Silica Nanoparticle Platform

C H A P T E R

20 Silica Nanoparticle Platform Jeffrey S. Souris, Nai-Tzu Chen†, Shih-Hsun Cheng†, Chin-Tu Chen and Leu-Wei Lo† 

Department of Radiology, The University of Chicago, Chicago, Illinois, U.S.A. †Division of Medical Engineering Research, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan O U T L I N E

Introduction

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Biocompatibility

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Nanoplatform Properties and Their Role in Biocompatibility Topology Hydrophilicity and Hydrophobicity Surface Charge Reactive Sites and Oxidation Protein Adsorption

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Silica Toxicity Cytotoxicity and Genotoxicity

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INTRODUCTION Cancer nanotheranostics employ multifunctional, nanometer-scale, organic and inorganic materials to extract diagnostic insight and deliver pathologytargeted therapy. The fundamental advantage of combining such seemingly disparate objectives is the ability to use patient-specific test results to tailor treatment programs that result in improved clinical outcomes, reduced costs, and minimal side effects. Such platforms also enable in vivo monitoring of both the nanomaterial’s biodistribution and fate and its therapeutic progress and efficacy throughout the course of treatment. Unlike most conventional diagnostic and chemotherapeutic agents, nanoparticles can be readily modified to provide extended circulation half-lives, passive accumulation in and near tumors via the enhanced permeability and retention (EPR) effect,

X. Chen and S. Wong (Eds): Cancer Theranostics. DOI: http://dx.doi.org/10.1016/B978-0-12-407722-5.00020-7

Administration and Exposure Route Significance

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Synthesis and Surface Modification of Silica Nanoparticles

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Silica Nanoparticle Nanotheranostics General Considerations Multifunctionalized Solitary Platforms Organic-Composite Hybrid Platforms Inorganic-Composite Hybrid Platforms

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Conclusions

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References

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active targeting of cancer cells, and minimal toxicity. With their easily manipulated, size-dependent physicochemistries, nanoparticles afford small diameters, enormous surface areas, and novel topologies that often give rise to unusual properties; properties that are generally quite unlike those of bulk material having the same composition and that result in bioactivities that heavily depend on the environment in which the particles are localized. In contrast to most other nanomaterials, however, silica nanoparticles do not acquire any new or unusual characteristics from the diminution of their size, other than a corresponding increase in surface area. Rather, their utility stems from the ease with which their surfaces can be functionalized and their ability to form both solid and porous structures, the latter of which greatly enhances the surface area and different topologies available for synergistic applications such as

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© 2014 Elsevier Inc. All rights reserved.

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protected codelivery of therapeutics (e.g., drugs, siRNAs, photosensitizers, radionuclides) and imaging contrast agents (e.g., fluorophores, radionuclides, Fe/Mn/Gd oxides). Silica nanoparticles are inexpensive and simple to synthesize, chemically inert, biocompatible and excretable, and easily dispersed in water. Silica nanoparticles are also effectively “transparent” at UV, visible, and near-infrared (NIR) wavelengths and unaffected by electric or magnetic fields—attributes that facilitate their external visualization and manipulation.

BIOCOMPATIBILITY Nanoparticle biomolecule interactions can be quite unlike those of bulk material of the same composition because the nanoparticle’s effective surface charge, stability, solubility, and hydration are greatly affected by the local milieu’s pH, ionic strength, temperature, and organic/molecular composition (e.g., proteins, lipids, carbohydrates, surfactants) [1]. Emerging studies, however, also suggest that a number of these unique attributes are likely to give rise to toxic sequelae, both acute and chronic, a situation further compounded by the typically inefficient targeting and inherently high stability that many nanomaterials exhibit in vivo [2 8]. Thus, a variety of schemata have been devised to minimize deleterious side effects, such as covering the nanoparticle’s surfaces with polymers, antioxidants, surfactants, or ligands or altering the nanoparticle’s size, shape, roughness, or surface charge [9 13]. Unfortunately, such measures often only afford partial remediation of toxicity and can themselves have undesired consequences, such as increasing a nanoparticle’s hydrodynamic diameter that then results in substantially altered biodistribution. Ultimately, to best minimize toxicity, nanoparticles should be degraded in situ into truly biocompatible subcomponents or excreted from the body once they have served their diagnostic and therapeutic purpose. Numerous studies have been published on the biocompatibility of a variety of nanoparticles and, to a lesser degree, their degradation by-products. But little consensus can be found among them as most studies cannot either be compared to one another (owing to variations in material synthesis, characterization, and/or toxicology) or simply contradict one another (for reasons undetermined) [2,4 6,11,14 19]. Even less has been published on the excretion of nanoparticles (or their subcomponents), reflecting, to some measure, a loss of traceability (e.g., disassociation/decomposition of fluorescent labels, or radioactive decay of radiolabels) as well as the intrinsic difficulty of directly visualizing them in situ. Nanoparticles whose in vivo imaging signals are

inherently stable (e.g., the fluorescence of quantum dots or the Raman spectroscopic signatures of carbon nanotubes) are few in number and possess chemistries that do not extrapolate to those of other nanoparticles. In addition, uniquely identifying biodegradation by-products in vivo can be exceedingly difficult. A few conclusions can be drawn from the clearance/excretion studies that do exist. When delivered intravenously, nanoparticle uptake occurs by one of five pathways—phagocytosis, macropinocytosis, caveolin-mediated, clathrin-mediated, and caveolin/ clathrin-independent endocytosis—at a rate that largely depends on the particle’s hydrophobicity, surface charge, and size [1,5,8,11,12,16,17,20 22]. Hydrophobic nanoparticles are rapidly removed from circulation by components of the reticuloendothelial system (RES), especially in the liver and spleen, and thus have short in vivo half-lives (from seconds to minutes) that often limit their clinical utility. Nanoparticles that carry significant surface charge tend to adsorb serum proteins (some in non-native conformation) that can affect their biodistribution, elicit immune response, and indiscriminately destabilize cell membranes and proteins. Nanoparticle size, however, most strongly correlates with clearance dynamics, with particles 3 nm in diameter and smaller extravasating tissues nonspecifically, those 3 to 8 nm in diameter undergoing renal clearance, those 30 to 80 nm in diameter being sequestered in lung and leaky vasculature (e.g., tumor and inflamed tissue via the enhanced permeation and retention effect), and particles larger than 80 nm becoming trapped by liver and spleen. Of course, uptake from circulation does not ensure excretion. Indeed, all studies to date of larger quantum dots (diameter .8 nm), regardless of surface coating, have shown relatively rapid sequestering by the RES of liver and spleen, but with none leaving those organs. Similar retention patterns have been observed with pristine single-walled carbon nanotubes, with rapid accumulations in liver, spleen, and lung, but with little subsequent excretion from those organs [19]. Even PEGylated gold nanospheres, with long blood circulation times (B30 h), have demonstrated a propensity to accumulate in the liver and spleen of mice as long as 7 days after their injection, leading to acute hepatic inflammation and apoptosis [4]. While rapid excretion may minimize the potential for toxic sequelae, it also mandates expedient and highly specific pathology targeting if it is to be of diagnostic or therapeutic benefit. For some applications, such efficacious targeting and elimination may either not be desired (e.g., particle residence time and numbers are insufficient to derive full diagnostic/therapeutic effect) or possible (e.g., particle has insufficient vascular access due to pathology, or low targeting affinity).

IV. THERANOSTIC PLATFORMS

NANOPLATFORM PROPERTIES AND THEIR ROLE IN BIOCOMPATIBILITY

In the following sections we discuss a few of the more salient aspects of silica theranostic biocompatibility, beginning with nanomaterial properties and biomolecular processes at the submicroscopic level, and followed by a macroscopic, toxicological/systems-level examination of their biocompatibility. More detailed analyses of these topics can be found in the recent literature [23 25].

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silica powders whose constituent particles varied in diameter from as small as 8 nm to as large as 260 nm, the amount of isolated silanol (IR adsorption band at 3750 cm21) was found to decrease as the particle diameter increased, nearly vanishing for the largest nanoparticle powders. By contrast, the ratio of optical absorbance of the hydrogen-bonded silanol IR band to that of the isolated silanol IR band was observed to increase as the particle diameter increased [31]. As such, the curvature of silica nanoparticles was found to significantly affect the ratio of isolated to interacting silanols, and thereby powder dispersibility in aqueous environments.

Topology One of the principal factors affecting the interaction of nanoparticles with biological materials is size; or, more precisely, relative size of the nanoparticle to that of proximal biomolecules. As the size of the nanoparticle and/or its surface features diminish, relative to those of nearby biomolecules, nanoparticle surface curvature plays an increasingly significant role in particle environment interactions. Particles possessing high curvature are typically able to accommodate larger amounts or numbers of adsorbed molecules per unit surface area due to decreased steric hindrance [26]. Nanoparticle curvature has also been observed to be a dominant factor in the conformational changes that occur to proteins during adsorption onto the particle [27]. In one set of studies, 15 nm diameter amorphous silica nanoparticles altered the secondary structure of a carbonic anhydrase (HCA I) considerably more than amorphous silica nanoparticles of 6 nm diameter [28]. In another surface curvature study, crystalline SiO2 nanoparticles possessing diameters of 5 to 100 nm were used to search for alterations in the phase transition of different chain-length, phosphotidylcholine bilayers that enveloped the SiO2 nanoparticles [29]. On the more highly curved surfaces of smaller nanoparticles, lipids formed bilayers in which the outer polar heads were more widely separated from one another, with more chains being interlocked with the outer layer of adsorbed lipids, to offset increased headgroup spacing. Physicochemical surface properties can exhibit strong dependence on particle surface curvature as well, leading to numerous other biologically interactive particle attributes such as hydrophilicity, hydrophobicity, surface charge (ζ-potential), and strength of hydrogen bonding. For example, silica surfaces frequently terminate with silanols (Si OH) and siloxanes (Si O Si). Silanols can be broadly classified as being (1) isolated, (2) interacting, or (3) geminal, whereas siloxane bridges form on condensation of two adjacent silanols [30]. In an FTIR study of amorphous

Hydrophilicity and Hydrophobicity Nanomaterial hydrophilicity largely reflects the presence and relative abundance of polar groups (e.g., Si-OH, Ti-OH) and/or under-coordinated metal ions (e.g., Ti31, Al31, Fe21/31) resident on the nanomaterial’s surface) [32,33]. The submicroscopic structure of a nanomaterial (e.g., crystalline vs. amorphous, crystalline phase/polymorph type, defect frequency, dopant presence) has also been found to play a significant role in the degree of the material’s wettability, solubility, and rate of dissolution. For example, the dissolution rate of amorphous silica is much greater than that of comparably-sized, ground crystalline quartz under similar environmental conditions, and large differences have been observed in the solubility of crystalline silica particles possessing different lattice symmetries [30]. Despite variations in Si O Si bond lengths and angles, low-pressure amorphous silicas share the same fundamental chemical unit with crystalline silicas: the silica tetrahedron. In contact with water, exposed silanol (Si OH) groups dissociate to yield negative surface charges that result in the formation of a hydration/solvation shell that inhibits particle aggregation, enabling aqueous dispersion and suspension. Reported dissolution rates for crystalline and amorphous SiO2 support the idea that crystalline polymorphs and amorphous silicas undergo the same hydrolysis reactions albeit at different rates that reflect the water’s access to the silica’s surface. Interestingly, the dissolution rates of both crystalline and the amorphous silicas are increased 50 to 100 times when alkaline or alkaline earth cations are introduced to otherwise pure solutions. This large enhancement has significant implications for the hydrophilicity and half-life of silica nanotheranostics in electrolytically variable, biological fluids. Indeed, the much higher solubility of amorphous silica in biofluids, with respect to crystalline silicas, is one of the primary factors purported to result in the former’s lower toxicity [34 37].

