cancer theranostics || silica nanoparticle platform

C H A P T E R

20

Silica Nanoparticle PlatformJeffrey 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 363

Biocompatibility 364

Nanoplatform Properties and Their Rolein Biocompatibility 365

Topology 365Hydrophilicity and Hydrophobicity 365Surface Charge 366Reactive Sites and Oxidation 366Protein Adsorption 366

Silica Toxicity 368Cytotoxicity and Genotoxicity 368

Administration and Exposure Route Significance 369

Synthesis and Surface Modificationof Silica Nanoparticles 371

Silica Nanoparticle Nanotheranostics 373General Considerations 373Multifunctionalized Solitary Platforms 373Organic-Composite Hybrid Platforms 377Inorganic-Composite Hybrid Platforms 381

Conclusions 386

References 387

INTRODUCTION

Cancer nanotheranostics employ multifunctional,nanometer-scale, organic and inorganic materials toextract diagnostic insight and deliver pathology-targeted therapy. The fundamental advantage of com-bining such seemingly disparate objectives is the abil-ity to use patient-specific test results to tailor treatmentprograms that result in improved clinical outcomes,reduced costs, and minimal side effects. Such plat-forms also enable in vivo monitoring of both the nano-material’s biodistribution and fate and its therapeuticprogress and efficacy throughout the course of treat-ment. Unlike most conventional diagnostic andchemotherapeutic agents, nanoparticles can be readilymodified to provide extended circulation half-lives,passive accumulation in and near tumors via theenhanced permeability and retention (EPR) effect,

active targeting of cancer cells, and minimal toxicity.With their easily manipulated, size-dependent physi-cochemistries, nanoparticles afford small diameters,enormous surface areas, and novel topologies thatoften give rise to unusual properties; properties thatare generally quite unlike those of bulk material hav-ing the same composition and that result in bioactiv-ities that heavily depend on the environment in whichthe particles are localized.

In contrast to most other nanomaterials, however,silica nanoparticles do not acquire any new or unusualcharacteristics from the diminution of their size, otherthan a corresponding increase in surface area. Rather,their utility stems from the ease with which theirsurfaces can be functionalized and their ability to formboth solid and porous structures, the latter of whichgreatly enhances the surface area and different topolo-gies available for synergistic applications such as

363X. Chen and S. Wong (Eds): Cancer Theranostics.

DOI: http://dx.doi.org/10.1016/B978-0-12-407722-5.00020-7 © 2014 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/B978-0-12-407722-5.00020-7

protected codelivery of therapeutics (e.g., drugs,siRNAs, photosensitizers, radionuclides) and imagingcontrast agents (e.g., fluorophores, radionuclides,Fe/Mn/Gd oxides). Silica nanoparticles are inexpen-sive and simple to synthesize, chemically inert, bio-compatible and excretable, and easily dispersed inwater. Silica nanoparticles are also effectively “trans-parent” at UV, visible, and near-infrared (NIR)wavelengths and unaffected by electric or magneticfields—attributes that facilitate their external visualiza-tion and manipulation.

BIOCOMPATIBILITY

Nanoparticle�biomolecule interactions can be quiteunlike those of bulk material of the same compositionbecause the nanoparticle’s effective surface charge,stability, solubility, and hydration are greatly affectedby 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 uniqueattributes are likely to give rise to toxic sequelae, bothacute and chronic, a situation further compoundedby the typically inefficient targeting and inherently highstability that many nanomaterials exhibit in vivo [2�8].Thus, a variety of schemata have been devised tominimize deleterious side effects, such as covering thenanoparticle’s surfaces with polymers, antioxidants,surfactants, or ligands or altering the nanoparticle’ssize, shape, roughness, or surface charge [9�13].Unfortunately, such measures often only afford partialremediation of toxicity and can themselves have unde-sired consequences, such as increasing a nanoparticle’shydrodynamic diameter that then results in substan-tially altered biodistribution.

Ultimately, to best minimize toxicity, nanoparticlesshould be degraded in situ into truly biocompatible sub-components or excreted from the body once they haveserved their diagnostic and therapeutic purpose.Numerous studies have been published on the biocom-patibility of a variety of nanoparticles and, to a lesserdegree, their degradation by-products. But little consen-sus can be found among them as most studies cannoteither be compared to one another (owing to variationsin material synthesis, characterization, and/or toxicol-ogy) or simply contradict one another (for reasonsundetermined) [2,4�6,11,14�19]. Even less has beenpublished on the excretion of nanoparticles (or their sub-components), reflecting, to some measure, a loss of trace-ability (e.g., disassociation/decomposition of fluorescentlabels, or radioactive decay of radiolabels) as well as theintrinsic difficulty of directly visualizing them in situ.Nanoparticles whose in vivo imaging signals are

inherently stable (e.g., the fluorescence of quantum dotsor the Raman spectroscopic signatures of carbon nano-tubes) are few in number and possess chemistries thatdo not extrapolate to those of other nanoparticles.In addition, uniquely identifying biodegradationby-products in vivo can be exceedingly difficult.

A few conclusions can be drawn from the clear-ance/excretion studies that do exist. When deliveredintravenously, nanoparticle uptake occurs by oneof five pathways—phagocytosis, macropinocytosis,caveolin-mediated, clathrin-mediated, and caveolin/clathrin-independent endocytosis—at a rate thatlargely depends on the particle’s hydrophobicity,surface charge, and size [1,5,8,11,12,16,17,20�22].Hydrophobic nanoparticles are rapidly removed fromcirculation by components of the reticuloendothelialsystem (RES), especially in the liver and spleen, andthus have short in vivo half-lives (from seconds to min-utes) that often limit their clinical utility. Nanoparticlesthat carry significant surface charge tend to adsorbserum proteins (some in non-native conformation) thatcan affect their biodistribution, elicit immune response,and indiscriminately destabilize cell membranes andproteins. Nanoparticle size, however, most stronglycorrelates with clearance dynamics, with particles3 nm in diameter and smaller extravasating tissuesnonspecifically, those 3 to 8 nm in diameter undergo-ing renal clearance, those 30 to 80 nm in diameterbeing sequestered in lung and leaky vasculature (e.g.,tumor and inflamed tissue via the enhanced perme-ation and retention effect), and particles larger than80 nm becoming trapped by liver and spleen.

Of course, uptake from circulation does not ensureexcretion. Indeed, all studies to date of larger quantumdots (diameter .8 nm), regardless of surface coating,have shown relatively rapid sequestering by the RESof liver and spleen, but with none leaving thoseorgans. Similar retention patterns have been observedwith pristine single-walled carbon nanotubes, withrapid accumulations in liver, spleen, and lung, butwith little subsequent excretion from those organs [19].Even PEGylated gold nanospheres, with long bloodcirculation times (B30 h), have demonstrated a pro-pensity to accumulate in the liver and spleen of miceas long as 7 days after their injection, leading to acutehepatic inflammation and apoptosis [4]. While rapidexcretion may minimize the potential for toxicsequelae, it also mandates expedient and highly spe-cific pathology targeting if it is to be of diagnostic ortherapeutic benefit. For some applications, such effica-cious targeting and elimination may either not bedesired (e.g., particle residence time and numbers areinsufficient to derive full diagnostic/therapeutic effect)or possible (e.g., particle has insufficient vascularaccess due to pathology, or low targeting affinity).

364 20. SILICA NANOPARTICLE PLATFORM

IV. THERANOSTIC PLATFORMS

In the following sections we discuss a few of themore salient aspects of silica theranostic biocompatibil-ity, beginning with nanomaterial properties and bio-molecular processes at the submicroscopic level, andfollowed by a macroscopic, toxicological/systems-levelexamination of their biocompatibility. More detailedanalyses of these topics can be found in the recentliterature [23�25].

NANOPLATFORM PROPERTIES ANDTHEIR ROLE IN BIOCOMPATIBILITY

Topology

One of the principal factors affecting the interactionof nanoparticles with biological materials is size; or,more precisely, relative size of the nanoparticle to thatof proximal biomolecules. As the size of the nanoparti-cle and/or its surface features diminish, relative tothose of nearby biomolecules, nanoparticle surfacecurvature plays an increasingly significant role inparticle�environment interactions. Particles possessinghigh curvature are typically able to accommodatelarger amounts or numbers of adsorbed molecules perunit surface area due to decreased steric hindrance[26]. Nanoparticle curvature has also been observed tobe a dominant factor in the conformational changesthat occur to proteins during adsorption onto the parti-cle [27]. In one set of studies, 15 nm diameter amor-phous silica nanoparticles altered the secondarystructure of a carbonic anhydrase (HCA I) consider-ably more than amorphous silica nanoparticles of 6 nmdiameter [28]. In another surface curvature study, crys-talline SiO2 nanoparticles possessing diameters of 5 to100 nm were used to search for alterations in the phasetransition of different chain-length, phosphotidylcho-line bilayers that enveloped the SiO2 nanoparticles[29]. On the more highly curved surfaces of smallernanoparticles, lipids formed bilayers in which theouter polar heads were more widely separated fromone another, with more chains being interlocked withthe outer layer of adsorbed lipids, to offset increasedheadgroup spacing.

