNs-CaP (♦); Inset: Images of excised tumors after 16 days of treatment; b) the tumor weights 16 days post-treatment by different materials. The DOX-equivalent dose is 10 mg/kg. The results represent the mean SDs (n=4); *P<0.05. (Reproduced from ref. [201] with permission of WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Figure 13. Different types of gatekeepers in temperature-responsive uncapping mechanism. (A) Controlled-drug release of Au-nanocage@mSiO2@PNIPAM nanocarriers under NIR laser irradiation. (B) Drug release and cell viability test; a) Photothermal curves of H2O and the Au-nanocage@mSiO2@PNIPAM (50 μg/mL) exposed to NIR laser irradiation, b) DOX release from the Au-nanocage@mSiO2@PNIPAM nanocarrier in PBS buffer (pH 7.4 and 5), with or without NIR laser irradiation, c) Viability of HeLa cells with or without NIR laser irradiation, under different concentration of carrier, DOX, and carrier + DOX, d) Micrographs of HeLa cells (trypan blue-stained) with or without NIR laser irradiation, with carrier and carrier + DOX. (Reproduced from ref. [205] with permission of American Chemical Society) (C) Temperature-triggered controlled-drug release of biotin-labeled DNA-MSNs nanocarriers. (Reproduced from ref. [213] with permission of WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) (D) Controlled-drug release of coiled-coil peptide-MSNs nanocarriers. (Reproduced from ref. [214] with permission of Royal Society of Chemistry)

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Figure 14. Enzyme-triggered release system based on MSNs for cancer therapy. (A) Formation of drug-loaded amine-functionalized protamine capped-MSNs (MSN–PRM) and subsequent enzyme-triggered release of payload. (B) Anticancer drug release from MSN–PRM nanoparticles at the proximity of trypsin overexpressing-cancer cells. (C) Fluorescent images of COLO 205 cells incubated with a) curcumin loaded-MSN–PRM nanoparticles and b) free curcumin in H2O. Drug release from the MSN–PRM nanoparticles leads to decreased fluorescence. (D) In vitro cell viability at a) different MSN–PRM concentrations, b) free curcumin and curcumin loaded-MSN–PRM nanoparticles. (Reproduced from ref. [82] with permission of WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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Figure 15. Light-triggered release system based on MSNs for cancer therapy. (A) Dox-loaded GO-wrapped MSNs (MSN-Dox@GO) bind with Cy5.5-As1411 aptamer and the corresponding intracellular drug release under NIR light. (B) Regulation of drug release under NIR light at different times; a) Temperature variation of MSN-Dox@GO solution upon 808 nm laser irradiation for six 10 min on/off cycles, b) Cumulative release of Dox by MSNDox@GO and MSN-Dox in the presence and absence of laser irradiation. (C) Fluorescent images of MCF-7 cells incubated with MSN-Dox@GO-Apt without and with laser. (Dox- red color, Cy5.5-As1411- green color and DAPI (nucleus staining)- blue color were recorded). (D) The synergistic dual-mode chemotherapy and PTT of MSNDox@GO-Apt; a) Cell viability of MCF-7 cells under MSN-Dox@GO-Apt, MSN@GO-Apt and control at different laser power densities, b) PTT and chemotherapy percentages of MSNDox@GO-Apt at different laser power densities, c) Fluorescent images of MCF-7 cells treated with MSN@GO-Apt or MSN-Dox@GO-Apt for 4 h followed by laser irradiation for 10 min, and incubated for 24 hours followed by staining with Live/Dead assay. (Cells irradiated only with laser served as control; Calcein (green, live cells) and PI (red, dead cells)). (Reproduced from ref. [221] with permission of Royal Society of Chemistry)