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Surface Charge As mentioned previously in regards to the solvation shells that develop from the hydrolysis of silica nanoparticle surfaces, the presence of polarized or charged moieties on the surface of silica nanoparticles greatly influences their interactions with the local environment. When a nanoparticle that possesses an electrically charged surface is immersed in an aqueous, electrolytic solution, an interfacial, electrical doublelayer immediately forms around the nanoparticle. The layer closest to the nanoplatform’s surface is stationary and comprised of adsorbed ions, a consequence of ion particle electrostatic attraction, hydrogen coordinative bonding, and/or van der Waals interactions. A second, more loosely associated layer of ions, termed the diffuse layer, envelopes the nanoparticle and first layer. Comprised of ions that are only weakly attracted to the underlying surface charge, due largely to the first layer’s electrical screening of the nanoparticle’s surface charge and “binding” competition with neighboring ions (ionic strength of biological fluids B150 nM), the ions of the diffuse layer are in a state of dynamic flux, the subject of thermal motion. The net electric charge in this diffuse layer is equal in magnitude to the net surface charge, but with the opposite polarity. The diffuse layer, or at least part of it, is able to move under the influence of tangential forces, with a slipping plane that separates mobile fluid from surface adsorbed fluid. The electric potential at this plane, a measure of the double-layer’s (and thus that of the underlying nanoparticle’s) charge, is defined as the nanoparticle’s ζ-potential. As such, calculated ζ-potentials are routinely used to assess nanoparticle colloidal stability in solution (i.e., resistance to flocculation) and have more recently been employed to ascertain the degree of protein adsorption onto the surface of silica nanoparticles [38].

Reactive Sites and Oxidation Nanoparticle surface charges, dangling bonds (i.e., surface-bound radicals), and poorly coordinated ions each have the potential to interact with nearby biomolecules. One of the more deleterious consequences of these interactions is the generation of reactive oxygen species (ROS•), a term that includes not only oxygenbased free radicals such as superoxide anion (O2•2), hydroxyl (HO•), and biomolecule-derived radicals such as alkylperoxyl (RO2•) and alkoxyl (RO•), but also nonradical species such as hydrogen peroxide (H2O2) and singlet oxygen (1O2) [39]. Although ROS play key roles in a number of cell signal transduction pathways, excess ROS are highly cytotoxic, damaging

proteins, lipids, and DNA, and a stressor that activates a number of redox-sensitive signaling pathways [40]. In general, nanoparticles can generate ROS either via direct mechanisms (surface-derived ROS) or indirect mechanisms (cell-derived ROS) that include alterations in mitochondrial function and immune system response [41 43]. Silica related ROS generation can be both particle surface and cell derived, and arises from the existence of either dangling bonds (e.g., undissociated silanols with potential to form strong hydrogen bonds) or exposed redox-reactive transition metals. As neither dangling bonds nor metal impurities are commonly present in synthetic amorphous silica (precipitated or pyrogenic silicas), amorphous silicas are generally incapable of directly inflicting oxidative damage [44]. However, crystalline silicas, especially when powderized, can possess large numbers of dangling bonds and are associated with both particle and cell-derived ROS pathologies, the latter including the inflammatory pulmonary disease silicosis [41,45 46].

Protein Adsorption Proteins display a high propensity to adsorb onto surfaces via electrostatic interactions, coordinative bonds, hydrogen bonds, and hydrophobic interactions. When nanoparticles come into contact with multicomponent biological fluids such as blood, plasma, and interstitial fluid, a bevy of suspended proteins immediately envelopes the particles. Indeed, as nanoparticles move onto or into cells, most nanoparticle cell interactions are strongly modulated by these proteins. The establishment and maintenance of this protein cloak, termed the protein corona, is a dynamic and competitive process governed by the avidity and relative concentration of proximal free proteins, the nanoparticle’s surface charge distribution and chemical reactivity, nanoparticle size relative to that of the adsorbing proteins, local pH and ionic strength, and temperature, as depicted in Figure 20.1 [47]. Proteins with greater mobility often adsorb first only to be subsequently replaced by less motile proteins that possess higher nanoparticle affinities. Lifetimes of these various particle ligand complexes range from microseconds to days and affected proteins may undergo conformational changes that result in the exposure of new epitopes of altered affinity and functionality. The encapsulation of nanoparticles with proteins also has significant implications for the nanoparticle’s surface charge (e.g., dispersion) and chemical reactivity (e.g., ROS generation). Several recent studies of silica nanoparticles have demonstrated that even modest, physiologically plausible increases in the degree of protein coverage can significantly affect both the magnitude

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NANOPLATFORM PROPERTIES AND THEIR ROLE IN BIOCOMPATIBILITY

Competitive binding interactions depend on protein concentration and body fluid composition

Characteristic protein on/off rates depend on material type and protein characteristics

Binding interactions release surface free energy, leading to surface reconstruction

e–

Particle e Surfac composition y it instabil

Protein binding accelerates dissolution of some materials

Available surface area, surface coverage, and angle of curvature determine adsorption profiles

Crystallinity

Electronic states

Hydrophobicity wettability

Size

Charge

Steric hindrance prevents binding

Hydrophilic or hydrophobic interactions Charge (for example, cationic binding)

(a)

Surface reconstruction

Electron-hole pairs lead to oxidative damage

O2′–

Release of surface free energy

SH SH

Protein conformational changes cause loss of enzyme activity

Surface opsonization/ liganding allows interaction with additional nano-bio interfaces

(b)

e– S S

O2

e– h+ Protein conformation, functional changes

Impact on protein structure and function

Protein fibrillation

Amyloid fibre

Exposure to cryptic epitopes

Protein crowding, layering, nucleation Immune recognition

FIGURE 20.1 Nanoparticle protein corona: cause and effect. (a) Numerous particle-dependent factors (e.g., topology, hydrophobicity, surface charge, reactive sites) contribute to the protein coronas that envelope nanoparticles following their exposure to biological fluids. Initial protein coronas are modified by their locally variable environments, rapidly evolving to lower free energy forms. (b) Consequences of protein adsorption onto nanoparticle surfaces can be diverse and lead to undesired properties such as cytotoxicity or hemolytic behaviors. Matured protein coronas of nanoparticles frequently exhibit alterations in surface charge, immune recognition, oxidative status, and enzymatic activity, depending on the moieties adsorbed. Source: Reproduced from [1] with kind permission of Macmillan Publishers Limited. Copyright 2009. All rights reserved.

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and polarity of the nanoparticle’s ζ potential [38,48 51]. As such, colloidal instabilities can potentially result from protein corona establishment and evolution and lead to nanoparticle aggregation and flocculation and protein clustering and fibrillation. While empirical studies suggest that protein coronas generally improve the colloidal stability of nanoparticle suspensions, counter examples do exist; functional proteins such as cytochrome c, DNase II, hemoglobin, and fibrinogen have been found to bridge SiO2 nanoparticles via electrostatic attraction and hydrogen bonding, and then aggregate the nanoparticles into coralloid forms [51].

SILICA TOXICITY Cytotoxicity and Genotoxicity As can be deduced from the forgoing discussion, nanoparticle biocompatibility is a complex and dynamic set of processes determined both by the nature of the particle and its environment; processes that can be either chemical or physical. Examples of chemical mechanisms include ROS production, dissolution and release of toxic ions, alteration of cell membrane electron and ion transport, catalytic oxidative damage, and lipid peroxidation. Physical mechanisms include disruption of protein folding and function, protein aggregation and fibrilation, and the disruption of membrane integrity, activity, and transport processes. ROS generation, and secondary processes to ROS generation (e.g., inflammatory cytokine response), are considered the principal chemical processes in nanotoxicology, although radical formation too can adversely effect cell viability [43,52 55]. Cellular uptake of molecules is frequently mediated by moiety-specific binding with membrane-bound receptors. In the absence of such receptors (e.g., viral infection), cellular uptake takes place via constitutive “adsorptive” endocytosis or fluid-phase pinocytosis [56,57]. Silica nanoparticles, by virtue of their surface silanol groups, have great affinity for a variety of phospholipid headgroups and are thus readily endocytosed [58]. Indeed, a number of research groups have demonstrated that mesoporous silica nanoparticles (MSNs) are rapidly (,30 min) endocytosed by a variety of mammalian cells including cancer cells (HeLa, CHO, lung, PANC-1), noncancer cells (neural glia, liver, endothelial), macrophages, and stem cells (3TL3, mesenchymal), to name but a few [59 65]. Nonfunctionalized, bare MSN uptake occurs primarily through clathrincoated endocytosis, though some surface functionalized MSNs, described in subsequent sections of this chapter, undergo cellular internalization via pinocytosis. Dissociation of the surface silanols of unfunctionalized

silica nanoparticles leaves the nanoplatforms with significant negative surface charge that, once endocytosed, triggers proton influx, osmotic swelling, permeation or rupture of the endosomal membrane (i.e., the protonsponge effect), and cytosolic release. Positive surface charge functionalization of silica nanoparticles, for example with amines, can also result in anion influx and osmotic swelling of the endosomal membrane, leading to membrane compromise and endosomal release of nanoparticles into the cytosol. Interestingly, amine and guanidinium (only) functionalized MSNs appear to enter cells via a clathrin and caveolae independent mechanism [61]. In a recent study examining the role of nanoparticle design in cellular toxicity and hemolytic activity, investigators exposed erythrocytes, macrophages, and cancer epithelial cells to differently charged, nonporous 115 nm diameter silica nanospheres, mesoporous 120 nm diameter silica nanospheres, and mesoporous silica nanorods with aspect ratios of 2 (80 3 200 nm), 4 (150 3 600 nm), and 8 (130 3 1000 nm) at doses of 100, 250, and 500 μg/ml [66]. Cancer epithelial cells (A549 adenocarcinomic alveolar basal) were generally found to be unharmed by the presence of SiO nanoparticles, whereas macrophages exhibited adverse, charge-dependent, and dose-dependent responses to porous and nonporous SiO nanoparticle exposure and postulated a reflection of the very different biological functions of the two cell types (i.e., protective boundary/barrier vs. phagocytic scavenger). In another study, smaller 30 nm diameter nonporous silica nanoparticles demonstrated macrophage toxicity at doses as low as 10 μg/ml [67]. Indeed, with macrophages serving as platforms in both innate (nonspecific) and adaptive (acquired) immunity, the nature and extent of macrophage nanoparticle interaction provides considerable insight as to nanotheranostic toxicity, especially given the serum protein opsonization that nanoparticles experience following their intravenous administration, encouraging additional receptor-mediated macrophage binding. Differences in the geometry of these silica nanoplatforms, however, appear to be of little consequence to either macrophage or epithelial cell toxicity [66]. In erythrocyte toxicity studies of mesoporous silica spheres and nanorods, bare or unmodified SiO nanoparticles demonstrated both porosity- and geometrydependent hemolytic activity at concentrations greater than 250 μg/ml, while amine-modified (for surface charge) mesoporous SiO nanoparticles exhibited significant hemolysis at particle concentrations as low as 100 μg/ml [68]. Correlation of particle (and pore) size with erythrocyte toxicity has been confirmed by others. In one study employing erythrocytes exposed to 50/100 μg/ml of either 100 nm diameter mesoporous (MCM-41 silica) or 600 nm diameter (SBA-15 silica)