Physicochemical surface properties can exhibitstrong dependence on particle surface curvature aswell, leading to numerous other biologically interactiveparticle attributes such as hydrophilicity, hydropho-bicity, surface charge (ζ-potential), and strength ofhydrogen bonding. For example, silica surfaces fre-quently terminate with silanols (�Si�OH) and silox-anes (Si�O�Si). Silanols can be broadly classified asbeing (1) isolated, (2) interacting, or (3) geminal,whereas siloxane bridges form on condensation of twoadjacent silanols [30]. In an FTIR study of amorphous

silica powders whose constituent particles varied indiameter from as small as 8 nm to as large as 260 nm,the amount of isolated silanol (IR adsorption band at3750 cm21) was found to decrease as the particle diam-eter increased, nearly vanishing for the largest nano-particle powders. By contrast, the ratio of opticalabsorbance of the hydrogen-bonded silanol IR bandto that of the isolated silanol IR band was observedto increase as the particle diameter increased [31].As such, the curvature of silica nanoparticles wasfound to significantly affect the ratio of isolated tointeracting silanols, and thereby powder dispersibilityin aqueous environments.

Hydrophilicity and Hydrophobicity

Nanomaterial hydrophilicity largely reflects thepresence 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 nanomater-ial’s surface) [32,33]. The submicroscopic structure of ananomaterial (e.g., crystalline vs. amorphous, crystal-line phase/polymorph type, defect frequency, dopantpresence) has also been found to play a significant rolein the degree of the material’s wettability, solubility,and rate of dissolution. For example, the dissolutionrate of amorphous silica is much greater than that ofcomparably-sized, ground crystalline quartz undersimilar environmental conditions, and large differenceshave been observed in the solubility of crystalline silicaparticles possessing different lattice symmetries [30].

Despite variations in Si�O�Si bond lengths andangles, low-pressure amorphous silicas share the samefundamental chemical unit with crystalline silicas: thesilica tetrahedron. In contact with water, exposed sila-nol (Si�OH) groups dissociate to yield negativesurface charges that result in the formation of ahydration/solvation shell that inhibits particle aggre-gation, enabling aqueous dispersion and suspension.Reported dissolution rates for crystalline and amor-phous SiO2 support the idea that crystalline poly-morphs and amorphous silicas undergo the samehydrolysis reactions albeit at different rates that reflectthe water’s access to the silica’s surface. Interestingly,the dissolution rates of both crystalline and the amor-phous silicas are increased 50 to 100 times whenalkaline or alkaline earth cations are introduced tootherwise pure solutions. This large enhancement hassignificant implications for the hydrophilicity andhalf-life of silica nanotheranostics in electrolyticallyvariable, biological fluids. Indeed, the much highersolubility of amorphous silica in biofluids, with respectto crystalline silicas, is one of the primary factorspurported to result in the former’s lower toxicity[34�37].

365NANOPLATFORM PROPERTIES AND THEIR ROLE IN BIOCOMPATIBILITY


Surface Charge

As mentioned previously in regards to the solvationshells that develop from the hydrolysis of silica nano-particle surfaces, the presence of polarized or chargedmoieties on the surface of silica nanoparticles greatlyinfluences their interactions with the local environ-ment. When a nanoparticle that possesses an electri-cally charged surface is immersed in an aqueous,electrolytic solution, an interfacial, electrical double-layer immediately forms around the nanoparticle.The layer closest to the nanoplatform’s surface is sta-tionary and comprised of adsorbed ions, a consequenceof ion�particle electrostatic attraction, hydrogen�coordinative bonding, and/or van der Waals interac-tions. A second, more loosely associated layer of ions,termed the diffuse layer, envelopes the nanoparticle andfirst layer. Comprised of ions that are only weaklyattracted to the underlying surface charge, due largelyto the first layer’s electrical screening of the nanoparti-cle’s surface charge and “binding” competition withneighboring ions (ionic strength of biological fluidsB150 nM), the ions of the diffuse layer are in a state ofdynamic flux, the subject of thermal motion. The netelectric charge in this diffuse layer is equal in magni-tude to the net surface charge, but with the oppositepolarity. The diffuse layer, or at least part of it, is ableto move under the influence of tangential forces,with a slipping plane that separates mobile fluid fromsurface adsorbed fluid. The electric potential at thisplane, a measure of the double-layer’s (and thus thatof the underlying nanoparticle’s) charge, is defined asthe nanoparticle’s ζ-potential. As such, calculatedζ-potentials are routinely used to assess nanoparticlecolloidal stability in solution (i.e., resistance to floccula-tion) and have more recently been employed to ascer-tain the degree of protein adsorption onto the surfaceof silica nanoparticles [38].

Reactive Sites and Oxidation

Nanoparticle surface charges, dangling bonds (i.e.,surface-bound radicals), and poorly coordinated ionseach have the potential to interact with nearby biomo-lecules. One of the more deleterious consequences ofthese interactions is the generation of reactive oxygenspecies (ROS•), a term that includes not only oxygen-based free radicals such as superoxide anion (O2•

2),hydroxyl (HO•), and biomolecule-derived radicalssuch as alkylperoxyl (RO2•) and alkoxyl (RO•), butalso nonradical species such as hydrogen peroxide(H2O2) and singlet oxygen (1O2) [39]. Although ROSplay key roles in a number of cell signal transductionpathways, excess ROS are highly cytotoxic, damaging

proteins, lipids, and DNA, and a stressor that activatesa number of redox-sensitive signaling pathways [40].

In general, nanoparticles can generate ROS eithervia direct mechanisms (surface-derived ROS) or indi-rect mechanisms (cell-derived ROS) that include altera-tions in mitochondrial function and immune systemresponse [41�43]. Silica related ROS generation can beboth particle surface and cell derived, and arises fromthe existence of either dangling bonds (e.g., undissoci-ated silanols with potential to form strong hydrogenbonds) or exposed redox-reactive transition metals.As neither dangling bonds nor metal impurities arecommonly present in synthetic amorphous silica (pre-cipitated or pyrogenic silicas), amorphous silicas aregenerally incapable of directly inflicting oxidativedamage [44]. However, crystalline silicas, especiallywhen powderized, can possess large numbers of dan-gling bonds and are associated with both particle andcell-derived ROS pathologies, the latter including theinflammatory pulmonary disease silicosis [41,45�46].

Protein Adsorption

Proteins display a high propensity to adsorb ontosurfaces via electrostatic interactions, coordinativebonds, hydrogen bonds, and hydrophobic interactions.When nanoparticles come into contact with multicom-ponent biological fluids such as blood, plasma, andinterstitial fluid, a bevy of suspended proteins immedi-ately envelopes the particles. Indeed, as nanoparticlesmove onto or into cells, most nanoparticle�cell inter-actions are strongly modulated by these proteins.The establishment and maintenance of this proteincloak, termed the protein corona, is a dynamic and com-petitive process governed by the avidity and relativeconcentration of proximal free proteins, the nanopar-ticle’s surface charge distribution and chemical reac-tivity, nanoparticle size relative to that of theadsorbing proteins, local pH and ionic strength, andtemperature, as depicted in Figure 20.1 [47]. Proteinswith greater mobility often adsorb first only to be sub-sequently replaced by less motile proteins that possesshigher nanoparticle affinities. Lifetimes of these vari-ous particle�ligand complexes range from microse-conds to days and affected proteins may undergoconformational changes that result in the exposure ofnew epitopes of altered affinity and functionality.

The encapsulation of nanoparticles with proteins alsohas significant implications for the nanoparticle’s sur-face charge (e.g., dispersion) and chemical reactivity(e.g., ROS generation). Several recent studies of silicananoparticles have demonstrated that even modest,physiologically plausible increases in the degree of pro-tein coverage can significantly affect both the magnitude



Competitive bindinginteractions dependon protein concentrationand body fluid composition

Binding interactions releasesurface free energy, leadingto surface reconstruction

Available surfacearea, surface coverage,and angle of curvaturedetermine adsorptionprofiles

e–

e–

e–

SHSH

S

S

h+

Characteristicprotein on/offrates depend onmaterial typeand proteincharacteristics

(a)

(b)

Electronicstates

Hydro-phobicitywettability

Charge

O2O2′–

Size

Crystallinity

Particlecomposition

Impact onprotein

structureand function

instabilitySurface

Protein bindingacceleratesdissolution ofsome materials

Hydrophilicor hydrophobicinteractions

Charge (for example, cationic binding)

Steric hindranceprevents binding

Surfacereconstruction

Release of surfacefree energy

Protein conformation,functional changes

Proteinfibrillation

Amyloidfibre

Proteincrowding,layering,nucleationExposure

to crypticepitopes

Surfaceopsonization/liganding allowsinteractionwith additionalnano-bio interfaces

Protein conformationalchanges cause loss ofenzyme activity

Electron-holepairs leadto oxidativedamage

Immunerecognition

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

367NANOPLATFORM PROPERTIES AND THEIR ROLE IN BIOCOMPATIBILITY


and polarity of the nanoparticle’s ζ potential [38,48�51].As such, colloidal instabilities can potentially resultfrom protein corona establishment and evolution andlead to nanoparticle aggregation and flocculation andprotein clustering and fibrillation. While empirical stud-ies suggest that protein coronas generally improve thecolloidal stability of nanoparticle suspensions, counterexamples do exist; functional proteins such as cyto-chrome c, DNase II, hemoglobin, and fibrinogen havebeen found to bridge SiO2 nanoparticles via electrostaticattraction and hydrogen bonding, and then aggregatethe nanoparticles into coralloid forms [51].