Figure 16. Co-delivery system with drugs and siRNA based on MSNs. (A) pH-responsive HMSNPs-assisted codelivery of targeted drug and siRNA. (B) Dox release profiles under varying different pH conditions (4.5 (■), 6.0 (▲), 7.4 (●)). (C) Confocal microscopy images of FA-MSNs-DOX-siRNA-treated HeLa (FA+) and MCF-7 (FA-) cells. FITC (green), Dox (red) and DAPI (violet). (D) MTT cytotoxicity assay of a) HeLa and b) MCF-7 cells after treatment by FA-MSNs+scrambled siRNA (■), FA-MSNs-DOX (●) and FA-MSNs-DOX+Bcl-2 siRNA (▲). (Reproduced from ref. [235] with permission of WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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Figure 17. Multifunctional MSNs for photothermo-/chemo-therapy and multimodal imaging. (A) a) and f) The structure of Gold capped-magnetic core/mesoporous silica shell nanoparticles (AuNRs-MMSNEs), b) TEM images of Fe2O3@SiO2@mSiO2, c-e) TEM images of AuNRs-MMSNEs. The arrow in the inset image depicts the PEG layer on the gold nanorod surface. (B) Infrared thermal images of an AuNRs-MMSNEs-injected tumor subjected to a) 1 Wcm-2 b) 2 Wcm-2 and c) PBS-injected tumor under 2 Wcm-2 laser irradiations. (C) In vivo MRI of a mouse before and after intratumor injection of AuNRs-MMSNEs. (D) Cell viability test of MCF-7 cells treated by AuNRsMMSNEs-NIR (purple), AuNRs-MMSNEs-DOX (red), AuNRs-MMSNEs-DOX-NIR (green) and the additive therapeutic efficacies (blue). (Reproduced from ref. [242] with permission of Elsevier Ltd.)

Table 1. The various modalities of MSNs in cancer therapy. Mesoporous Silica Nanoparticles in Cancer Therapy Early Cancer Detection and Drug Delivery systems Diagnosis Imaging Contrast Agents

Anti-MDR systems

MSN-Based

Passive delivery system

Simultaneous delivery of multiple drugs Mesoporous Nano Silica Chips Active delivery system Co-delivery of gens and anticancer drugs Fluorescent Silica Nanoparticles Controlled-release drug delivery Multiple combination delivery for Optical Imaging systems systems

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Table 2. Examples of ligands conjugated to the surface of MSNs Ligand type Small molecule

Ligand name Folic acid (FA)

Anti-cancer drug Camptothecin

Camptothecin Paclitaxel Doxorubicin

Methotrexate FA+Dexamethasone (DEX) Antibody

Anti-TRC105 Anti-HER2/neu Anti-ME1 Anti-ErbB2 (AB2428)

Peptide

K4YRGD Transferrin Cyclic-RGD RGD TAT

cRGDyK c(RGDfE) RGDFFFFC K7RGD c-RGDFK N3GPLGRGRGDK-Ad K8(RGD)2 pHLIP Protein

EGF Transferrin

PEI-assisted gene delivery MTX Doxorubicin Doxorubicin

TNF-α Doxorubicin Camptothecin Doxorubicin Doxorubicin Camptothecin Sunitinib Gemcitabine Doxorubicin

Doxorubicin Doxorubicin Doxorubicin

Doxorubicin Doxorubicin & Paclitaxel

Cell/Tumor type U2Os SK-BR-3, MCF-7, MCF POF Panc-1, MiaPaCa-2 Panc-1

Ref [162] [13] [14] [165]

HeLa KB HeLa

[160, 163] [164] [165]

HeLa HeLa

[166] [167]

HUVEC, MCF-7 MCF-7 NIH 3T3, MCF7, BT-474 MM, A549 MCF-7

[168] [154] [169] [170] [171]

HepG2 Panc-1, HFF, BF549, MDA-MB435 U87 MG & COS 7 HeLa MCF-7/ADR HeLa, A549 U87MG BxPC-3 U-87 MG, COS7 HeLa

[172] [173]

SCC-7 , HT-29 U87 MG MCF-7/ADR, mA549, U20S, H1299, HepG2

[181] [182] [183]

HuH7tub HeLa, MRC-5 U-87 MG-luc2

[164] [184] [156]

[174] [155] [175] [176] [177] [178] [179] [180]

Aptamer

AS1411 Sgc8 TBA A15

Doxorubicin Doxorubicin Docetaxel

MCF-7 HeLa HeLa

[185] [157] [186]

Saccharides

Galactose

Camptothecin

HCT-116, MDA-MB-231, Capan1

[144]

Mannose

TPE-PDT

MCF-7, MDA-MB-231, HCT-116

[18, 187]

Hyaluronic acid

Camptothecin

MCF-7, L929

[188]

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Table 3. A timeline of evolution of MSNs and their cancer theranostics. Year

I.