IV. THERANOSTIC PLATFORMS

SILICA TOXICITY

nanospheres, researchers observed no adverse effects with the former but significant, induced membrane deformation and hemolysis in the latter [69]. Endothelial cells also exhibit increases in toxicity with increasing particle size. In studies using 16 to 304 nm diameter porous silica nanoparticles, exposure of human umbilical cord endothelial cells (HUVEC) to 212 nm nanoparticles at 30,000 particles/cell demonstrated a 50% decrease in cell viability, while the same cytotoxicity was observed following exposure to 304 nm particles at 15,000 particles/cell [70]. Even more dramatic, however, is the demonstrably greater cytotoxicity associated with the use of nonporous silica nanoparticles. In studies comparing small 25 nm diameter nanospheres, those comprised of nonporous silica displayed a 72% decrease in erythrocyte viability relative to those comprised of mesoporous silica, with hemolysis occurring in the nonporous SiO nanoparticles for concentrations as low as 20 μg/ml [71]. Other studies with epithelial, neuronal, stem cells, lymphocyte, and fibroblast cells also exhibit particle(size, shape, structure, charge, dose) and celldependent cytotoxicities. For example, exposure of human embryonic kidney cells (HEK-293) to 20 to 760 nm diameter nonporous silica nanospheres at doses of 20 to 2000 μg/ml revealed much greater toxicity for smaller particles than for larger particles (e.g., 50% decreased viability at doses .80 μg/ml of 20 nm particles vs. doses .140 μg/ml of 50 nm or larger particles) [72]. Kidney epithelium cells (HeLa), exposed to 70 nm diameter nonporous silica nanospheres at a dose of 50 μg/ml, displayed no cytotoxicity in the presence of serum but high cytotoxicity in the absence of serum, while no toxicity was found in 200 nm and 500 nm diameter nonporous silica nanospheres similarly dosed either with or without serum [73]. Studies of fibroblasts (3T3) exposed to 38 nm diameter nonporous silica nanoparticles showed cell viability decreased by 50% at particle concentrations greater than 50 μg/ml, while porous silica nanoparticles were found to have little adverse effect on human T-cell lymphoma (Jurkat) cells [74,75]. Taken together these in vitro studies strongly suggest porous silica, for a given dose and particle size, shape, and charge, to be much less cytotoxic than comparable nonporous silica. On a submicroscopic level, much of the observed cytotoxicity arises from the disruption of membrane integrity and function. However, as noted earlier, a number of smaller silica nanoparticles (especially porous) readily undergo endocytotic incorporation and subsequent endosomal release into the cytosol. Once inside the cell such nanoparticles are potentially proteotoxic and/or genotoxic because, similar to

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proteins, nucleic acids also exhibit a high tendency to adsorb onto surfaces via electrostatic interactions and coordinative bonds, hydrogen bonds, and hydrophobic interactions. While interference with protein synthesis might take place in the cytosol, direct genotoxicity mandates DNA access, either via the nanoparticle crossing the nuclear membrane or its proximity during mitosis (either in binding directly to nucleic acids or interfering with mitotic spindles). One reported example of the direct genotoxicity of small silica nanoparticles is that of 40 to 70 nm diameter nonporous SiO nanoparticles contacting the DNA strands of human epithelial cells (HEP-2) during mitosis [76]. To date, though, such findings of genotoxicity triggered by the direction interaction of inorganic nanoparticles with nucleic acids are rare. Genotoxicity is much more likely to arise indirectly, generally from the production of oxidative stress on cells [77,78]. As briefly described earlier, ROS generation by nanoparticles can give rise to the oxidation of DNA bases, the breaking of DNA strands, and the derivation of DNA adducts from lipid peroxidation (e.g., via malondialdehyde reacting with deoxyadenosine and deoxyguanosine). The nanoparticles themselves can stimulate mitochondria to produce ROS that destabilizes DNA, while the liberation of metal ions from nanoparticles can modulate the permeability of nuclear membranes. Even macrophage nanoparticle interactions can lead to inflammatory scenarios that overwhelm intracellular and extracellular defenses against oxidative stress, leading to indirect genotoxicity. Although very few studies of silica nanoparticle genotoxicity have been reported in the literature, the general consensus among those that do exist is that the numerous dangling bonds present in both nonporous and crystalline silica nanoparticles are likely responsible for indirectly eliciting the response [79].

Administration and Exposure Route Significance It is extraordinarily difficult if not impossible to extrapolate, for a given nanoplatform, the findings of static, well-controlled in vitro cytotoxicity studies to those of dynamic, environmentally variable, and diverse in vivo exposures: the intrinsic complexity, variety, and number of potential particle and environment interactions makes such inferences of nanotoxicology intractable. Fortunately, however, preclinical studies of nanoparticle biodistribution and host response, as well as analyses of accidental human exposures, provide some insight as to the general mechanisms involved and sequelae expected. As physiological responses are typically dose related and access (anatomical) dependent, the route by which nanoparticles gain entry into the body (e.g., injection,

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inhalation, ingestion, skin contact) is of great relevance to the toxicity they pose. Unfortunately such toxicological data for silica nanoparticles, as well as for many other insoluble inorganic nanoplatforms, are sparse. As discussed previously, intravenous introduction of untargeted silica nanoparticles typically results in their immediate opsonization by serum proteins and that further expedites their endosomal uptake into cells, exploiting the same cellular machinery that viruses employ. With time and in the absence of abnormal or leaky vasculature, silica nanoparticles tend to accumulate in the liver and spleen, after which they may undergo hepatobiliary excretion and/or degradation into biocompatible orthosilicic acid (Si(OH)4). Murine model based studies of the biodistribution, biocompatibility, and clearance of mesoporous silica nanoparticles have shown both charge and geometry dependent elimination, with positively charged spherical moieties experiencing the most rapid hepatobiliary excretion, shorter nanorods being quickly sequestered and cleared by the liver, and longer nanorods becoming trapped within the spleen [68]. In contrast to in vivo behavior of mesoporous silica nanoparticle, intravenously delivered nonporous silica nanoparticles typically show significant delays in particle clearance [80]. Considerably more toxicological data have been collected on the inhalation of aerosolized silica nanomaterials, owing to its use in a number of industries and its mobility when airborne. When exposed to lung parenchyma, silica nanoparticles often result in size and morphology dependent toxicities, with smaller, more charged particles displaying the greatest effect. Studies of humans routinely exposed to nonporous or crystalline SiO nanoparticles like those in ground quartz dust often exhibit a spectrum of respiratory disorders that include silicosis, interstitial fibrosis, industrial bronchitis, small airway disease, and emphysema [81 85]. In one study of 7 patients accidentally exposed to aerosolized paint bearing silica nanoparticles and suffering from multifactorial pulmonary disease (pleural effusions, progressive pulmonary fibrosis), 20 nm diameter largely nonporous silica nanoparticles were discovered in vascular endothelial cells, macrophages, and pulmonary and lymphatic microvessels less than 3 months post-exposure [85]. After 15 months, epithelial cells and macrophages of the afflicted were found to posses relatively few silica nanoparticles, though some patient biopsies demonstrated the existence of 70 nm long nanostructures and agglomerates within the nucleus and cytoplasm of alveolar epithelial cells. Nanoparticle exposure dosages could not be determined and, after 18 months, 2 patients had died of pulmonary failure and 2 suffered severe disability. Pulmonary exposure to aerosolized

amorphous and porous silica nanoparticles generally appears to be less toxic, perhaps reflecting their relative paucity of dangling bonds (i.e., undissociated silanols). However, toxicological studies of pulmonary exposures to amorphous and porous silica nanoparticles have shown increases in reactive oxygen species concentrations, reductions in glutathione levels, and induction of proinflammatory, inflammatory, and oxidative stress responses both in vitro and in vivo [85]. At present there are no systematic toxicological studies of the effects of skin contact with silica nanoparticles. Although surface hydroxyl groups of silica nanoparticles tend to increase their hydrophilicity, the isoelectric point for silica is between 2.3 and 2.8 while that of skin is between 3.5 and 4.8, making both surfaces negatively charged (unless intentionally modified) and thus repulsive under normal physiological conditions [86]. The brick-and-mortar-like, circuitous structure of intact stratum corneum—comprised of corneocytes filled with keratin, cerimides, cholesterol, and fatty acids—and intrinsically low water content, generally precludes particles possessing diameters .20 nm from passage to the capillaries of the dermis [87]. Exceptions to such impermeability, however, can occur in regions of comprised skin integrity (e.g., abrasions, lacerations, burns) or via shunting along comparatively sparse transappendageal routes such as along hair follicles and sweat ducts. As a result of the skin’s barrier qualities, a number of silica nanoparticlebased therapies have been approved for the treatment of dermatological conditions such as acne and rosacea. Ingestion provides yet another route of silica nanoparticle administration and exposure; one on which numerous preclinical drug development studies have focused their attention, given the great stability of silica in low pH environments. In one such study, nonporous silica nanoparticles having diameters of 70, 300, and 1000 nm were orally administered to mice at a maximum dose of 100 mg/kg [88]. 70 nm SiO nanoparticles proved lethal to mice at doses greater than 20 mg/kg, while 300 and 1000 nm nanoparticles demonstrated no adverse reactions. Subsequent examination of the harvested spleens, kidneys, and lungs of mice subjected to 300 and 1000 nm SiO nanoparticle oral administration revealed no apparent toxicity. However, degenerative necrosis of hepatocytes was discovered in mouse livers shortly following the administration of 70 nm silica nanoparticles, and liver fibrosis accompanying longer-term exposure to 70 nm silica nanoparticles. Taken together, these findings appear to support those of cell studies: that, all other things being equal, the relatively larger surface area to volume ratios of smaller nanoparticles correlates with increases in cytotoxicity. Interest in the ingestion of porous silica nanoparticles has recently garnered

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SYNTHESIS AND SURFACE MODIFICATION OF SILICA NANOPARTICLES

considerable interest as well, due to availability of their large pore volumes and enormous surface areas for drug conjugation. Mesoporous silica nanoparticles in particular have proven especially appealing for such applications as their periodic and highly adjustable pore geometry, which lends itself to protected and controllable delivery of large quantities of otherwise water insoluble drugs or cytotoxic drugs [89]. Controlled toxicological studies of ingested mesoporous silica nanoparticles generally report little if any observed toxicity. Rabbits and dogs orally administered 200 to 1000 nm diameter mesoporous silica nanoparticles exhibited no signs of toxicity [90]. Noteworthy, however, is that all studies conducted to date have examined otherwise healthy individuals; those with ulcerative and inflammatory GI conditions were excluded.

SYNTHESIS AND SURFACE MODIFICATION OF SILICA NANOPARTICLES Silica nanoparticles currently under development for nanotheranostic applications can structurally, and to some extent therefore functionally, be classified as either nonporous or porous. Though compositionally indistinguishable, structural differences between nonporous and porous silica nanoparticles are marked as the forgoing discussion notes, with significant implications both for their application and biocompatibility. Synthesis of nonporous silica nanoparticles is typically conducted by one of two approaches: via the Sto¨ber process or reverse microemulsions. The Sto¨ber process, developed in 1968, is a sol-gel method that generates monodispersed spherical silica particles [91]. This technique relies on the controlled hydrolysis and condensation of a silica precursor, most often the alkoxide terraethyl orthosilicate (Si(OC2H5)4, abbreviated TEOS, taking place in the presence of excess water and a low molar-mass alcohol such as ethanol, with ammonia serving as a catalyst. Particle size and, to a lesser degree, morphology, can be adjusted during synthesis via changes in water silane ratio, catalyst or solvent type, and temperature. For example, methanol solutions result in the smallest diameter particles and narrowest diameter distributions, while both particle diameter and diameter distribution increase with the use of longer-chain alcohols. In general, the diameters of nonporous silica particles derived from Sto¨ber processes range from 20 to 2000 nm, with mutual electrostatic repulsion of their negatively charged surfaces preventing their flocculation in aqueous solutions. Sto¨ber processes have also been used to create nonporous silica coatings around smaller seed and core