SILICA TOXICITY

Cytotoxicity and Genotoxicity

As can be deduced from the forgoing discussion,nanoparticle biocompatibility is a complex anddynamic set of processes determined both by thenature of the particle and its environment; processesthat can be either chemical or physical. Examples ofchemical mechanisms include ROS production, disso-lution and release of toxic ions, alteration of cell mem-brane electron and ion transport, catalytic oxidativedamage, and lipid peroxidation. Physical mechanismsinclude disruption of protein folding and function,protein aggregation and fibrilation, and the disruptionof membrane integrity, activity, and transport pro-cesses. ROS generation, and secondary processes toROS generation (e.g., inflammatory cytokine response),are considered the principal chemical processes innanotoxicology, although radical formation too canadversely effect cell viability [43,52�55].

Cellular uptake of molecules is frequently mediatedby moiety-specific binding with membrane-boundreceptors. In the absence of such receptors (e.g., viralinfection), cellular uptake takes place via constitutive“adsorptive” endocytosis or fluid-phase pinocytosis[56,57]. Silica nanoparticles, by virtue of their surfacesilanol groups, have great affinity for a variety of phos-pholipid headgroups and are thus readily endocytosed[58]. Indeed, a number of research groups have demon-strated that mesoporous silica nanoparticles (MSNs) arerapidly (,30 min) endocytosed by a variety of mamma-lian cells including cancer cells (HeLa, CHO, lung,PANC-1), noncancer cells (neural glia, liver, endothe-lial), macrophages, and stem cells (3TL3, mesenchymal),to name but a few [59�65]. Nonfunctionalized,bare MSN uptake occurs primarily through clathrin-coated endocytosis, though some surface functionalizedMSNs, 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 sig-nificant negative surface charge that, once endocytosed,triggers proton influx, osmotic swelling, permeation orrupture of the endosomal membrane (i.e., the proton-sponge effect), and cytosolic release. Positive surfacecharge functionalization of silica nanoparticles, forexample with amines, can also result in anion influxand osmotic swelling of the endosomal membrane, lead-ing to membrane compromise and endosomal release ofnanoparticles into the cytosol. Interestingly, amine andguanidinium (only) functionalized MSNs appear toenter cells via a clathrin and caveolae independentmechanism [61].

In a recent study examining the role of nanoparticledesign in cellular toxicity and hemolytic activity, investi-gators exposed erythrocytes, macrophages, and cancerepithelial cells to differently charged, nonporous 115 nmdiameter silica nanospheres, mesoporous 120 nm diame-ter silica nanospheres, and mesoporous silica nanorodswith aspect ratios of 2 (803 200 nm), 4 (1503 600 nm),and 8 (1303 1000 nm) at doses of 100, 250, and500 μg/ml [66]. Cancer epithelial cells (A549 adenocarci-nomic alveolar basal) were generally found to beunharmed by the presence of SiO nanoparticles,whereas macrophages exhibited adverse, charge-depen-dent, and dose-dependent responses to porous and non-porous SiO nanoparticle exposure and postulated areflection of the very different biological functions of thetwo cell types (i.e., protective boundary/barrier vs.phagocytic scavenger). In another study, smaller 30 nmdiameter nonporous silica nanoparticles demonstratedmacrophage toxicity at doses as low as 10 μg/ml [67].Indeed, with macrophages serving as platforms in bothinnate (nonspecific) and adaptive (acquired) immunity,the nature and extent of macrophage�nanoparticleinteraction provides considerable insight as to nanother-anostic toxicity, especially given the serum protein opso-nization that nanoparticles experience following theirintravenous administration, encouraging additionalreceptor-mediated macrophage binding. Differences inthe geometry of these silica nanoplatforms, however,appear to be of little consequence to either macrophageor epithelial cell toxicity [66].

In erythrocyte toxicity studies of mesoporous silicaspheres and nanorods, bare or unmodified SiO nano-particles demonstrated both porosity- and geometry-dependent hemolytic activity at concentrations greaterthan 250 μg/ml, while amine-modified (for surfacecharge) mesoporous SiO nanoparticles exhibited signif-icant hemolysis at particle concentrations as low as100 μg/ml [68]. Correlation of particle (and pore) sizewith erythrocyte toxicity has been confirmed by others.In one study employing erythrocytes exposed to50/100 μg/ml of either 100 nm diameter mesoporous(MCM-41 silica) or 600 nm diameter (SBA-15 silica)



nanospheres, researchers observed no adverse effectswith the former but significant, induced membranedeformation and hemolysis in the latter [69].Endothelial cells also exhibit increases in toxicity withincreasing particle size. In studies using 16 to 304 nmdiameter porous silica nanoparticles, exposure ofhuman umbilical cord endothelial cells (HUVEC) to212 nm nanoparticles at 30,000 particles/cell demon-strated a 50% decrease in cell viability, while the samecytotoxicity was observed following exposure to304 nm particles at 15,000 particles/cell [70].

Even more dramatic, however, is the demonstrablygreater cytotoxicity associated with the use of nonpo-rous silica nanoparticles. In studies comparing small25 nm diameter nanospheres, those comprised ofnonporous silica displayed a 72% decrease in eryth-rocyte viability relative to those comprised of meso-porous silica, with hemolysis occurring in thenonporous SiO nanoparticles for concentrations as lowas 20 μg/ml [71].

Other studies with epithelial, neuronal, stem cells,lymphocyte, and fibroblast cells also exhibit particle-(size, shape, structure, charge, dose) and cell-dependent cytotoxicities. For example, exposure ofhuman embryonic kidney cells (HEK-293) to 20 to760 nm diameter nonporous silica nanospheres atdoses of 20 to 2000 μg/ml revealed much greatertoxicity for smaller particles than for larger particles(e.g., 50% decreased viability at doses .80 μg/ml of20 nm particles vs. doses .140 μg/ml of 50 nm orlarger particles) [72]. Kidney epithelium cells (HeLa),exposed to 70 nm diameter nonporous silica nano-spheres at a dose of 50 μg/ml, displayed no cytotoxic-ity in the presence of serum but high cytotoxicity inthe absence of serum, while no toxicity was found in200 nm and 500 nm diameter nonporous silica nano-spheres similarly dosed either with or without serum[73]. Studies of fibroblasts (3T3) exposed to 38 nmdiameter nonporous silica nanoparticles showed cellviability decreased by 50% at particle concentrationsgreater than 50 μg/ml, while porous silica nanopar-ticles were found to have little adverse effect onhuman T-cell lymphoma (Jurkat) cells [74,75]. Takentogether these in vitro studies strongly suggest poroussilica, for a given dose and particle size, shape, andcharge, to be much less cytotoxic than comparablenonporous silica.

On a submicroscopic level, much of the observedcytotoxicity arises from the disruption of membraneintegrity and function. However, as noted earlier, anumber of smaller silica nanoparticles (especiallyporous) readily undergo endocytotic incorporationand subsequent endosomal release into the cytosol.Once inside the cell such nanoparticles are potentiallyproteotoxic and/or genotoxic because, similar to

proteins, nucleic acids also exhibit a high tendency toadsorb onto surfaces via electrostatic interactions andcoordinative bonds, hydrogen bonds, and hydrophobicinteractions. While interference with protein synthesismight take place in the cytosol, direct genotoxicitymandates DNA access, either via the nanoparticlecrossing the nuclear membrane or its proximity duringmitosis (either in binding directly to nucleic acids orinterfering with mitotic spindles). One reported exam-ple of the direct genotoxicity of small silica nanoparti-cles is that of 40 to 70 nm diameter nonporous SiOnanoparticles contacting the DNA strands of humanepithelial cells (HEP-2) during mitosis [76]. To date,though, such findings of genotoxicity triggered by thedirection interaction of inorganic nanoparticles withnucleic acids are rare.

Genotoxicity is much more likely to arise indirectly,generally from the production of oxidative stress oncells [77,78]. As briefly described earlier, ROS genera-tion by nanoparticles can give rise to the oxidation ofDNA bases, the breaking of DNA strands, and the der-ivation of DNA adducts from lipid peroxidation (e.g.,via malondialdehyde reacting with deoxyadenosineand deoxyguanosine). The nanoparticles themselvescan stimulate mitochondria to produce ROS that desta-bilizes DNA, while the liberation of metal ions fromnanoparticles can modulate the permeability of nuclearmembranes. Even macrophage�nanoparticle interac-tions can lead to inflammatory scenarios that over-whelm intracellular and extracellular defenses againstoxidative stress, leading to indirect genotoxicity.Although very few studies of silica nanoparticle geno-toxicity have been reported in the literature, the gen-eral consensus among those that do exist is that thenumerous dangling bonds present in both nonporousand crystalline silica nanoparticles are likely responsi-ble for indirectly eliciting the response [79].

Administration and Exposure Route Significance

It is extraordinarily difficult if not impossible toextrapolate, for a given nanoplatform, the findings ofstatic, well-controlled in vitro cytotoxicity studies tothose of dynamic, environmentally variable, anddiverse in vivo exposures: the intrinsic complexity,variety, and number of potential particle and environ-ment interactions makes such inferences of nanotoxi-cology intractable. Fortunately, however, preclinicalstudies of nanoparticle biodistribution and hostresponse, as well as analyses of accidental humanexposures, provide some insight as to the generalmechanisms involved and sequelae expected. As phys-iological responses are typically dose related andaccess (anatomical) dependent, the route by whichnanoparticles gain entry into the body (e.g., injection,

369SILICA TOXICITY


inhalation, ingestion, skin contact) is of great relevanceto the toxicity they pose.