Structural Evolution of MSNs

Applications

Reference

Synthesis of MSNs, their structural modifications and functionalizations

1990 1992 1997 1999

Mesoporous silica materials first synthesized using Stöber process Mesoporous silica material with hexagonal porous structure First submicron MCM-41 particles using a modified Stöber process Initiation of functionalization of MSNs with various organic functional groups

2001

Synthesis of MCM-41 particles using a dilute surfactant solution (100 nm)

[249]

2004

Synthesis of MSNs using a double surfactant system or dialysis process (below 50 nm) Synthesis of MSNs surface functionalised with second generation (G2) PAMAM dendrimers Chemical modifications on MSNs with Small molecules (Folic acid and Mannose), Biomacromolecules (Proteins and Antibodies) and Bio-oligosomes (Peptides and with aptamers) Metal-core MSNs (embedded gold, silver or iron oxide)

[250]

2004 2006 onwards 2009 2009 onwards

2013 2012 2012

2013

Functionalisation of MSNs with various organic functional groups, e.g. PEI-coated MSNs PEG-coated MSNs PEI-FA functionalised MSNs MSNs with PEI-PEG copolymer coating Hollow-type and rattle-type MSNs Synthesis of hollow silica/titania nanoparticles (HNPs) incorporating the monoclonal antibody Herceptin Synthesis of core/shell magnetic MSN (MMSN) encapsulating nanodiamonds, gold nanoparticles or graphitic carbon as the core of nanoparticles

Loading of drugs into pores of MSNs by co-condensation and post-synthetic grafting method

[245] [31, 246] [247] [248]

[251] Trigger receptor-mediated endocytosis by selective binding on cell surface receptors Provide additional functionalities like, antimicrobial activity, plasmonic effect or MRI capabilities Targeting of cancer cells, drug delivery loading of drugs into pores of MSNs by covalent attachment - by electrostatic interaction, to adsorb siRNA and DNA constructs in vitro

See Table 1 [84, 252, 253] [13, 152, 159, 235, 254-258]

Interior drug loading for reduced cancer cell viability.

[75, 87, 93] [259]

Theranostic applications, combined photothermal therapy and chemotherapy for IR-light induced controlled drug release

[84, 88, 89, 242, 243, 260-263]

2013

Synthesis of MSNs with different configurations of mesopores with tunable pore diameters - raspberry, stellate, worm-like Au-capped MSNs

[264] For enzyme and substrate co-delivery

2013

CdS QD-capped photosensitive nano-gated MSNs

For CMT drug delivery

[11, 193, 265] [190, 266]

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II.

Mechanized controlled- and trigger responsive- drug release systems

2003

Release of cargo from mesopores by breaking of covalent bonds between molecules (e.g. coumarin) Release of cargo from mesopores by shrinking and swelling of polymer chains Nanovalves: Delivery of cargo from the mesopores by the Shuttling of a cyclic molecule (e.g. [2]rotaxane)

2003 2007 onwards

Mechanized nanovalves for drug delivery Mechanized nanovalves for drug delivery

[267] [268]

pH-sensitive nanovalve for trapping drug molecules inside the MSN mesopores, and drug delivery (DOX) from MSNs. Also help in overcoming cancer MDR

[253, 269-275]

2007 onwards 2010 onwards

Nanoimpellers: Delivery of cargo from the mesopores by the wagging motion of azobenzene. Cargo release assisted by bulky groups (e.g. Au, Fe3O4 nanoparticles, CdS nanocrystals, rotaxanes, dendrimers, polymers, proteins, coordination compounds) over the pore openings

In vitro delivery of anti-cancer drug for tumor shrinking; NIR light-induced drug delivery to cancer cells Surface functionalised MSN-based multifunctional theranostic supramolecular assemblies.