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particles [92]. Reverse microemulsion, also known as water-in-oil, approaches to silica nanoparticle synthesis were developed in the 1990s and are used to generate 20 to 100 nm diameter, highly monodisperse spherical particles [93]. In this technique, ammonia-catalyzed polymerization of TEOS takes place in nanometersized water droplets (termed nanoreactors) that are stabilized by surfactants and dispersed in a continuous domain of oil. It is a thermodynamically stable, isotropic, highly tunable system in which the size of the micelles, and subsequently of the silica nanoparticles, can be adjusted by altering the ratio of water to organic surfactant. Nonporous silica nanoparticles can be readily functionalized for theranostic applications via the noncovalent entrapment of hydrophilic functional molecules within the particle’s silica framework. An example of entrapment functionalization is the embedding of fluorescent dyes such as fluorescein isothiocyanate (FITC) and ATTO 647N directly within the silica matrix of the nanoparticle. Spectroscopic analyses of these constructs demonstrate significantly enhanced photostability and brightness relative to free dye molecules, arising primarily from increases in local fluorophore number density and reductions in fluorophore selfaggregation, inner cell effects, and oxygen/pH quenching. Functional moieties can also be integrated within the nanoparticle’s silica matrix via the use of organoalkoxysilane derivatives during nanoparticle synthesis [94]. As the siloxane linkers remain randomly or uniformly distributed throughout the growth media during silica condensation, homogeneous small molecule functionalization can be realized throughout the resulting nanoparticle. Functional entities that are incompatible with the chemistry of nonporous silica nanoparticle synthesis must be “grafted” onto the particle’s surface subsequently, generally by exposing the silica nanoparticles to trialkoxysilane derivatives. Porous silica is also frequently used in theranostic applications, owing to its high level of biocompatibility, ease of functionalization, and unique and adjustable topology, with mesoporous silica being the most common form. Synthesis of mesoporous silica nanoparticles can proceed via one of several modified Sto¨ber processes, with the formation of liquid crystalline mesophases of amphiphilic surfactants that operate as structure-directing templates for the polymerization of silica precursors (e.g., TEOS). Under acidic or basic conditions, once the silica nanoparticles have grown to their desired size, the template surfactants are removed to generate pores, either by calcination or solvent extraction in acids or alcohol mixtures for cationic surfactants such as cetyltrimethylammonium bromide (CTAB) or alcohols for neutral surfactants such as n-dodecylamine. Calcination promotes

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condensation of unreacted silanol groups (and thus reduces attachment sites for post-synthesis functionalization) and many functional entities are destroyed at calcination temperatures (400 550 C), so solvent extractions are most often employed. Moreover, with the existence of different liquid crystalline mesophases and surfactant morphologies, surfactant-templated synthesis of mesoporous silica nanoparticles can be used to derive a variety of isoforms that include permutations of mesostructure (e.g., disordered, wormhole-like, hexagonal, cubic, and lamellar mesophases) and morphology (e.g., spheres, hollow spheres, fibers, tubules, gyroids, helical fibers, crystals, and many hierarchical structures), simply by controlling the reaction conditions (e.g., temperature, pH, surfactant concentration, silica source). Pore diameters are largely dictated by template characteristics, however; for example, varying the alkyl chain length of homologs of quaternary ammonium surfactants can result in minor modification (1.8 to 2.3 nm). Additional pore expansion, needed for peptide, aptamer, gene, or protein delivery, can be achieved through the use of “swelling agents” such as N,N-dimethylhexadecylamine (DMHA) and 1,3,5-trimethylbenzene (TMB). Pore expansion via such agents, however, often results in less uniform, wormhole-like pore structures. Largersized pores can also be obtained through the use of block polymers as templates [95]. Alternative approaches to achieving large pore diameter mesoporous silica nanoparticles include the use of block polymers as templates or the incorporation of multiple surfactants of different molecular weight, the latter of which can also be used to generate dual-mesoporous materials. Surface modification of mesoporous silica nanoparticles for subsequent molecule attachment are generally accomplished either by cocondensation (direct incorporation) during synthesis or by post-synthesis grafting. In cocondensation, tetra/tri-alkoxysilane is condensed into the pores of the silica nanoparticle during synthesis, resulting in the homogeneous incorporation of the functional moiety throughout the silica. Organoalkoxysilanes can also be cocondensed into the growing nanoparticle to produce an inorganic-organic hybrid for subsequent, additional functionalization. The choice of alkoxysilane precursor is constrained to those that are soluble in the aqueous phase of synthesis and can survive the great variations in pH encountered during particle polymerization and surfactant extraction. Surprisingly large numbers of functional groups can be accommodated via cocondensation techniques without significantly affecting pore topology. Anionic organoalkoxysilanes, such as thiolate-, carboxylate-, and sulfonate-containing organoalkoxysilane, have been used to electrostatically preclude phase

separation of precursors and cleavage of Si-C bonds during sol-gel synthesis and ensuing surfactant removal [96]. Cocondensation of two organoalkoxysilanes can bestow both acidic and basic functional groups onto nanoparticle frameworks, resulting in novel chemical properties and applications [96,97]. Post-synthesis grafting onto mesoporous silica nanoparticles exploits the presence of surface silanol groups (Si-OH), which can occur with abundance if not calcinated, that act as convenient anchoring points for organic functionalization. Grafting is commonly carried out by silylation on free (Si OH) and geminal silanol (Si(OH)2) groups, though hydrogen-bonded silanol groups are less accessible to modification as they readily form hydrophilic networks with one another. The original particle morphology and pore structure remain intact with grafting, though the technique often results in nonuniform distributions of organic functional groups. Such inhomogeneous coverage with functional groups is especially problematic within pores as these surface silanols are less directly accessible to grafting moieties. To address this problem as well as enable independent functionalization of the outermost and innermost surfaces of the silica nanoparticle, researchers selectively and alternately passivate the silanols in one domain while grafting onto those of the other domain; for example, by not extracting surfactant templates from pores until after first grafting functional groups onto the particle’s exterior, then extracting templates and reacting only the remaining unaffected, pore-lining silanols. Despite exhibiting greater functionalization inhomogeneity than cocondensation approaches, post-synthetic grafting is the most popular approach for covalently incorporating organic functionalities onto mesoporous silica. The forgoing description of surface modifications of silica nanoparticles largely reflects the versatility of silanol/organosilanol chemistry. With the introduction of organosilanes bearing amino, thiol, or carboxy reactive moieties, either during or following nanoparticle synthesis, covalent conjugation of a wide variety of biomolecules can be rendered. Disulfide-modified oligonucleotides can be linked to thiol-functionalized silica nanoparticles by disulfide-coupling chemistry. Carbodiimide reagents can be used to couple aminebearing biomolecules to carboxy-modified silica nanoparticles. In addition, a wide array of amino reactive biological constructs (e.g., enzymes, antibodies, aptamers) can be attached via succinimidyl esters and iso (thio)cyanates to silica nanoparticles possessing amino groups. Intrinsic electrostatic interactions between the biomoieties and nanoparticles can also be used for bioconjugation, though carboxy and amino group introductions can significantly alter a nanoparticle’s overall surface charge with unintended consequences, such as

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particle aggregation. Physical adsorption (physisoption) arising from van der Waals interactions can also be used to form silica nanoparticle bioconjugates, though such moieties are often transitory under biological settings due to the weakness of attraction. Lipids and polyethylene glycol (PEG) coatings have also been used to alter the surface properties of silica nanoparticles, though primarily to enhance nanoplatform biocompatibility. As will shortly become evident, the availability of these diverse surface modifications of silica, both porous and nonporous, has resulted in multifunctional platforms with a wide variety of applications in biotechnology and biomedicine.

SILICA NANOPARTICLE NANOTHERANOSTICS General Considerations Recently there has been considerable interest in employing nanometer-sized biocompatible materials that are both diagnostic and therapeutic. Termed nanotheranostics, the motivation for such entities arises from a desire to monitor disease status. Election to provide both diagnostic and therapeutic functions in a single moiety also stems from the desire to circumvent differences in biodistribution and selectivity that frequently accompany the employment of distinct diagnostic and therapeutic agents, and to exploit existent overlapping functionalities (e.g., both diagnostics and therapeutics mandate specific targeting of pathology). Silica nanoparticles, by virtue of their high biocompatibility, in vivo stability, tailorability, diverse morphology, and ease of functionalization are especially well suited for theranostic applications. In situ diagnosis and, in some cases, staging of disease relies on the incorporation of a suitable contrast agent (or agents) within the nanotheranostic for the clinical imaging technique (or techniques) chosen. Current clinical imaging modalities and contrast agents for which silica nanoparticles are being developed as nanotheranostic agents include (1) magnetic resonance imaging (MRI), via the use of gadolinium, manganese, and iron oxide; (2) positron emission tomography (PET), via the use of the radiolabeled glucose analog fluorodeoxyglucose (18F-FDG); (3) single photon emission computed tomography (SPECT), via the use of radionuclides such as metastable technetium-99 (99mTc), iodine-123 (123I), and iodine-131 (131I); (4) computed tomography (CT), via the use of gold, iodine, and barium; and (5) optical imaging/diffuse optical tomography (OI/DOT), via the use of fluorophores such as indocyanine green (ICG), FITC, and Alexa Fluor or ATTO dyes (as well as

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substrates of bioluminescent enzymes such as luciferase, in animal studies). Therapeutic functionalization of silica nanoparticle theranostics for oncological applications are being tailored to the specific form and presentation or stage of cancer and include (1) nucleic acid therapy (NAT), via the use of siRNA, DNA plasmids, ribo/DNA-zymes, and antisense oligonucleotides; (2) chemotherapy, via the use of doxorubicin, paclitaxel, and cisplatin; (3) photodynamic therapy (PDT), via the use of palladium-porphyrin and zinc(II) phthalocyanine photosensitizers; and (4) radioisotope therapy (RIT), via the use of radionuclides such as yttrium-90 (90Y), iodine-131, and strontium-89 (89Sr). To be clinically useful, nanotheranostics must demonstrate specific accumulation at the site of interest, either through active or passive targeting. Passive targeting in oncology relies on the nonspecific accumulation of nanoplatforms in the leaky vasculature of tumors via the enhanced extravasation, permeation, and retention (EPR) effect. As noted earlier, however, such passive targeting is extremely dependent on the nanoplatform’s size, shape, reactivity, and surface charge, making the reduction of nonspecific binding a challenging, problematic task. Nanotheranostic diagnostic and therapeutic utility are each significantly strengthened through the use of active targeting, whereby targeting moieties (e.g., small peptides, antibodies, aptamers, protein fragments), attached to the nanotheranostic’s exterior, specifically associate with receptors at the disease site. Such incorporation of pathology targeting ligands can enable true molecular imaging of disease location and status. Labeling nanoparticles with multiple copies of a given targeting ligand substantially increases particle-pathology avidity, while employment of multiple different ligands on the same nanoplatform can permit the nanotheranostic to grapple with dynamic variations in targeted receptor expression, as often occurs in cancers.