Unfortunately such toxicological data for silicananoparticles, as well as for many other insoluble inor-ganic nanoplatforms, are sparse. As discussed previ-ously, intravenous introduction of untargeted silicananoparticles typically results in their immediate opso-nization by serum proteins and that further expeditestheir endosomal uptake into cells, exploiting the samecellular machinery that viruses employ. With time andin the absence of abnormal or leaky vasculature, silicananoparticles tend to accumulate in the liver andspleen, after which they may undergo hepatobiliaryexcretion and/or degradation into biocompatibleorthosilicic acid (Si(OH)4). Murine model based studiesof the biodistribution, biocompatibility, and clearanceof mesoporous silica nanoparticles have shown bothcharge and geometry dependent elimination, with pos-itively charged spherical moieties experiencing themost rapid hepatobiliary excretion, shorter nanorodsbeing quickly sequestered and cleared by the liver,and longer nanorods becoming trapped within thespleen [68]. In contrast to in vivo behavior of mesopor-ous silica nanoparticle, intravenously delivered nonpo-rous silica nanoparticles typically show significantdelays in particle clearance [80].

Considerably more toxicological data have been col-lected on the inhalation of aerosolized silica nanoma-terials, owing to its use in a number of industries andits mobility when airborne. When exposed to lungparenchyma, silica nanoparticles often result in sizeand morphology dependent toxicities, with smaller,more charged particles displaying the greatest effect.Studies of humans routinely exposed to nonporous orcrystalline SiO nanoparticles like those in groundquartz dust often exhibit a spectrum of respiratory dis-orders that include silicosis, interstitial fibrosis, indus-trial bronchitis, small airway disease, and emphysema[81�85]. In one study of 7 patients accidentallyexposed to aerosolized paint bearing silica nanoparti-cles and suffering from multifactorial pulmonary dis-ease (pleural effusions, progressive pulmonaryfibrosis), 20 nm diameter largely nonporous silicananoparticles were discovered in vascular endothelialcells, macrophages, and pulmonary and lymphaticmicrovessels less than 3 months post-exposure [85].After 15 months, epithelial cells and macrophages ofthe afflicted were found to posses relatively few silicananoparticles, though some patient biopsies demon-strated the existence of 70 nm long nanostructuresand agglomerates within the nucleus and cytoplasm ofalveolar epithelial cells. Nanoparticle exposure dosagescould not be determined and, after 18 months,2 patients had died of pulmonary failure and 2 sufferedsevere disability. Pulmonary exposure to aerosolized

amorphous and porous silica nanoparticles generallyappears to be less toxic, perhaps reflecting their rela-tive paucity of dangling bonds (i.e., undissociated sila-nols). However, toxicological studies of pulmonaryexposures to amorphous and porous silica nanoparti-cles have shown increases in reactive oxygen speciesconcentrations, reductions in glutathione levels, andinduction of proinflammatory, inflammatory, and oxi-dative stress responses both in vitro and in vivo [85].

At present there are no systematic toxicologicalstudies of the effects of skin contact with silica nano-particles. Although surface hydroxyl groups of silicananoparticles tend to increase their hydrophilicity, theisoelectric point for silica is between 2.3 and 2.8 whilethat of skin is between 3.5 and 4.8, making both sur-faces negatively charged (unless intentionally modi-fied) and thus repulsive under normal physiologicalconditions [86]. The brick-and-mortar-like, circuitousstructure of intact stratum corneum—comprised ofcorneocytes 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, canoccur in regions of comprised skin integrity (e.g., abra-sions, lacerations, burns) or via shunting along com-paratively sparse transappendageal routes such asalong hair follicles and sweat ducts. As a result of theskin’s barrier qualities, a number of silica nanoparticle-based therapies have been approved for the treatmentof dermatological conditions such as acne and rosacea.

Ingestion provides yet another route of silica nano-particle administration and exposure; one on whichnumerous preclinical drug development studies havefocused their attention, given the great stability ofsilica in low pH environments. In one such study, non-porous silica nanoparticles having diameters of 70,300, and 1000 nm were orally administered to mice ata maximum dose of 100 mg/kg [88]. 70 nm SiO nano-particles proved lethal to mice at doses greater than20 mg/kg, while 300 and 1000 nm nanoparticles dem-onstrated no adverse reactions. Subsequent examina-tion of the harvested spleens, kidneys, and lungs ofmice subjected to 300 and 1000 nm SiO nanoparticleoral administration revealed no apparent toxicity.However, degenerative necrosis of hepatocytes wasdiscovered in mouse livers shortly following theadministration of 70 nm silica nanoparticles, and liverfibrosis accompanying longer-term exposure to 70 nmsilica nanoparticles. Taken together, these findingsappear to support those of cell studies: that, all otherthings being equal, the relatively larger surface area tovolume ratios of smaller nanoparticles correlates withincreases in cytotoxicity. Interest in the ingestion ofporous silica nanoparticles has recently garnered



considerable interest as well, due to availability oftheir large pore volumes and enormous surface areasfor drug conjugation. Mesoporous silica nanoparticlesin particular have proven especially appealing forsuch applications as their periodic and highlyadjustable pore geometry, which lends itself to pro-tected and controllable delivery of large quantities ofotherwise water insoluble drugs or cytotoxic drugs[89]. Controlled toxicological studies of ingested meso-porous silica nanoparticles generally report little if anyobserved toxicity. Rabbits and dogs orally adminis-tered 200 to 1000 nm diameter mesoporous silicananoparticles exhibited no signs of toxicity [90].Noteworthy, however, is that all studies conducted todate have examined otherwise healthy individuals;those with ulcerative and inflammatory GI conditionswere excluded.

SYNTHESIS AND SURFACEMODIFICATION OF SILICA

NANOPARTICLES

Silica nanoparticles currently under developmentfor nanotheranostic applications can structurally, andto some extent therefore functionally, be classified aseither nonporous or porous. Though compositionallyindistinguishable, structural differences between non-porous and porous silica nanoparticles are marked asthe forgoing discussion notes, with significant implica-tions both for their application and biocompatibility.

Synthesis of nonporous silica nanoparticles is typi-cally conducted by one of two approaches: via theStober process or reverse microemulsions. The Stoberprocess, developed in 1968, is a sol-gel method thatgenerates monodispersed spherical silica particles [91].This technique relies on the controlled hydrolysis andcondensation of a silica precursor, most often the alk-oxide terraethyl orthosilicate (Si(OC2H5)4, abbreviatedTEOS, taking place in the presence of excess water anda low molar-mass alcohol such as ethanol, with ammo-nia serving as a catalyst. Particle size and, to a lesserdegree, morphology, can be adjusted during synthesisvia changes in water�silane ratio, catalyst or solventtype, and temperature. For example, methanol solu-tions result in the smallest diameter particles and nar-rowest diameter distributions, while both particlediameter and diameter distribution increase with theuse of longer-chain alcohols. In general, the diametersof nonporous silica particles derived from Stober pro-cesses range from 20 to 2000 nm, with mutual electro-static repulsion of their negatively charged surfacespreventing their flocculation in aqueous solutions.Stober processes have also been used to create non-porous silica coatings around smaller seed and core

particles [92]. Reverse microemulsion, also known aswater-in-oil, approaches to silica nanoparticle synthesiswere developed in the 1990s and are used to generate20 to 100 nm diameter, highly monodisperse sphericalparticles [93]. In this technique, ammonia-catalyzedpolymerization of TEOS takes place in nanometer-sized water droplets (termed nanoreactors) that are sta-bilized by surfactants and dispersed in a continuousdomain of oil. It is a thermodynamically stable, isotro-pic, highly tunable system in which the size of themicelles, and subsequently of the silica nanoparticles,can be adjusted by altering the ratio of water toorganic surfactant.

Nonporous silica nanoparticles can be readily func-tionalized for theranostic applications via the noncova-lent entrapment of hydrophilic functional moleculeswithin the particle’s silica framework. An example ofentrapment functionalization is the embedding of fluo-rescent dyes such as fluorescein isothiocyanate (FITC)and ATTO 647N directly within the silica matrix ofthe nanoparticle. Spectroscopic analyses of theseconstructs demonstrate significantly enhanced photo-stability and brightness relative to free dye molecules,arising primarily from increases in local fluorophorenumber density and reductions in fluorophore self-aggregation, inner cell effects, and oxygen/pH quench-ing. Functional moieties can also be integrated withinthe nanoparticle’s silica matrix via the use of organoalk-oxysilane derivatives during nanoparticle synthesis [94].As the siloxane linkers remain randomly or uniformlydistributed throughout the growth media during silicacondensation, homogeneous small molecule functionali-zation can be realized throughout the resulting nanopar-ticle. Functional entities that are incompatible with thechemistry of nonporous silica nanoparticle synthesismust be “grafted” onto the particle’s surface subse-quently, generally by exposing the silica nanoparticlesto trialkoxysilane derivatives.