[276-278]

2010-11

Temperature sensitive MSN-Drug configurations pH-sensitive MSN-Drug configurations UV light-sensitive MSN-Drug configurations

Trigger-responsive nanovalves for drug delivery

[199-201, 203-205, 207210, 220, 252, 270, 271, 284, 285]

2007 onwards

Various designs for light-responsive triggers - Rotaxanes Cyclodextrins, Azobenzene-based nanoimpellers

Drug delivery from MSN materials for PDT treatment

[286-289]

III.

[163, 190196, 279-282]

Multifunctionalised MSNs for multifunctional applications

2011

Combination of QDs and MSNs

Affinity for multi-therapeutic drugs

[244]

2012

Gold capped-magnetic core/mesoporous silica shell nanoparticles (AuNRs-MMSNEs)

Combined photothermo-/chemo-therapy and multimodal imaging

[242]

2012

DOX-loaded nanoellipsoids consisting of ellipsiodal Fe3O4 cores, and PEGylated gold nanorods conjugated-mesoporous silica shells

A multifunctional platform enabling- Chemotherapy, Phototherapy, T2-wieghted MRI, Infrared thermal imaging and dark-field optical imaging

2013

CPT drug loaded-MSN mesopores capped with CdS nanoparticles

Nanodevice for combination anti-cancer therapy

[266]

2010, 2012

Gold nanocrystal-coated MSNs containing gold nanorods

[291, 292]

2010

Development of first trifunctionalised MSNs, incorporating a fluorescent reporter, a peptide, and a photosensitizer

Plasmon-induced thermotherapy, TPE of tracers, and deep tissue drug release Tracking, targeting α2β3 integrin expression, and for photodyanamic therapy

[290]

[293]

Page 67 of 69

2010

Coating of PEGylated-phospholipids onto hydrophobic, silane-modified MSNs, combined with FITC and folate ligands Magnetic, pH-sensitive, Fe3O4 nanoparticle-capped MSNs

Improve water suspension stability of MSNs and decrease nonspecific protein binding, for imaging tracer, and for targeting ligand Smart drug delivery platforms that prevent premature release of conveyed cargo

[294]

2012

MSNs embedded with iron oxide nanocrystals, and surface functionalized with thermo-responsive copolymer PEI/NIPAM

For treatment of MDR cancers where drug release is controlled by external, alternating magnetic field.

[297]

2012

A triple core/shell nanomaterial containing Fe3O4@SiO2@α-NaYF/Yb,Er, with a nanorattle structure, and a middle hollow interior loaded with DOX. Mesoporous silica coated with NaYF4:Yb,Er upconversion fluorescent nanoparticles (UCNs)

In vivo magnetic drug delivery system, and cancer targeting based on applied magnetic field Demonstration of NIR-light induced PDT treatment by in vitro and in vivo studies

[298]

2011-13

2012

IV.

[280, 295, 296]

[299]

siRNA and pDNA delivery by MSN-based drug carriers

2010-11

PEI-coated MSNs

Adsorption of siRNA and DNA constructs in vitro

[254, 256]

2009, 2011

Amino acid-functionalised layer porous MSNs

Adsorption of plasmid DNA with lower cytotoxicity

[300, 301]

2011, 2013

Fe3O4-inner core magnetic MSN

DNA adsorption/desorption and for siRNA delivery after surface modification of PEI and KALA peptides.

[302-304]

2012

DOX-loaded, TAT functionalised MSN nanoparticles

Targeted drug delivery to the nuclei of cancer cells

[305]

2014

2013

PEI-coated functionalized MSNs

2014

Histidine-functionalised MSN

Targeted drug delivery to the nuclei and mitochondria of cancer cells Simultaneous delivery of DOX and BCL-2 targeted siRNA into cancer cells Delivery of DOX and Pgp siRNA to cancer cells (both in vitro and in vivo) pDNA transfection

[236]

2009

MSN-loaded with topotecan, and mitochondria-targeting moiety conjugated to an antibiotic peptide. siRNA/DOX MSN-based nanocarrier

[233] [234, 256, 306] [307]

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