Multifunctionalized Solitary Platforms Multifunctionalized solitary silica nanoparticles, both porous and nonporous, have found numerous applications as nanotheranostics, one of which is for photodynamic therapy (PDT). PDT is frequently used in clinics to treat dermatologic disorders such as acne, rosacea, and psoriasis, ophthalmological diseases such as wet macular degeneration, and several forms of lung and esophageal cancer. It is an intrinsically focal therapy, far less invasive than surgical approaches, and with far fewer side effects than pharmacological interventions. PDT involves the pathology’s uptake of photosensitizers (PS)—often porphyrins, chlorophylls, or dyes—whose external photoirradiation produces locally cytotoxic, reactive oxygen species (ROS). Many

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PSs are also intrinsically fluorescent, enabling their use in diagnostic OI as well. The photosensitizer, in its ground (singlet) state, absorbs a photon that promotes the PS to a short-lived excited singlet state. Because the excited singlet state is so short lived, the PS has little opportunity to either transfer energy or electrons to other molecules nearby and instead undergoes intersystem crossing to leave it in an excited, much longerlived, triplet state. As the PS subsequently decays back to its initial ground (singlet) state, it transfers energy to nearby ground (triplet) state molecular oxygen, raising the latter to its first excited singlet state. The so-generated singlet oxygen molecules (and other ROSs) are especially efficacious cytotoxic agents that rapidly induce apoptosis or necrosis. The overwhelming majority of clinically available PSs, however, possess significant limitations that constrain their wider use, which include high hydrophobicity, substantial self-aggregation, and poor tumor selectivity. A, considerable effort has gone into developing compounds with improved “deliverability.” In one recent study investigators described the use of the organically modified silica (ormosil) nonporous nanomaterials for PDT [98]. The researchers integrated 2-[1 hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH)—a lipophilic, second-generation, chlorinbased photosensitizer currently in phase II clinical trials (Photochlor)—into silica matrices. The resulting constructs demonstrated that HPPH incorporation within silica significantly enhances the quantum efficiency of HPPH (as well as ROS generation and local cytotoxicity) relative to free HPPH as intravenously delivered. Unfortunately HPPH, though better suited than first-generation PSs such as porfimer sodium (an oligomer mixture of ether and ester-linked porphyrin units, commercially available as Photofrin), can only be used for PDT to a tissue depth of a few millimeters, due to the inherent scattering and absorption of photons in most mammalian tissues at visible wavelengths. To enhance PDT efficiency at greater tissue depths, more recent efforts have focused on working at longer, near-infrared (NIR) wavelengths—where photon absorption and scattering in tissues are greatly reduced—with either NIR dyes or combinations of two-photon absorbing dyes with PSs. In vitro and in vivo OI and PDT studies using 105 nm diameter nonporous silica nanoparticles in which the cationic NIR dye methylene blue was entrapped, revealed considerable gains in detection sensitivity and therapeutic efficacy [99]. In another study, researchers coencapsulated HPPH and the two-photon absorbing dye 9,10-bis[40-[400 aminostyryl]styryl]anthracene (BDSA) within nonporous silica nanoparticles, revealing PDT activation via BDSA upconversion of NIR light with

partial energy transfer to HPPH [100]. Silica coating of highly porous, nanometer-sized iron-carboxylate metal-organic framework (MOF) particles, postsynthesis covalently modified with the fluorescent dye 1,3,5,7-tetramethyl-4,4-difluoro-8-bromomethyl-4bora-3a,4a-diaza-s-indacene (Br-BODIPY) and the anticancer drug cisplatin, have also been developed for theranostic applications [101]. Mesoporous silica nanoparticles (MSNs) have also been developed as highly efficient PDT nanotheranostics. In one study investigators sequentially functionalized the nanoplatform’s 3 topologically distinct domains with contrast agents for traceable imaging of particle targeting, payloads for therapeutic intervention, and biomolecular ligands for highly-targeted particle delivery, as shown in Figure 20.2 [102]. Traceable imaging of nanoparticles was accomplished by directly incorporating (cocondensing) the NIR fluorophore ATTO 647N into the MSN’s silica framework to exploit the relative transparency of most tissues at NIR wavelengths and maximize MSN surface area available for the subsequent conjugating drugs and targeting ligands. An oxygen-sensing, palladium-porphyrin based photosensitizer (Pd-porphyrin; PdTPP) was incorporated into the MSN’s nanochannels to enable PDT. And cRGDyK peptides, tiling the outermost surfaces of MSNs, were used for targeting αvβ3 integrin over-expression of cancer cells and to ensure the internalization of the photosensitizer PdTPP. In vitro cell evaluation of the theranostic platform demonstrated not only excellent targeting specificity and minimal collateral damage, but highly potent therapeutic effect as well. Human MCF-7 breast cancer (αvβ3 integrin negative) and U87-MG glioblastoma (αvβ3 integrin positive) cells were used with flow cytometry and confocal microscopy to assess the functionalized MSN’s targeting specificity and uptake efficiency, while propridium iodide staining and WST-1 assay enabled determination of PDT response prior to and following 532 nm wavelength laser irradiation. These in vitro studies revealed that the trifunctionalized MSN nanotheranostics possessed not only excellent targeting specificity but highly potent therapeutic effect as well. Members of the same group subsequently developed MSNs that coencapsulated twophoton absorption dyes and photosensitizers, thus enabling high-energy transfer rates for two-photon activated PDT [103]. With the judicious tailoring of two-photon donor-acceptor ratios, the well-ordered mesoporous structure of MSNs demonstrated energy transfer rates up to an unprecedented 93%. Intracellular energy transfer proved extremely efficient as well in the nanoplatform, with dramatic singlet oxygen induced cytotoxicity observed in both in vitro and in vivo breast cancer models. In another study employing porous silica for deep tissue PDT, Gary-Bobo et al.

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(a)

(b)

FIGURE 20.2 Three domain (silica framework, mesopore, particle exterior) functionalization of mesoporous silica nanoparticles (MSNs). (a) MSNs were synthesized via conventional sol-gel chemistry so as to possess the near-infrared fluorophores ATTO 647N cocondensed within their silica frameworks. Following extraction of templates, palladium-based photosensitizers were loaded into the MSN pores. The nanoparticle was then PEGylated and targeted for chemotherapy via conjugation of c(RGDyK) peptides. (b) Operational schematic of the trifunctionalized theranostic MSNs for traceable, targeted, photodynamic therapy. Source: Reproduced from [102] with kind permission of The Royal Society of Chemistry. Copyright 2010. All rights reserved.

used mannose-functionalized mesoporous silica particles for tumor-targeted two-photon PDT, observing 70% regression of tumor size after a single treatment in athymic mice bearing tumor xenografts [104]. Silica-based nanoparticles have garnered considerable interest for their use as nanotheranostic platforms in the delivery of drugs and nucleic acids as well. Mesoporous silica nanoparticles, in particular, make for appealing delivery vehicles as they can convey considerably more drug within their environmentally protected pores than can be accommodated on the surfaces of comparably sized, nonporous silica nanoparticles. Porous and mesoporous silica constructs, unlike their nonporous and crystalline silica counterparts, do not require that the conveyed drug be covalently conjugated to the nanoparticle’s surface; this is significant in that many drugs cannot tolerate covalent attachment without adversely affecting their structure or function. Lastly, as will be described shortly, MSNs

can be synthesized so that their pores are capped with gatekeeping molecules that enable finely controlled drug release either via internal or external stimuli. Ashley et al. developed MSN-supported lipid bilayers, termed protocells, as theranostic platforms via liposome fusion and encapsulation of fluorescent MSNs that bore with various therapeutic agents (e.g., drugs, siRNA, protein toxins, quantum dots), as illustrated in Figure 20.3 [105]. The investigators demonstrated that the lipid membranes of these MSNs retain both high in-plane two-dimensional fluidity and high stability against destabilization in the presence of blood components or leakage of drug cargos from the silica core. Post-synthesis modifications of protocells— to promote cell targeting, endosomal escape, and nuclear accumulation—resulted in 10,000-fold greater nanoplatform affinities for human hepatocellular carcinoma cells than for hepatocytes, endothelial cells, or immune cells, enabling individual protocells, loaded

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FIGURE 20.3 Mesoporous silica-supported lipid bilayer constructs, termed protocells, for multitherapeutic cancer treatment. Fusion of liposomes onto previously synthesized spherical MSNs, and subsequent targeting ligand association, yielded 10,000-fold greater affinity for human hepatocarcinoma than for hepatocytes, endothelial cells, or immune cells, a manifestation of the enhanced targeting efficacy afforded by the fluid supported lipid bilayer. Incorporation of PEG into the bilayer improves colloidal stability and decreases nonspecific interactions. Source: Reproduced from [105] with kind permission of Macmillan Publishers Limited. Copyright 2011. All rights reserved.

with a drug cocktail, to kill individual drug-resistant human hepatocellular carcinoma cells. This represented a 106-fold improvement over comparable liposomes. Recently, Lu et al. reported the synthesis of MSNbased theranostic particles that contain the fluorophore FITC embedded (cocondensed) within the silica framework, the anticancer therapeutic camptothecin (CPT) condensed within the nanopores, and folic acid grafted onto the MSN’s exterior for cancer targeting [106]. These studies were primarily focused on the toxicity, biodistribution, clearance, and therapeutic properties of their nanotheranostics, with human breast cancer cell lines (MCF-7, MCF10F, and SK-BR-3) used for in vitro analyses and six-week-old female BALB/ cAnNCrj-nu nude mice for in vivo studies, some of which bore MCF-7 xenografts. Optical imaging and inductively coupled plasma mass spectroscopy (for silica content) were used to monitor the nanotheranostic’s biodistribution and excretion, respectively. Fluorescence imaging revealed that their 100 to 130 nm diameter MSNs preferentially localized to the tumor, kidneys, and liver. The tumors in the mice treated with CPT-loaded MSNs were virtually eliminated at the end of experiments although, surprisingly,

conjugation with folic acid on the MSN’s surface seemed to produce little additional antitumor effect when compared to untargeted CPT-loaded MSNs, a finding postulated to be a reflection of rather modest upregulation of folate receptors in the MCF-7 xenografts and significant EPR effect. Also noteworthy was the near complete excretion of MSNs from mice: B95% of Si was excreted out of body through urine and feces, consistent with observations of Souris et al. [107]. Lee et al. developed theranostic MSNs in which the anticancer drug doxorubicin (dox) was conjugated onto the particle’s pore walls through the use of finely tuned, pH-sensitive linkers [108]. Initially the NIR fluorophore ATTO 647N was cocondensed into the MSN’s silica framework during nanoparticle synthesis. Following template extraction and pore expansion (to 5 nm) of the 100 nm diameter nanoparticle, the pore walls and dox were separately modified so as to possess pH-sensitive hydrazone bonds. When MSNs had been loaded with drug, the pH-sensitive linkers possessed two hydrazone bonds that were cleavable at endosomal/lysosomal pHs (4 6) but stable in circulation (pH 7.4). pH sensitive hydrazone linkers are generally compatible with drugs that have functional

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ketones or aldehydes, such as cerubidine and idarubicin. Fluorescence spectroscopy and imaging were used to quantitate the MSN’s uptake and release of dox, as well as the nanoparticle’s therapeutic efficacy, with human hepatoma cells (Hep-G2) in vitro. Because the emission spectra of dox overlapped with the absorption spectra of ATTO 647N, and the average distance between donor and acceptor was shorter than 10 nm, efficient Fo¨rster resonance energy transfer (FRET) took place prior to the release of the intrinsically fluorescent dox from the MSN’s constituent ATTO 647N, a phenomenon the investigators also used to monitor dox release in situ. Murine biodistribution studies of ATTO 647N-MSN-hydrazone-dox, conducted using in vivo fluorescence imaging and subsequently confirmed by transmission electron microscopy (TEM) of harvested tissues, demonstrated hepatic endosome and lysosome colocalization. He et al. developed yet another form of pH-responsive MSNs capable of overcoming MDR [109]. Their composite nanoparticles consisted of MSN carriers of dox that still contained the porogen cetyltrimethylammoniumbromide (CTAB), where the surfactant served as a chemosensitizer that both circumvented MDR and enhanced drug efficacy. The cytotoxicity of these particles was evaluated using MCF-7 and MCF-7/ADR human breast cancer cell lines and demonstrated that MSN-dox-CTAB significantly enhanced dox intracellular accessibility of MCF7/ADR with much greater effect than free drug against both cell lines. Multitherapeutic conveyances by MSNs, such as the codelivery of chemotherapeutic drugs and siRNAs targeting cellular survival mechanisms, have also been reported recently. Chen et al. presented convincing evidence that MSNs, carrying both dox and siRNA and targeted against mRNA encoding Bcl-2 protein in multidrug resistant (MDR) cancer cells, significantly increased the cytotoxicity of dox by B132-fold relative to free drug via a pronounced increased apoptosis [110]. Mesoporous silica nanoparticle shells, with and without cores, have also been devised as theranostic nanoplatforms for the protected delivery of drugs and organics monomers and polymers. One example is that of Kim et al. who synthesized monodisperse core shell constructs (45, 60, 90, and 105 nm diameter) in which a layer of mesoporous silica enveloped a single iron oxide (IO) Fe3O4 nanocrystal of either 15 or 22 nm diameter, as shown in Figure 20.4 [111]. While the IO core provided MRI contrast, investigators elected to also fluorescently label the silica shell for optical and microscopic tracking of their nanoplatform, via the covalent incorporation of FITC and rhodamine B within the silica mesopores. The IO-mesoporous silica (MS) shell was then PEGylated for improved in vivo stability and loaded with the anticancer agent dox.