Porous silica is also frequently used in theranosticapplications, owing to its high level of biocompatibil-ity, ease of functionalization, and unique andadjustable topology, with mesoporous silica being themost common form. Synthesis of mesoporous silicananoparticles can proceed via one of several modifiedStober processes, with the formation of liquid crystal-line mesophases of amphiphilic surfactants thatoperate as structure-directing templates for the poly-merization of silica precursors (e.g., TEOS). Underacidic or basic conditions, once the silica nanoparticleshave grown to their desired size, the template surfac-tants are removed to generate pores, either by calcina-tion or solvent extraction in acids or alcohol mixturesfor cationic surfactants such as cetyltrimethylammo-nium bromide (CTAB) or alcohols for neutral surfac-tants such as n-dodecylamine. Calcination promotes

371SYNTHESIS AND SURFACE MODIFICATION OF SILICA NANOPARTICLES


condensation of unreacted silanol groups (and thusreduces attachment sites for post-synthesis functionali-zation) and many functional entities are destroyedat calcination temperatures (400�550�C), so solventextractions are most often employed. Moreover, withthe existence of different liquid crystalline mesophasesand surfactant morphologies, surfactant-templatedsynthesis of mesoporous silica nanoparticles can beused to derive a variety of isoforms that include per-mutations of mesostructure (e.g., disordered, worm-hole-like, hexagonal, cubic, and lamellar mesophases)and morphology (e.g., spheres, hollow spheres, fibers,tubules, gyroids, helical fibers, crystals, and many hier-archical structures), simply by controlling the reactionconditions (e.g., temperature, pH, surfactant concen-tration, silica source). Pore diameters are largely dic-tated by template characteristics, however; forexample, varying the alkyl chain length of homologs ofquaternary ammonium surfactants can result in minormodification (1.8 to 2.3 nm). Additional pore expan-sion, needed for peptide, aptamer, gene, or proteindelivery, can be achieved through the use of “swellingagents” such as N,N-dimethylhexadecylamine(DMHA) and 1,3,5-trimethylbenzene (TMB). Poreexpansion via such agents, however, often resultsin less uniform, wormhole-like pore structures. Larger-sized pores can also be obtained through the use ofblock polymers as templates [95]. Alternativeapproaches to achieving large pore diameter mesopor-ous silica nanoparticles include the use of block poly-mers as templates or the incorporation of multiplesurfactants of different molecular weight, the latter ofwhich can also be used to generate dual-mesoporousmaterials.

Surface modification of mesoporous silica nanopar-ticles for subsequent molecule attachment are gener-ally accomplished either by cocondensation (directincorporation) during synthesis or by post-synthesisgrafting. In cocondensation, tetra/tri-alkoxysilane iscondensed into the pores of the silica nanoparticle dur-ing synthesis, resulting in the homogeneous incorpo-ration of the functional moiety throughout the silica.Organoalkoxysilanes can also be cocondensed into thegrowing nanoparticle to produce an inorganic-organichybrid for subsequent, additional functionalization.The choice of alkoxysilane precursor is constrained tothose that are soluble in the aqueous phase of synthe-sis and can survive the great variations in pH encoun-tered during particle polymerization and surfactantextraction. Surprisingly large numbers of functionalgroups can be accommodated via cocondensation tech-niques without significantly affecting pore topology.Anionic organoalkoxysilanes, such as thiolate-, carbox-ylate-, and sulfonate-containing organoalkoxysilane,have been used to electrostatically preclude phase

separation of precursors and cleavage of Si-C bondsduring sol-gel synthesis and ensuing surfactantremoval [96]. Cocondensation of two organoalkoxysi-lanes can bestow both acidic and basic functionalgroups onto nanoparticle frameworks, resulting innovel chemical properties and applications [96,97].

Post-synthesis grafting onto mesoporous silica nano-particles exploits the presence of surface silanol groups(Si-OH), which can occur with abundance if not calci-nated, that act as convenient anchoring points fororganic functionalization. Grafting is commonly car-ried out by silylation on free (�Si�OH) and geminalsilanol (Si(OH)2) groups, though hydrogen-bondedsilanol groups are less accessible to modification asthey readily form hydrophilic networks with oneanother. The original particle morphology and porestructure remain intact with grafting, though the tech-nique often results in nonuniform distributions oforganic functional groups. Such inhomogeneous cover-age with functional groups is especially problematicwithin pores as these surface silanols are less directlyaccessible to grafting moieties. To address this problemas well as enable independent functionalization of theoutermost and innermost surfaces of the silica nano-particle, researchers selectively and alternately passivatethe silanols in one domain while grafting onto those ofthe other domain; for example, by not extracting sur-factant templates from pores until after first graftingfunctional groups onto the particle’s exterior, thenextracting templates and reacting only the remain-ing unaffected, pore-lining silanols. Despite exhibitinggreater functionalization inhomogeneity than cocon-densation approaches, post-synthetic grafting is themost popular approach for covalently incorporatingorganic functionalities onto mesoporous silica.

The forgoing description of surface modifications ofsilica nanoparticles largely reflects the versatility ofsilanol/organosilanol chemistry. With the introductionof organosilanes bearing amino, thiol, or carboxy reac-tive moieties, either during or following nanoparticlesynthesis, covalent conjugation of a wide variety ofbiomolecules can be rendered. Disulfide-modifiedoligonucleotides can be linked to thiol-functionalizedsilica nanoparticles by disulfide-coupling chemistry.Carbodiimide reagents can be used to couple amine-bearing biomolecules to carboxy-modified silica nano-particles. In addition, a wide array of amino reactivebiological constructs (e.g., enzymes, antibodies, apta-mers) can be attached via succinimidyl esters and iso(thio)cyanates to silica nanoparticles possessing aminogroups. Intrinsic electrostatic interactions between thebiomoieties and nanoparticles can also be used for bio-conjugation, though carboxy and amino group intro-ductions can significantly alter a nanoparticle’s overallsurface charge with unintended consequences, such as



particle aggregation. Physical adsorption (physisop-tion) arising from van der Waals interactions can alsobe used to form silica nanoparticle bioconjugates,though such moieties are often transitory under bio-logical settings due to the weakness of attraction.Lipids and polyethylene glycol (PEG) coatings havealso been used to alter the surface properties of silicananoparticles, though primarily to enhance nanoplat-form biocompatibility. As will shortly become evident,the availability of these diverse surface modificationsof silica, both porous and nonporous, has resulted inmultifunctional platforms with a wide variety of appli-cations in biotechnology and biomedicine.

SILICA NANOPARTICLENANOTHERANOSTICS

General Considerations

Recently there has been considerable interest inemploying nanometer-sized biocompatible materialsthat are both diagnostic and therapeutic. Termednanotheranostics, the motivation for such entities arisesfrom a desire to monitor disease status. Election toprovide both diagnostic and therapeutic functions in asingle moiety also stems from the desire to circumventdifferences in biodistribution and selectivity that fre-quently accompany the employment of distinct diag-nostic and therapeutic agents, and to exploit existentoverlapping functionalities (e.g., both diagnostics andtherapeutics mandate specific targeting of pathology).Silica nanoparticles, by virtue of their high biocompati-bility, in vivo stability, tailorability, diverse morphol-ogy, and ease of functionalization are especially wellsuited for theranostic applications.

In situ diagnosis and, in some cases, staging of dis-ease relies on the incorporation of a suitable contrastagent (or agents) within the nanotheranostic for theclinical imaging technique (or techniques) chosen.Current clinical imaging modalities and contrastagents for which silica nanoparticles are being devel-oped as nanotheranostic agents include (1) magneticresonance imaging (MRI), via the use of gadolinium,manganese, and iron oxide; (2) positron emissiontomography (PET), via the use of the radiolabeled glu-cose analog fluorodeoxyglucose (18F-FDG); (3) singlephoton emission computed tomography (SPECT),via the use of radionuclides such as metastabletechnetium-99 (99mTc), iodine-123 (123I), and iodine-131(131I); (4) computed tomography (CT), via the useof gold, iodine, and barium; and (5) optical imagi-ng/diffuse optical tomography (OI/DOT), via the useof fluorophores such as indocyanine green (ICG),FITC, and Alexa Fluor or ATTO dyes (as well as

substrates of bioluminescent enzymes such as lucifer-ase, in animal studies). Therapeutic functionalizationof silica nanoparticle theranostics for oncological appli-cations are being tailored to the specific form and pre-sentation or stage of cancer and include (1) nucleicacid therapy (NAT), via the use of siRNA, DNA plas-mids, ribo/DNA-zymes, and antisense oligonucleo-tides; (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) radioisotopetherapy (RIT), via the use of radionuclides such asyttrium-90 (90Y), iodine-131, and strontium-89 (89Sr).

To be clinically useful, nanotheranostics must dem-onstrate specific accumulation at the site of interest,either through active or passive targeting. Passive tar-geting in oncology relies on the nonspecific accumu-lation of nanoplatforms in the leaky vasculature oftumors via the enhanced extravasation, permeation,and retention (EPR) effect. As noted earlier, however,such passive targeting is extremely dependent on thenanoplatform’s size, shape, reactivity, and surfacecharge, making the reduction of nonspecific binding achallenging, problematic task. Nanotheranostic diag-nostic and therapeutic utility are each significantlystrengthened through the use of active targeting,whereby targeting moieties (e.g., small peptides, anti-bodies, aptamers, protein fragments), attached to thenanotheranostic’s exterior, specifically associate withreceptors at the disease site. Such incorporation ofpathology targeting ligands can enable true molecularimaging of disease location and status. Labeling nano-particles with multiple copies of a given targetingligand substantially increases particle-pathology avid-ity, while employment of multiple different ligands onthe same nanoplatform can permit the nanotheranosticto grapple with dynamic variations in targeted recep-tor expression, as often occurs in cancers.

Multifunctionalized Solitary Platforms

Multifunctionalized solitary silica nanoparticles,both porous and nonporous, have found numerousapplications as nanotheranostics, one of which is forphotodynamic therapy (PDT). PDT is frequently usedin clinics to treat dermatologic disorders such as acne,rosacea, and psoriasis, ophthalmological diseases suchas wet macular degeneration, and several forms oflung and esophageal cancer. It is an intrinsically focaltherapy, far less invasive than surgical approaches,and with far fewer side effects than pharmacologicalinterventions. PDT involves the pathology’s uptake ofphotosensitizers (PS)—often porphyrins, chlorophylls,or dyes—whose external photoirradiation produceslocally cytotoxic, reactive oxygen species (ROS). Many

373SILICA NANOPARTICLE NANOTHERANOSTICS


PSs are also intrinsically fluorescent, enabling their usein diagnostic OI as well. The photosensitizer, in itsground (singlet) state, absorbs a photon that promotesthe PS to a short-lived excited singlet state. Becausethe excited singlet state is so short lived, the PS haslittle opportunity to either transfer energy or electronsto other molecules nearby and instead undergoes inter-system crossing to leave it in an excited, much longer-lived, triplet state. As the PS subsequently decays backto its initial ground (singlet) state, it transfers energyto nearby ground (triplet) state molecular oxygen,raising the latter to its first excited singlet state.The so-generated singlet oxygen molecules (and otherROSs) are especially efficacious cytotoxic agents thatrapidly induce apoptosis or necrosis. The overwhelm-ing majority of clinically available PSs, however, pos-sess significant limitations that constrain their wideruse, which include high hydrophobicity, substantialself-aggregation, and poor tumor selectivity. A, consid-erable effort has gone into developing compoundswith improved “deliverability.”

In one recent study investigators described the useof the organically modified silica (ormosil) nonporousnanomaterials for PDT [98]. The researchers integrated2-[1�hexyloxyethyl]-2-devinyl pyropheophorbide-a(HPPH)—a lipophilic, second-generation, chlorin-based photosensitizer currently in phase II clinicaltrials (Photochlor)—into silica matrices. The resultingconstructs demonstrated that HPPH incorporationwithin silica significantly enhances the quantum effi-ciency of HPPH (as well as ROS generation and localcytotoxicity) relative to free HPPH as intravenouslydelivered. Unfortunately HPPH, though better suitedthan first-generation PSs such as porfimer sodium(an oligomer mixture of ether and ester-linked porphy-rin units, commercially available as Photofrin), canonly be used for PDT to a tissue depth of a few milli-meters, due to the inherent scattering and absorptionof photons in most mammalian tissues at visiblewavelengths.

To enhance PDT efficiency at greater tissue depths,more recent efforts have focused on working atlonger, near-infrared (NIR) wavelengths—where pho-ton absorption and scattering in tissues are greatlyreduced—with either NIR dyes or combinations oftwo-photon absorbing dyes with PSs. In vitro andin vivo OI and PDT studies using 105 nm diameternonporous silica nanoparticles in which the cationicNIR dye methylene blue was entrapped, revealed con-siderable gains in detection sensitivity and therapeuticefficacy [99]. In another study, researchers coencap-sulated HPPH and the two-photon absorbing dye9,10-bis[40-[400�aminostyryl]styryl]anthracene (BDSA)within nonporous silica nanoparticles, revealing PDTactivation via BDSA upconversion of NIR light with

partial energy transfer to HPPH [100]. Silica coatingof highly porous, nanometer-sized iron-carboxylatemetal-organic framework (MOF) particles, post-synthesis covalently modified with the fluorescentdye 1,3,5,7-tetramethyl-4,4-difluoro-8-bromomethyl-4-bora-3a,4a-diaza-s-indacene (Br-BODIPY) and the anti-cancer drug cisplatin, have also been developed fortheranostic applications [101].

Mesoporous silica nanoparticles (MSNs) have alsobeen developed as highly efficient PDT nanotheranos-tics. In one study investigators sequentially functiona-lized the nanoplatform’s 3 topologically distinctdomains with contrast agents for traceable imaging ofparticle targeting, payloads for therapeutic intervention,and biomolecular ligands for highly-targeted particledelivery, as shown in Figure 20.2 [102]. Traceable imag-ing of nanoparticles was accomplished by directly incor-porating (cocondensing) the NIR fluorophore ATTO647N into the MSN’s silica framework to exploit the rel-ative transparency of most tissues at NIR wavelengthsand maximize MSN surface area available for the subse-quent conjugating drugs and targeting ligands. An oxy-gen-sensing, palladium-porphyrin based photosensitizer(Pd-porphyrin; PdTPP) was incorporated into theMSN’s nanochannels to enable PDT. And cRGDyK pep-tides, tiling the outermost surfaces of MSNs, were usedfor targeting αvβ3 integrin over-expression of cancercells and to ensure the internalization of the photosensi-tizer PdTPP. In vitro cell evaluation of the theranosticplatform demonstrated not only excellent targetingspecificity and minimal collateral damage, but highlypotent therapeutic effect as well. Human MCF-7 breastcancer (αvβ3 integrin negative) and U87-MG glioblas-toma (αvβ3 integrin positive) cells were used with flowcytometry and confocal microscopy to assess the func-tionalized MSN’s targeting specificity and uptake effi-ciency, while propridium iodide staining and WST-1assay enabled determination of PDT response prior toand following 532 nm wavelength laser irradiation.

These in vitro studies revealed that the trifunctiona-lized MSN nanotheranostics possessed not only excel-lent targeting specificity but highly potent therapeuticeffect as well. Members of the same group subse-quently developed MSNs that coencapsulated two-photon absorption dyes and photosensitizers, thusenabling high-energy transfer rates for two-photonactivated PDT [103]. With the judicious tailoring oftwo-photon donor-acceptor ratios, the well-orderedmesoporous structure of MSNs demonstrated energytransfer rates up to an unprecedented 93%.Intracellular energy transfer proved extremely efficientas well in the nanoplatform, with dramatic singlet oxy-gen induced cytotoxicity observed in both in vitro andin vivo breast cancer models. In another study employ-ing porous silica for deep tissue PDT, Gary-Bobo et al.



used mannose-functionalized mesoporous silica parti-cles for tumor-targeted two-photon PDT, observing70% regression of tumor size after a single treatmentin athymic mice bearing tumor xenografts [104].

Silica-based nanoparticles have garnered consider-able interest for their use as nanotheranostic platformsin the delivery of drugs and nucleic acids as well.Mesoporous silica nanoparticles, in particular, makefor appealing delivery vehicles as they can convey con-siderably more drug within their environmentallyprotected pores than can be accommodated on the sur-faces of comparably sized, nonporous silica nanoparti-cles. Porous and mesoporous silica constructs, unliketheir nonporous and crystalline silica counterparts,do not require that the conveyed drug be covalentlyconjugated to the nanoparticle’s surface; this is signifi-cant in that many drugs cannot tolerate covalentattachment without adversely affecting their structureor function. Lastly, as will be described shortly, MSNs

can be synthesized so that their pores are capped withgatekeeping molecules that enable finely controlleddrug release either via internal or external stimuli.

Ashley et al. developed MSN-supported lipidbilayers, termed protocells, as theranostic platforms vialiposome fusion and encapsulation of fluorescentMSNs that bore with various therapeutic agents (e.g.,drugs, siRNA, protein toxins, quantum dots), as illus-trated in Figure 20.3 [105]. The investigators demon-strated that the lipid membranes of these MSNs retainboth high in-plane two-dimensional fluidity and highstability against destabilization in the presence ofblood components or leakage of drug cargos from thesilica core. Post-synthesis modifications of protocells—to promote cell targeting, endosomal escape, andnuclear accumulation—resulted in 10,000-fold greaternanoplatform affinities for human hepatocellular carci-noma cells than for hepatocytes, endothelial cells, orimmune cells, enabling individual protocells, loaded

(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 withintheir silica frameworks. Following extraction of templates, palladium-based photosensitizers were loaded into the MSN pores. The nanoparti-cle was then PEGylated and targeted for chemotherapy via conjugation of c(RGDyK) peptides. (b) Operational schematic of the trifunctiona-lized theranostic MSNs for traceable, targeted, photodynamic therapy. Source: Reproduced from [102] with kind permission of The Royal Society ofChemistry. Copyright 2010. All rights reserved.



with a drug cocktail, to kill individual drug-resistanthuman hepatocellular carcinoma cells. This repre-sented a 106-fold improvement over comparableliposomes.