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Fluorescence and confocal laser scanning microscopy of human breast cancer cells (MCF-7) revealed excellent endocytotic internalization of the nanoparticles in cells within 30 minutes of administration. In vitro T2weighted MRI of IO-MSshell-PEG with a 1.5T scanner revealed r1 and r2 relaxivities of 3.40 and 245 mM21 s21, respectively. In vitro T2-weighted MRI of MCF-7 cells showed concentration-dependent uptake of the nanoparticles, in agreement with fluorescence microscopy of the same specimens. To assess the untargeted IO-MSshell-PEG construct’s in vivo utility, human breast cancer cells (SK-BR-3) were subcutaneously injected into the dorsal shoulders of nude mice. Subsequent in vivo and ex vivo fluorescence and MR imaging of the resulting xenografts, following IOMSshell-PEG intravenous injection, showed significant EPR effect passive accumulation of nanoparticles within tumors, confirmed by TEM ex vivo. The same group subsequently developed dox-loaded MSNs in which the fluorophore FITC (or TRITC) had been covalently attached to the MSN’s pore walls and IO nanoparticles chemically attached to the MSN’s exterior surface, to yield dramatically enhanced T2-weighted MRI contrast: with r2 relaxivities of 76.2 mM21 s21 for the IO-capped MSNs compared to 26.8 mM21 s21 for free IO [112].

Organic-Composite Hybrid Platforms To further enhance the biofunctionality of silicabased nanotheranostics, considerable effort has gone into the development of organic-composite hybrids whose inner and/or outer surfaces contain organic polymers such as polyethylene glycol (PEG), polyethylene imine (PEI), or one of a variety of pH-sensitive polymers. While organic hybridization of porous and nonporous silica nanoparticles can be active (e.g., such as in enabling nanoparticles to react to changes in their local environment), to date it has more frequently served static functions such as stabilizing payloads for conveyance or enhancing their delivery via increasing circulatory lifetimes and proteolytic resistance, while decreasing their immunogenicity and/or antigenicity. For drug delivery theranostic applications, mesoporous silica platforms are the dominant solid support platform in organic-composite hybrids, due to their higher payload capacity than nonporous silica moieties. For example, Wang et al. coated PEGylatedphospholipids onto hydrophobic, silane-modified MSNs that possessed FITC for optical tracking and folate for targeting, observing significantly inhibited flocculation and reduced nonspecific protein binding [113]. Another polymer, PEI, has been found to greatly enhance drug delivery efficiency of silica nanoparticles via its unique proton-sponge and endosome

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FIGURE 20.4 Untargeted mesoporous silica shell iron oxide core theranostic nanoplatform for combined MR/fluorescence imaging and drug delivery via EPR effect. (A) TEM of different diameter (g: 45 nm; h: 60 nm; i: 90 nm; j: 105 nm) mesoporous silica shells that had been synthesized around individual 15 nm diameter iron oxide (Fe3O4) nanocrystal cores. Very high shell uniformity was achieved via only varying the concentration of iron oxide nanocrystals, with nanoparticle diameter increasing as core size decreased. Similar methods were used to coat mesoporous silica onto 25 nm MnO nanocrystals (k) and one-dimensional α-FeOOH nanotubes (l). (B) In vivo T2-weighted MR (a) and fluorescence (b) imaging of mice with/without subcutaneous injection (dorsal shoulder) of MCF-7 cells labeled with/without F3O4 mesoporous silica shells at 10 μg Fe mL21. In vivo T2-weighted MR imaging (c) of mice before/after intravenous injection (tail vein) of MCF-7 cells labeled with F3O4 mesoporous silica shells at 5 mg Fe kg21. Source: Reproduced from [111] with kind permission of Wiley-VCH Verlag GmbH & Co. Copyright 2008. All rights reserved.

buffering properties that destabilize lysosomal membranes and promote endosomal escape of their contents [114,115]. Rosenholm et al. developed onionlike, core-shell-shell fluorescent mesoporous silica

nanoplatforms that provide targeted, traceable delivery of therapeutic compounds [116]. In their design, a biodegradable mesoporous silica matrix core, in which the therapeutic payload is stored, is surrounded by a

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Disulfide reducing agent light saccharides

N

Insulin Br

O

H2N

O HO

S S

O

O

O B Nanoparticles (Au-, CdS, Fe3O4, PAMAM, Insulin)

NO2 HN

O

R S MSN

Au-NP

Guest Molecule MSN

FIGURE 20.5 Gatekeeping moieties used with MSNs. Both “hard” (e.g., gold and iron oxide nanoparticles, CdS quantum dots) and “soft” (e.g., antibodies, dendrimers, insulin) gates have been attached to MSNs via stimuli-responsive linkages. Such constructs prevent premature dispersal of nanoparticle contents from pores and permit external control of their therapeutic payloads, for optimal treatment timing and pathology colocalization. Source: Reproduced from [167] with kind permission of Wiley-VCH Verlag GmbH & Co. Copyright 2010. All rights reserved.

PEI-layer that serves to simultaneously increase colloidal stability and promote endosomal escape into the cytoplasm. Folic acid targeting ligands decorated the outermost exposed surfaces of this cancer theranostic. Recent interest in organic hybrid MSNs has arisen not only from their enhancement of endosomal escape [116 118], light sensing [119 120], pH sensing [121 123], and water solubility [113], but also due to the ability of these nanomaterials to codeliver more than two types of payload in an additive fashion. Meng et al. designed a dual-drug delivery system using PEI-coated MSNs that were functionalized to efficiently codeliver the chemotherapeutic agent dox and P-glycoprotein (Pgp) siRNA to multidrug resistant (MDR) cancer cells for synergistic cytotoxicity [118]. Enhanced synergistic cytotoxicity of their platform was initially suggested by the observation of greatly increased intracellular and intranuclear levels of dox following theranostic administration: levels far exceeding that found from either conventionally delivered free dox or dox delivered by MSNs that were devoid of Pgp siRNA. Subsequent in vivo studies, using an MDR tumor xenograft model, convincingly demonstrated the synergism of dox and Pgp codelivery. Interestingly, the investigators also observed significant Pgp knockdown at heterogeneous tumor sites that corresponded to the regions where dox was released intracellularly and induced apoptosis [124]. Flexible organic molecules have also been used as “gatekeepers” of the pores on MSNs. Mal et al. developed MSNs that exploited the reversible

photodimerization of coumarin to controllably open and close the pores of MSNs in organic solvents [125]. MSN pores were functionalized with 7-[(3 triethoxysilyl)propoxy]coumarin. Casasus et al. developed an ionically-controlled MSN gatekeeper comprised of diaminoethylenepropylsilane derivatives grafted immediately outside pore entrances and mercaptopropyl groups positioned proximally inside pores [126]. As illustrated in Figure 20.5, more elaborate gatekeeper constructs for theranostic payload release, perhaps more appropriately termed nanomachines, have also been derived using organic-composite hybridization with underlying silica nanoplatforms [127]. Radu et al. reported dendrimer-capped MSNs that can be used both for drug delivery and gene transfection system, using a generation 2 poly(amidoamine) dendrimer (G2-PAMAM), as shown in Figure 20.6 [62,127]. Hernandez et al. developed organic supramolecular gatekeepers termed nanovalves that consist of a series of electrochemical redox-controlled pseudorotaxanes attached to mesoporous silica [128]. In the earliest versions of this construct, [2]-pseudorotaxane [DNPDCBPQT]4 1 was used as a gatekeeper, of which 1,5dioxynaphtalene derivative (DNPD) served as the particle-attached gatepost and cyclobis-(paraquat-pphenylene) (CBPQT4 1 ) served as the gate. Following the same redox-controlled release approach, the investigators refined their system by developing a reversible [2]-rotaxane R4 1 nanovalve, comprised of two separate recognition sites in a dumbbell structure

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FIGURE 20.6 MSN codelivery of DNA and drug. As proof of concept, positively charged PANAM dendrimer caps were placed at MSN pore orifices and used to both seal in the nanochannel’s drug contents (substituted, in this case, with the fluorescent dye Texas Red) and attach and protect plasmid DNA encoded for enhanced green fluorescent protein (EGFP). Gene transfection, visible in the fluorescence microscopy insert, was reported to be more efficient than that obtained via commercially available transfection kits. Source: Reproduced from [167] with kind permission of Wiley-VCH Verlag GmbH & Co. Copyright 2010. All rights reserved.

with CBPQT4 1 once again serving as the gate and recognition sites, consisting of tetrathiafulvalene (TTF) and 1,5-dioxynaphtalene (DNP), which were separated from one another by an oligoethyleneglycol chain [129]. Another pseudorotaxane-based MSN controlled release system was reported by Park et al. using PEI as the fixed motif and α- or γ-cyclodextrin (CD) as the movable ring [130]. At high pH, the CD ring was observed to thread into the PEI base thereby closing the pore, while at low pH, PEI-CD irreversibly dissociated, with CD diffusing away from the nanoplatform and the pore becoming permanently open. For in vivo (aqueous) applications, CDs and cucurbit[6]uril (CB[6]) are often used in the construction of MSN pore gatekeepers, owing to their ability to form inclusion complexes that can reversibly dissociate in response to external stimuli and their high intrinsic biocompatibility [131]. Using CB[6]-containing [2]pseudorotaxanes, CB[6] rings reversibly respond to deprotonation of bisammonium stalks by dethreading, unblocking the pores for the release of MSN payloads. Biologically relevant, autonomous operation of these constructs (e.g., for lysosomal release) was achieved through the use of trisammonium stalks that contain one anilinium and two (CH2NH21CH2) centers in which the anilinium nitrogen atom (unlike the other two nitrogen atoms) is not protonated at neutral pH [132]. At neutral pH the

CB[6] rings reside on the two (NH2(CH2)4NH21) recognition sites, blocking the nanopore. Tuning the pKa of the anilinium nitrogen, via varying the parasubstituent on the aryl rings, permits precise control of the pore opening rate and pH value of operation. Inverse conformations of gatekeeper, with mobile stalks and stationary rings, have also been synthesized. Zhao et al. have used β-CD rings arranged to form cylindrical cavities in which rhodamine B/benzidine stalks move as pistons in response to changes in local pH value [133]. These short cylindrical β-CD cavities extend radially outward from the MSN’s surface and self-assemble so as to maintain alignment with the MSN’s pores. As such, these nanoparticles can also be used to deliver larger moieties since the β-CD ring linkages to the nanoparticle contain cleavable imine double bonds that are hydrolyzed under acidic conditions causing the ring-MSN dissociation and payload dispersal. Alternatively this construct could be used as a dualdrug platform to deliver both large and small molecules, tuned for release at different pH values. Additional organic-composite silica motifs have been devised for the externally controlled release of therapeutic compounds from MSN pores. Patel et al. used α-CD tori that encircled PEG stalks whose esterlinked adamantyl endcap, embedded within MSN pore orifices, can be enzymatically (irreversibly) cleaved to release MSN payloads [134]. Light activation is another