Recently, Lu et al. reported the synthesis of MSN-based theranostic particles that contain the fluorophoreFITC embedded (cocondensed) within the silica frame-work, the anticancer therapeutic camptothecin (CPT)condensed within the nanopores, and folic acid graftedonto the MSN’s exterior for cancer targeting [106].These studies were primarily focused on the toxicity,biodistribution, clearance, and therapeutic propertiesof their nanotheranostics, with human breast cancercell lines (MCF-7, MCF10F, and SK-BR-3) used forin vitro analyses and six-week-old female BALB/cAnNCrj-nu nude mice for in vivo studies, some ofwhich bore MCF-7 xenografts. Optical imaging andinductively coupled plasma mass spectroscopy (forsilica content) were used to monitor the nanothera-nostic’s biodistribution and excretion, respectively.Fluorescence imaging revealed that their 100 to 130 nmdiameter MSNs preferentially localized to the tumor,kidneys, and liver. The tumors in the mice treatedwith CPT-loaded MSNs were virtually eliminatedat the end of experiments although, surprisingly,

conjugation with folic acid on the MSN’s surfaceseemed to produce little additional antitumor effectwhen compared to untargeted CPT-loaded MSNs, afinding postulated to be a reflection of rather modestupregulation of folate receptors in the MCF-7 xeno-grafts and significant EPR effect. Also noteworthy wasthe near complete excretion of MSNs from mice:B95% of Si was excreted out of body through urineand feces, consistent with observations of Souris et al.[107].

Lee et al. developed theranostic MSNs in which theanticancer drug doxorubicin (dox) was conjugatedonto the particle’s pore walls through the use of finelytuned, pH-sensitive linkers [108]. Initially the NIRfluorophore ATTO 647N was cocondensed into theMSN’s silica framework during nanoparticle synthesis.Following template extraction and pore expansion(to 5 nm) of the 100 nm diameter nanoparticle, thepore walls and dox were separately modified so as topossess pH-sensitive hydrazone bonds. When MSNshad been loaded with drug, the pH-sensitive linkerspossessed two hydrazone bonds that were cleavable atendosomal/lysosomal pHs (4�6) but stable in circula-tion (pH 7.4). pH sensitive hydrazone linkers are gen-erally compatible with drugs that have functional

FIGURE 20.3 Mesoporous silica-supported lipid bilayer constructs, termed protocells, for multitherapeutic cancer treatment. Fusion ofliposomes onto previously synthesized spherical MSNs, and subsequent targeting ligand association, yielded 10,000-fold greater affinity forhuman hepatocarcinoma than for hepatocytes, endothelial cells, or immune cells, a manifestation of the enhanced targeting efficacy affordedby 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.



ketones or aldehydes, such as cerubidine and idarubi-cin. Fluorescence spectroscopy and imaging were usedto quantitate the MSN’s uptake and release of dox, aswell as the nanoparticle’s therapeutic efficacy, withhuman hepatoma cells (Hep-G2) in vitro. Because theemission spectra of dox overlapped with the absorp-tion spectra of ATTO 647N, and the average distancebetween donor and acceptor was shorter than 10 nm,efficient Forster resonance energy transfer (FRET) tookplace prior to the release of the intrinsically fluorescentdox from the MSN’s constituent ATTO 647N, a phe-nomenon the investigators also used to monitor doxrelease in situ. Murine biodistribution studies of ATTO647N-MSN-hydrazone-dox, conducted using in vivofluorescence imaging and subsequently confirmedby transmission electron microscopy (TEM) of har-vested tissues, demonstrated hepatic endosome andlysosome colocalization. He et al. developed yetanother form of pH-responsive MSNs capable of over-coming MDR [109]. Their composite nanoparticles con-sisted of MSN carriers of dox that still contained theporogen cetyltrimethylammoniumbromide (CTAB),where the surfactant served as a chemosensitizer thatboth circumvented MDR and enhanced drug efficacy.The cytotoxicity of these particles was evaluated usingMCF-7 and MCF-7/ADR human breast cancer celllines and demonstrated that MSN-dox-CTAB signifi-cantly enhanced dox intracellular accessibility of MCF-7/ADR with much greater effect than free drug againstboth cell lines. Multitherapeutic conveyances byMSNs, such as the codelivery of chemotherapeuticdrugs and siRNAs targeting cellular survival mechan-isms, have also been reported recently. Chen et al. pre-sented convincing evidence that MSNs, carrying bothdox and siRNA and targeted against mRNA encodingBcl-2 protein in multidrug resistant (MDR) cancercells, significantly increased the cytotoxicity of doxby B132-fold relative to free drug via a pronouncedincreased apoptosis [110].

Mesoporous silica nanoparticle shells, with andwithout cores, have also been devised as theranosticnanoplatforms for the protected delivery of drugs andorganics monomers and polymers. One example is thatof Kim et al. who synthesized monodisperse core�shell constructs (45, 60, 90, and 105 nm diameter) inwhich a layer of mesoporous silica enveloped a singleiron oxide (IO) Fe3O4 nanocrystal of either 15 or 22 nmdiameter, as shown in Figure 20.4 [111]. While the IOcore provided MRI contrast, investigators elected toalso fluorescently label the silica shell for optical andmicroscopic tracking of their nanoplatform, via thecovalent incorporation of FITC and rhodamine Bwithin the silica mesopores. The IO-mesoporous silica(MS) shell was then PEGylated for improved in vivostability and loaded with the anticancer agent dox.

Fluorescence and confocal laser scanning microscopyof human breast cancer cells (MCF-7) revealed excel-lent endocytotic internalization of the nanoparticles incells within 30 minutes of administration. In vitro T2-weighted MRI of IO-MSshell-PEG with a 1.5T scannerrevealed r1 and r2 relaxivities of 3.40 and 245 mM21

s21, respectively. In vitro T2-weighted MRI of MCF-7cells showed concentration-dependent uptake of thenanoparticles, in agreement with fluorescence micros-copy of the same specimens. To assess the untargetedIO-MSshell-PEG construct’s in vivo utility, humanbreast cancer cells (SK-BR-3) were subcutaneouslyinjected into the dorsal shoulders of nude mice.Subsequent in vivo and ex vivo fluorescence and MRimaging of the resulting xenografts, following IO-MSshell-PEG intravenous injection, showed significantEPR effect passive accumulation of nanoparticleswithin tumors, confirmed by TEM ex vivo. The samegroup subsequently developed dox-loaded MSNs inwhich the fluorophore FITC (or TRITC) had been cova-lently attached to the MSN’s pore walls and IO nano-particles chemically attached to the MSN’s exteriorsurface, to yield dramatically enhanced T2-weightedMRI contrast: with r2 relaxivities of 76.2 mM21 s21 forthe IO-capped MSNs compared to 26.8 mM21 s21 forfree IO [112].

Organic-Composite Hybrid Platforms

To further enhance the biofunctionality of silica-based nanotheranostics, considerable effort has goneinto the development of organic-composite hybridswhose inner and/or outer surfaces contain organicpolymers such as polyethylene glycol (PEG), polyeth-ylene imine (PEI), or one of a variety of pH-sensitivepolymers. While organic hybridization of porous andnonporous silica nanoparticles can be active (e.g., suchas in enabling nanoparticles to react to changes in theirlocal environment), to date it has more frequentlyserved static functions such as stabilizing payloads forconveyance or enhancing their delivery via increasingcirculatory lifetimes and proteolytic resistance, whiledecreasing their immunogenicity and/or antigenicity.For drug delivery theranostic applications, mesopor-ous silica platforms are the dominant solid supportplatform in organic-composite hybrids, due to theirhigher payload capacity than nonporous silica moie-ties. For example, Wang et al. coated PEGylated-phospholipids onto hydrophobic, silane-modifiedMSNs that possessed FITC for optical trackingand folate for targeting, observing significantly inhib-ited flocculation and reduced nonspecific protein bind-ing [113]. Another polymer, PEI, has been found togreatly enhance drug delivery efficiency of silica nano-particles via its unique proton-sponge and endosome



buffering properties that destabilize lysosomal mem-branes and promote endosomal escape of their con-tents [114,115]. Rosenholm et al. developed onion-like, core-shell-shell fluorescent mesoporous silica

nanoplatforms that provide targeted, traceable deliveryof therapeutic compounds [116]. In their design, a bio-degradable mesoporous silica matrix core, in whichthe therapeutic payload is stored, is surrounded by a

FIGURE 20.4 Untargeted mesoporous silica shell�iron oxide core theranostic nanoplatform for combined MR/fluorescence imaging anddrug 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 beensynthesized around individual 15 nm diameter iron oxide (Fe3O4) nanocrystal cores. Very high shell uniformity was achieved via only varyingthe concentration of iron oxide nanocrystals, with nanoparticle diameter increasing as core size decreased. Similar methods were used to coatmesoporous silica onto 25 nm MnO nanocrystals (k) and one-dimensional α-FeOOH nanotubes (l). (B) In vivo T2-weighted MR (a) and fluores-cence (b) imaging of mice with/without subcutaneous injection (dorsal shoulder) of MCF-7 cells labeled with/without F3O4 mesoporous silicashells 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 withF3O4 mesoporous silica shells at 5 mg Fe kg21. Source: Reproduced from [111] with kind permission of Wiley-VCH Verlag GmbH & Co. Copyright2008. All rights reserved.



PEI-layer that serves to simultaneously increase colloi-dal stability and promote endosomal escape into thecytoplasm. Folic acid targeting ligands decorated theoutermost exposed surfaces of this cancer theranostic.