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avenue of considerable interest for controlled drug release. Photoactive azobenzene nanoimpellers—so termed from their original application of expelling MSN cargo via cyclic cis/trans configuration changes induced by UV/visible light switching [135]—have been modified for aqueous applications by Ferris et al. [136]. In their design, MSN surfaces are functionalized with azobenzene-containing stalks (in the trans configuration) that are complexed by β-CD (or pyrenemodified β-CD) rings, sealing the pores shut. After photoirradiation at 351 nm, the isomerization of transto-cis azobenzene units leads to dissociation of β-CD (or pyrene-modified β-CD) rings from the stalks, thereby opening the nanogates and releasing pore payloads. Thermoresponsive gatekeepers for MSNs have also been realized through the use of organic supramolecular assemblies. Baeza et al. developed a multidrug nanoplatform for the treatment of MDR cancers whose therapeutic release was remotely controlled via an externally controlled, alternating magnetic field, nominally operating at 100 kHz [117]. In their nanotheranostic, superparamagnetic iron oxide (γ-Fe2O3) nanocrystals were entrapped within the silica matrix of MSNs, after which a thermoresponsive copolymer shell, comprised of poly(ethyleneimine)-b-poly(Nisopropylacrylamide) (PEI/NIPAM), was applied. The thermoresponsive copolymer shell functioned not only as a temperature-sensitive gatekeeper for release of drugs entrapped within MSN pores, but also as a temperature-responsive retention system for conjugated therapeutic proteins. At lower temperatures, entrapped drugs and conjugated proteins remain tightly bound to the theranostic platform. Application of a strong alternating magnetic field to the nanotheranostic induced nanoscale motion of iron nanocrystals, with the MSN’s silica framework imposing a restoring force in opposition that elevated the nanoplatform’s temperature several degrees Celsius. In vitro studies of PEI/NIPAM-entrapped proteins revealed a protein release profile similar to that observed with direct heating of the sample, consistent with the hypothesis that the polymer collapses and changes to a more hydrophobic surface, causing the increase in the release rate of the attached protein. Increases in MSN temperature also resulted in the enlargement of MSN pores, an effect that could be used to alter the rate of drug release from the pores relative to the rate of thermoresponsive copolymer shell retained therapeutics. Coordinated, sequential delivery and release of combinatorial drugs is essential for the optimization of multidrug synergism. For instance, in organic hybrid MSN codelivery of Pgp siRNA and dox described earlier, initial Pgp knockdown ensures that subsequently released dox has sufficient time within the cell to reach

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the cell’s nucleus and intercalate its DNA. Without previous Pgp knockdown, most of the dox released within drug-resistant cancer cells will undergo rapid efflux from the cell, greatly diminishing therapeutic efficacy.

Inorganic-Composite Hybrid Platforms Due to their own unique physicochemical properties, discrete inorganic nanoparticles such as quantum dots (QDs), superparamagnetic iron oxide nanoparticles, and gold nanocrystals have each also garnered considerable interest for use in functionalizing silica nanoplatforms, resulting in constructs that may be termed inorganic-composite hybrid platforms. For example, narrow-band spectral emissions of QDs enable concurrent or mutiplexed operation of differently functionalized or targeted silica nanoplatforms within the same animal model [137 139]. Gold nanocrystal decorated MSNs and mesoporous silica shell enveloped gold nanorods permit externally controlled twophoton excitation of contrast agents, stimulation of drug release, and plasmon-induced thermotherapy in deep tissues [140 142]. Iron oxide nanoparticles, grafted onto mesoporous silica nanoparticles, can be used for platform tracking by MRI as well as local hyperthermia (thermotherapy) and controlled release of platform contents via oscillating, external magnetic fields. As with their organic-composite hybrid siblings, inorganic-composite hybrid nanoplatforms designed for theranostic applications frequently employ mesoporous silica as their therapeutic conveyance superstructure due to its larger payload capacity, more easily tuned and functionalized pore topology, and generally higher biocompatibility than nonporous/ crystalline silica constructs. Thus we will focus our attention on inorganic nanoparticles that have been encapsulated with either mesoporous silica or MSNs onto which exogenous inorganic nanoparticles have been attached. Encapsulation with silica is an especially simple, benign means of stabilizing and/or surface functionalizing inorganic nanoparticles. Qian et al. developed a novel therapeutic nanodevice in which NaYF4 upconverting nanocrystals were encapsulated within a uniform mesoporous silica shell that contained a zinc phthalocyanine photosensitizer [143]. NIR laser irradiation of the encased nanocrystals activated the incorporated photosensitizer generating reactive singlet oxygen for cytotoxic effect. Ji et al. synthesized nanotheranostic gold nanoshells in which an internal, amorphous silica layer was used as a dielectric with a superparamagnetic iron oxide-silica core for both MR imaging and photothemal therapy [144]. For this

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FIGURE 20.7 Alternate approach to conveyance of DNA using MSNs. (a) 40 to 70 nm diameter spherical mesoporous silica shells were synthesized around 15 nm diameter iron oxide (Fe3O4) nanocrystal cores. MSN pores were expanded up to 6 nm in diameter. Through the use of an externally applied magnetic field, up to 89.5% of DNA (salmon) double-strands were adsorbed within the confines of the MSN’s mesopores. (b) Enlarged view of a DNA-loaded mesopore. Adsorbed DNA loadings as high as 121.6 mg DNA/g MSN were reported. Source: Reproduced from [145] with kind permission of the American Chemical Society. Copyright 2011. All rights reserved.

work, a superparamagnetic iron oxide nanocrystal was first coated with a layer of amorphous silica via conventional sol-gel techniques, then functionalized with amine groups. Gold nanocrystal seeds (2 3 nm) were then attached to the amorphous silica surface to nucleate the growth of a gold shell on the silica. The middle layer of silica containing hybrid nanoparticles provided a dielectric interface for shifting the plasmonic resonance to the NIR region, enabling both MRI contrast and photothermal therapy in the same construct. Chen et al. synthesized gold nanorods with aspect ratios of B4 that, when subsequently encased within amorphous silica, proved to be considerably strengthened against surface plasmon induced thermal deformation when optically pumped [142]. With a repetitively pulsed Ti:sapphire laser operating at 808 nm and delivering the human maximum permissible exposure (MPE) of 31.12 mJ/cm2 for 90 seconds, the researchers observed 0.7 db and 3.0 db reductions in the photoacoustic signals of phantoms bearing silica encased and bare gold nanorods, respectively. TEM studies, as well as optical absorption spectra of postirradiated samples, revealed significant reshaping of bare, unencased nanorods toward a spherical topology, but little change in the shape of the amorphous silica encased nanorods. As such, amorphous silica coating can permit much longer-lived, higher-contrast photoacoustic imaging than previously was possible. Further functionalization of the construct, for example, by enveloping the inorganic-composite structure with a thermoresponsive copolymer, could enable controlled

delivery and release of small molecules and peptides embedded within the copolymer encasement. Mesoporous silica encapsulation of iron oxide nanoparticles has been used to both add magnetic properties to mesoporous silica and add targeted, payload conveyance to iron oxide nanoplatforms, with applications as diverse as enhanced particle separation [145], magnetically triggered drug release [117], and MRI traceability [111,146 148]. Li et al. [145] and Zhang et al. [146] synthesized 40 to 70 nm diameter magnetic MSNs that possess 15 nm diameter Fe3O4 inner cores within mesoporous silica shells possessing poreexpanded (6.1 nm), radially aligned pores, as depicted in Figure 20.7. Using short salmon DNA, investigators adsorbed a maximum of 121.6 mg/g DNA/MSN in chaotropic salt and demonstrated that their magnetic MSNs provided an easily manipulated platform for DNA adsorption and desorption processes via external magnetic fields. Fe3O4 magnetic MSNs have also been used to convey siRNA. Li et al. developed a magnetic mesoporous silica nanoparticle (M-MSNs) based, polyelectrolyte (PEI) and fusogenic peptide (KALA) functionalized siRNA delivery system— dubbed M-MSN_siRNA@PEI-KALA—that was shown to be highly effective at initiating target gene silencing both in vitro and in vivo [149]. M-MSN_siRNA@PEIKALA construction began with the magnetically induced adsorption of siRNA within the mesopores of M-MSNs, followed by PEI coating the nanoparticle’s exterior. KALA peptides were then grafted onto the outer surfaces of the PEI coating. Cytoplasmic delivery

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of the nanoparticle M-MSN_siRNA@PEI-KALA was observed to initiate via electrostatic membrane association and subsequent endosomal internalization. Once MSN-bearing endosomes matured to become endolysosomes, the lower pH (B5) of the latter triggered a conformational change in the nanoparticles’ KALA peptides—from α-helix to random coil—that simultaneously destroyed the endolysosome membrane and enabled the release of loaded siRNA as free entities within the cytoplasm (nanoparticles not labeled with KALA peptides were observed to remain within intact mature endolysosomes, with no siRNA release). In vitro knockdown studies of enhanced green fluorescent protein (EGFP) and vascular endothelial growth factor (VEGF) in tumor cells demonstrated high efficiency, while in vivo experiments employing intratumorally injected M-MSN-VEGF siRNA@PEI-KALA revealed significant inhibition of tumor growth, likely the result of the suppression of neovascularization. Ma et al. developed highly versatile, inorganiccomposite hybrid nanotheranostics for multimodal imaging and multimodal therapy [150]. In this construct, ellipsoidal Fe3O4 cores were encased within mesoporous silica shells via conventional sol-gel chemistry. Post-synthesis, the IO-MSNs were decorated with PEGylated gold nanorods and the mesoporous silica nanochannels loaded with dox. As such, the nanotheranostic enabled, in a single particle, chemotherapy, photothermotherapy, T2-weighted MR imaging, and dark-field optical and infrared thermal imaging. Considerable in vivo MRI contrast enhancement was observed, while infrared thermal imaging revealed in vivo heating efficacy of these nanomaterials to be substantial; following 5 mm diameter 808 nm laser irradiation of tumors previously injected with nanoellipsoids, tissue temperature increases of 3 C at 1 W/cm2 and 18 C at 2 W/cm2 irradiation were recorded, the latter causing irreparable tumor damage. Wu et al. developed similar tumbler-like multimodal MSNs that employed both T2-weighted MRI and optical reporters—termed Mag-Dye@MSNs—by combining amorphous silica coated Fe3O4 cores and organic fluorescent dyes with an ammonia solution that contained dilute TEOS and low concentration of C16TAB surfactant [151,152]. In a somewhat similar approach, Liong et al. fabricated dye-doped MSNs that incorporated magnetite nanoparticles and folic acid (FA) targeting ligands to obtain an inorganic-composite hybrid nanoplatform for simultaneous drug delivery, MR and fluorescence imaging, and magnetic manipulation [153]. These nanoparticles underwent greater than twofold increased cellular uptake in pancreatic cancer cells than did comparable, untargeted nanoparticles.

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Loading of these magnetic dye-doped FA-targeting MSNs with the hydrophobic anticancer drug camptothecin demonstrated greatly increased cytotoxicity with pancreatic cancer cells but not with fibroblast (control) cells. Although a variety of inorganic nanoconstructs can be encapsulated in silica shells to fabricate multifunctional silica nanoparticles, such encapsulation is not always straightforward or without consequence, such as the morphological disruption of neighboring nanopore structure. Such difficulties can, however, occasionally be circumvented via the decoration of inorganic nanoconstructs onto the surface of previously synthesized silica nanoparticles to yield yet another inorganic-composite hybrid class. Chelebaeva et al. designed hybrid structures that combined twophoton fluorescent dye-doped MSNs with coordination polymer nanoparticles (CPNs)—the latter a form of magnetic inorganic nanoparticle—for multimodal imaging [154]. Lee et al. synthesized highly versatile, composite nanoparticles via decorating the outermost surfaces of mesoporous dye-doped silica nanoparticles with multiple superparamagnetic magnetite nanocrystals [112,155]. Integration of numerous magnetite nanocrystals on the silica nanoparticle’s surface yielded highly synergistic magnetic effects (e.g., specific nuclear relaxivity (r2) of 80 mM21s21) that resulted in remarkable 2.8-fold enhancement of T2-weighted MR signals. In addition to serving as fluorescence and MRI contrast agents, these nanoplatforms also incorporated hydrazone-linked dox for time-dependent/pH-sensitive drug release following nanoparticle endocytosis, revealing only 4% dox release at pH 7.4 but 78% dox release at pH 4.0, at 56 hours exposure. Cellular uptake of dox hydrazone-MSN-FITC-Fe3O4-PEG was verified by confocal laser scanning microscopy. Another function of exogenous inorganic nanoconstructs associated with the exteriors of porous silica nanoparticles is that of gatekeepers in stimuliresponsive delivery systems. By capping nanochannel entrances with environmental (e.g., pH, osmolarity, photoactivated) sensing inorganic nanoentities that prevent the premature release of conveyed cargo, MSNs have been designed to operate as “smart” drug delivery platforms [131,156]. Gan et al. developed magnetic (Fe3O4) nanoparticle-capped MSNs by using reversible acid-labile 1,3,5-triazaadamantane units for pH-sensitive endosomal delivery and release [157]. In this design the functionalized Fe3O4 nanoparticles served directly as nanogates, to regulate dosing. In vitro payload release studies revealed that the pHsensitive MSN ensembles quickly release the nanochannel contents between pH values of 5.0 and 6.0,

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but gave no detectable release whatsoever at physiological pH 7.4. Sun et al. developed gold nanoparticlecapped MSNs (Au-MSNs) for intracellular codelivery of enzymes and substrates, with no apparent loss of bioactivity [158]. In this construct, Au-MSN nanochannels were loaded with bioluminescent substrate luciferin and their PEGylated exteriors embedded with the bioluminescent enzyme luciferase. When exposed to disulfide-reducing antioxidants, the gold nanoparticle caps dissociated from the MSNs’ pores, enabling the release of the MSN interior’s luciferin. After proximal dispersal, enzyme and substrate linkage arose, as evidenced by the chemiluminescent signal observed. Photosensitive nanogated MSNs for drug delivery have also been developed by Knezevic and Lin. [159]. In these constructs, magnetite nanoparticle incorporated MSNs were loaded with anticancer drug camptothecin. Mesopore entrances were then capped with CdS quantum dots through photocleavable o-nitrobenzyl based carbamate linkages. Camptothecin release was successfully triggered by UV photoirradiation, while the nanoplatform’s position was manipulated via a variable, external, magnetic field. Lai et al. recently created a redox-controlled drug delivery system based on cadmium sulfide (CdS) nanoparticles capping of MSNs [160]. In this system, a disulfide linker was used to chemically attach CdSs to MSNs, cleavable with various disulfide reducing agents such as dithiothreitol and mercaptoethanol. Giri et al. developed an inorganic-composite redoxcontrolled drug delivery system that could be positioned via externally applied magnetic fields [64]. This design employed 3-(propyldisulfanyl)propionic acidfunctionalized MSNs whose mesopores were capped by 3-aminopropyltriethoxysilyl-functionalized superparamagnetic iron oxide nanoparticles (Fe3O4). Incorporation of surface ferrite nanoparticles has also been used to enhance the operation of organic nanovalves situated at mesopore entrances. Thomas et al. demonstrated such enhanced nanovalve performance by encapsulating zinc-doped iron oxide nanoparticles within mesoporous silica and capping the pores with cyclic cucurbit[6]uril nanovalve to derive a magnetically activated release system [161]. Nanochannel release of particle payload in this system was achieved by application of an external oscillating magnetic field whose interaction with the magnetic nanoparticles resulted in thermal energy generation sufficient enough to break the electrostatically bound nanovalve molecules. The introduction of zinc doping to iron oxide nanocrystals provided a fourfold increase in

hyperthermic effects compared to undoped iron oxide nanocrystals. To increase the payload capacity of mesoporous silica nanotheranostics further, and produce new topological domains for additional functionalization, silica nanorattles have recently been developed by a number of investigators. These hybrid constructs are generally inorganic-composite silica structures that consist of a porous silica shell whose hollow interior is filled with a suspension of therapeutics (e.g., hydrophobic and hydrophilic drugs, siRNA, proteins, peptides) and/or contrast agents (e.g., quantum dots, iron oxide nanoparticles, dye-doped silica nanosphere) [162 164]. Liu et al. fabricated B150 nm diameter multifunctional silica nanorattles that had been encapsulated within PEGylated, thin, gold nanoshells for a combination of remotely controlled photothermal therapy (via plasmonic heating) and chemotherapy (via docetaxel), as shown in Figure 20.8 [165]. In vitro cell (HepG2) studies and in vivo (hepatoma H22) small animal studies demonstrated the dual therapeutic moiety to possess markedly higher therapeutic efficacy and lower systemic toxicity than free docetaxel chemotherapy, although not with the hyperthermia-enhanced cytotoxicity characteristic of conventional, combined docetaxelthermotherapy. Zhang et al. developed mesoporous multifunctional upconverting luminescent and magnetic nanorattles for targeted optical imaging and chemotherapy, as described in Figure 20.9 [166]. In this system, nanorattles comprised of porous hydrophilic, rareearthdoped (Yb,Er) NaYF4 shells, each containing a loose magnetic nanoparticle, were synthesized via an ion-exchange process. The region between the magnetic core and the NaYF4:Yb,Er shell was void, making it possible to load the nanorattle with significant quantities of therapeutic agents. To preclude the leakage of iron in biologically acidic environments such as endosomes and lysosomes, mesoporous silica was used to encase the constituent magnetic Fe3O4 nanoparticle cores. After 2-photon NIR excitation, the resulting visible luminescence enabled nanoplatform localization within tumors, while external magnetic fields were used to position the nanorattles to regions of interest. Incubation of nanorattles with human hepatoma cells (QGY-7703), followed by MTT (3 4,5 dimethylthiazol 2 yl)-2,5-diphenyltetrazolium bromide) assay and fluorescence microscopy, showed the nanorattles to have low cytotoxicity and excellent cell imaging properties. In vivo experiments yielded substantial tumor shrinkage when nanorattles void spaces were loaded the antitumor drug dox, and the nanoparticles magnetically navigated toward the pathology.

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FIGURE 20.8 Combined nanoplatform for photothermal therapy and chemotherapy. (A) Cross-sectional structure of core and shell interface inside silica nanorattle (a). Schematic representation of structure of PEGylated gold shell-enveloped silica nanorattle, loaded with drug. TEM images of (c) silica nanorattle, (d) gold-seeded silica nanorattle, in preparation for gold-shell growth, (e) complete gold shell-encased silica nanorattle, and (f) PEGylated gold shell-encased silica nanorattle. (B) Thermal imaging of excised hepatoma 22 (H22) solid tumors under laser irradiation, showing temperature rise ( C) as a function of exposure time. Source: Reproduced from [165] with kind permission of Wiley-VCH Verlag GmbH & Co. Copyright 2011. All rights reserved.

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CONCLUSIONS

L

FIGURE 20.9 Mesoporous upconversion luminescent and magnetic nanorattles for targeted chemotherapy. (A) Construct synthesis began with 20 nm diameter Fe3O4 nanocrystal cores onto which was grown a SiO2 shell until B90 nm in diameter to prevent iron dissolution in acidic biological conditions. Particle was then coated with a layer of amorphous Y/Yb,Er(OH)CO3-H2O and heated to derive 20 nm thick Y2O3 cubic phase polycrystalline shell, termed magnetic upconverting oxide nanospheres (MUC-O-NS). Ion exchange in the presence of HF and NaF resulted in a 115 nm diameter, porous α-NaYF4:Yb,Er optically upconverting shell (20 nm thick, 4.8 nm diameter pores) whose hollow interior retained the iron oxide core encased in a now 10 nm thick silica shell, termed magnetic upconverting fluoride nanorattle (MUC-F-NR). (B) (a) TEM and SEM (inset) of Fe3O4@SiO2 nanospheres; (b) HTREM after growth of Y2O3/Yb,Er layer; (c) SEM and (d) TEM of MUC-F-NR; (e) HTREM of the α-NaYF4:Yb,Er shell, with

Nanotheranostics seek to enhance both diagnostic insight and treatment outcome through the use of multifunctional, nanometer-sized, organic/inorganic constructs. Ideally nanotheranostics also enable in vivo monitoring of agent biodistribution and fate, and provide feedback as to disease progression/regression. The easily manipulated, size-dependent physicochemistries of these nanoplatforms often give rise to unusual physical, chemical, and biological properties quite unlike those of bulk material having the same composition, with bioactivities that depend heavily on the environment in which the nanoparticles find themselves—some therapeutically beneficial, others potentially toxic. Silica nanostructures, however, do not acquire any additional or unusual physicochemical characteristics as a result of their diminutive size other than a corresponding increase in surface area. Rather, much of the utility of silica nanoplatforms in theranostic applications originates from their ease of surface functionalization and ability to form solid, hollow, and porous structures—the last two of which provide even greater, topologically distinct surface areas for further functionalization. Silica nanoparticles are also effectively “transparent” at UV, visible, NIR, and RF wavelengths and unaffected by presence of internal or external electric or magnetic fields. Moreover, silica nanoparticles are easily synthesized, chemically inert, inexpensive, biocompatible and excretable, and hydrophilic, making them especially well suited to biomedical applications, as is evident from the current literature [131,167 173]. Despite the enormous number and variety of silica nanotheranostics developed to date, a number of fundamental challenges remain to be addressed prior to their widespread clinical use. First, and perhaps foremost among them, are improvements in nanoplatform targeting specificity. PEGylation generally reduces reticuloendothelial system (RES) recognition of nanoplatforms significantly, and can increase circulation times 2 to 10 times that over identical particles that are not PEGylated. However, the paucity of suitable biomarkers for pathology-specific targeting—the bane of all current forms of molecular imaging—inevitably results in significant nonspecific binding and/or uptake by the RES, frequently followed by entrapment in the liver, spleen, and bone marrow. Indeed, a significant source of

energy dispersive Xray (EDX) of a single MUC-F-NR; (f) MUC-F-NR whose silica has been completely etched away to reveal wall structure. Source: Reproduced from [166] with kind permission of the American Chemical Society. Copyright 2011. All rights reserved.

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REFERENCES

nanoplatform-induced toxicity, aside from that potentially inflicted by components of the nanoplatform itself, arises simply from RES accumulation of nanoparticles. In vivo aggregation of nanoplatforms often leads not only to more probable RES sequestration but also to loss of nanotheranostic functionality and occlusion of capillaries in the liver, lungs, kidneys, and spleen. Dosing is also problematic in the clinical translation of nanotheranostics as the dose typically required for therapeutic efficacy is generally much higher than that required for a diagnostic effect. For example, a free drug may need to be delivered at mg/kg of body weight levels whereas a radioactive tracer for a PET/ SPECT scan requires much less than 1 μg/kg body weight. Lastly, and unique to theranostic compounds, is the difference in agent dwell times needed for diagnosis versus treatment. Although diagnostics and therapeutics each require highly specific targeting, imaging mandates that the region of interest (i.e., pathology) have markedly higher signal than that of surrounding tissue during image acquisition (i.e., contrast). Consequently most conventional contrast agents for imaging are designed to clear from the blood as quickly as possible, usually within minutes to hours. Rapid clearance of contrast agents also enables perfusion (dynamic) study of the pathology and permits earlier “follow-up” imaging. A therapeutic approach, however, demands that the nanotheranostics have longer circulation times, to permit not only accumulation at the pathology but for drug release as well. Notwithstanding such seemingly formidable impediments, silica-based nanotheranostics, with their intrinsically high biocompatibility and tunability of both form and function, are especially well positioned among nanomaterials to succeed in accommodating the numerous platform-pathology specific optimizations required.

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IV. THERANOSTIC PLATFORMS