Recent interest in organic hybrid MSNs has arisennot only from their enhancement of endosomal escape[116�118], light sensing [119�120], pH sensing[121�123], and water solubility [113], but also due tothe ability of these nanomaterials to codeliver morethan two types of payload in an additive fashion.Meng et al. designed a dual-drug delivery systemusing PEI-coated MSNs that were functionalized toefficiently codeliver the chemotherapeutic agent doxand P-glycoprotein (Pgp) siRNA to multidrug resistant(MDR) cancer cells for synergistic cytotoxicity [118].Enhanced synergistic cytotoxicity of their platformwas initially suggested by the observation of greatlyincreased intracellular and intranuclear levels of doxfollowing theranostic administration: levels far exceed-ing that found from either conventionally deliveredfree dox or dox delivered by MSNs that were devoidof Pgp siRNA. Subsequent in vivo studies, using anMDR tumor xenograft model, convincingly demon-strated the synergism of dox and Pgp codelivery.Interestingly, the investigators also observed signifi-cant Pgp knockdown at heterogeneous tumor sites thatcorresponded to the regions where dox was releasedintracellularly and induced apoptosis [124].

Flexible organic molecules have also been usedas “gatekeepers” of the pores on MSNs. Mal et al.developed MSNs that exploited the reversible

photodimerization of coumarin to controllablyopen and close the pores of MSNs in organicsolvents [125]. MSN pores were functionalized with7-[(3�triethoxysilyl)propoxy]coumarin. Casasus et al.developed an ionically-controlled MSN gatekeepercomprised of diaminoethylenepropylsilane derivativesgrafted immediately outside pore entrances andmercaptopropyl groups positioned proximally insidepores [126].

As illustrated in Figure 20.5, more elaborate gate-keeper constructs for theranostic payload release, per-haps more appropriately termed nanomachines, havealso been derived using organic-composite hybrid-ization with underlying silica nanoplatforms [127].Radu et al. reported dendrimer-capped MSNs that canbe used both for drug delivery and gene transfectionsystem, using a generation 2 poly(amidoamine) den-drimer (G2-PAMAM), as shown in Figure 20.6[62,127]. Hernandez et al. developed organic supramo-lecular gatekeepers termed nanovalves that consist of aseries of electrochemical redox-controlled pseudorotax-anes attached to mesoporous silica [128]. In the earliestversions of this construct, [2]-pseudorotaxane [DNPD-CBPQT]41 was used as a gatekeeper, of which 1,5-dioxynaphtalene derivative (DNPD) served as theparticle-attached gatepost and cyclobis-(paraquat-p-phenylene) (CBPQT41 ) served as the gate. Followingthe same redox-controlled release approach, theinvestigators refined their system by developing areversible [2]-rotaxane R41 nanovalve, comprised oftwo separate recognition sites in a dumbbell structure

Disulfide reducing agentlight

saccharides

InsulinNBr

O

HO

O

OHN

MSN

BO

NO2OO

RS

SS

H2N

MSN Au-NP

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

Guest Molecule

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 prematuredispersal of nanoparticle contents from pores and permit external control of their therapeutic payloads, for optimal treatment timing andpathology colocalization. Source: Reproduced from [167] with kind permission of Wiley-VCH Verlag GmbH & Co. Copyright 2010. All rights reserved.



with CBPQT41 once again serving as the gate andrecognition sites, consisting of tetrathiafulvalene (TTF)and 1,5-dioxynaphtalene (DNP), which were sepa-rated from one another by an oligoethyleneglycolchain [129].

Another pseudorotaxane-based MSN controlledrelease system was reported by Park et al. using PEI asthe fixed motif and α- or γ-cyclodextrin (CD) as themovable ring [130]. At high pH, the CD ring wasobserved to thread into the PEI base thereby closingthe pore, while at low pH, PEI-CD irreversibly dissoci-ated, with CD diffusing away from the nanoplatformand 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 gate-keepers, owing to their ability to form inclusion com-plexes that can reversibly dissociate in response toexternal stimuli and their high intrinsic biocompatibil-ity [131]. Using CB[6]-containing [2]pseudorotaxanes,CB[6] rings reversibly respond to deprotonation ofbisammonium stalks by dethreading, unblocking thepores for the release of MSN payloads. Biologically rel-evant, autonomous operation of these constructs (e.g.,for lysosomal release) was achieved through the use oftrisammonium stalks that contain one anilinium andtwo (CH2NH2

1CH2) centers in which the aniliniumnitrogen atom (unlike the other two nitrogen atoms) isnot protonated at neutral pH [132]. At neutral pH the

CB[6] rings reside on the two (NH2(CH2)4NH21) recog-

nition sites, blocking the nanopore. Tuning the pKa ofthe anilinium nitrogen, via varying the parasubstituenton the aryl rings, permits precise control of the poreopening rate and pH value of operation. Inverse con-formations of gatekeeper, with mobile stalks and sta-tionary rings, have also been synthesized. Zhao et al.have used β-CD rings arranged to form cylindrical cav-ities in which rhodamine B/benzidine stalks move aspistons in response to changes in local pH value [133].These short cylindrical β-CD cavities extend radiallyoutward from the MSN’s surface and self-assembleso as to maintain alignment with the MSN’s pores.As such, these nanoparticles can also be used todeliver larger moieties since the β-CD ring linkages tothe nanoparticle contain cleavable imine double bondsthat are hydrolyzed under acidic conditions causingthe ring-MSN dissociation and payload dispersal.Alternatively this construct could be used as a dual-drug platform to deliver both large and small mole-cules, tuned for release at different pH values.

Additional organic-composite silica motifs havebeen devised for the externally controlled release oftherapeutic compounds from MSN pores. Patel et al.used α-CD tori that encircled PEG stalks whose ester-linked adamantyl endcap, embedded within MSN poreorifices, can be enzymatically (irreversibly) cleaved torelease MSN payloads [134]. Light activation is another

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



avenue of considerable interest for controlled drugrelease. Photoactive azobenzene nanoimpellers—sotermed from their original application of expellingMSN cargo via cyclic cis/trans configuration changesinduced by UV/visible light switching [135]—havebeen modified for aqueous applications by Ferris et al.[136]. In their design, MSN surfaces are functionalizedwith azobenzene-containing stalks (in the trans config-uration) that are complexed by β-CD (or pyrene-modified β-CD) rings, sealing the pores shut. Afterphotoirradiation at 351 nm, the isomerization of trans-to-cis azobenzene units leads to dissociation of β-CD(or pyrene-modified β-CD) rings from the stalks,thereby opening the nanogates and releasing porepayloads.

Thermoresponsive gatekeepers for MSNs have alsobeen realized through the use of organic supramolecu-lar assemblies. Baeza et al. developed a multidrugnanoplatform for the treatment of MDR cancers whosetherapeutic release was remotely controlled via anexternally controlled, alternating magnetic field, nomi-nally operating at 100 kHz [117]. In their nanothera-nostic, superparamagnetic iron oxide (γ-Fe2O3)nanocrystals were entrapped within the silica matrixof MSNs, after which a thermoresponsive copolymershell, comprised of poly(ethyleneimine)-b-poly(N-isopropylacrylamide) (PEI/NIPAM), was applied.The thermoresponsive copolymer shell functioned notonly as a temperature-sensitive gatekeeper for releaseof drugs entrapped within MSN pores, but also asa temperature-responsive retention system for conju-gated therapeutic proteins. At lower temperatures,entrapped drugs and conjugated proteins remaintightly bound to the theranostic platform. Applicationof a strong alternating magnetic field to the nanothera-nostic induced nanoscale motion of iron nanocrystals,with the MSN’s silica framework imposing a restoringforce in opposition that elevated the nanoplatform’stemperature several degrees Celsius. In vitro studies ofPEI/NIPAM-entrapped proteins revealed a proteinrelease profile similar to that observed with directheating of the sample, consistent with the hypothesisthat the polymer collapses and changes to a morehydrophobic surface, causing the increase in therelease rate of the attached protein. Increases in MSNtemperature also resulted in the enlargement of MSNpores, an effect that could be used to alter the rate ofdrug release from the pores relative to the rate of ther-moresponsive copolymer shell retained therapeutics.

Coordinated, sequential delivery and release ofcombinatorial drugs is essential for the optimization ofmultidrug synergism. For instance, in organic hybridMSN codelivery of Pgp siRNA and dox described ear-lier, initial Pgp knockdown ensures that subsequentlyreleased dox has sufficient time within the cell to reach

the cell’s nucleus and intercalate its DNA. Withoutprevious Pgp knockdown, most of the dox releasedwithin drug-resistant cancer cells will undergo rapidefflux from the cell, greatly diminishing therapeuticefficacy.

Inorganic-Composite Hybrid Platforms

Due to their own unique physicochemical proper-ties, discrete inorganic nanoparticles such as quantumdots (QDs), superparamagnetic iron oxide nanoparti-cles, and gold nanocrystals have each also garneredconsiderable interest for use in functionalizing silicananoplatforms, resulting in constructs that may betermed inorganic-composite hybrid platforms. For exam-ple, narrow-band spectral emissions of QDs enableconcurrent or mutiplexed operation of differently func-tionalized or targeted silica nanoplatforms within thesame animal model [137�139]. Gold nanocrystal deco-rated MSNs and mesoporous silica shell envelopedgold nanorods permit externally controlled two-photon excitation of contrast agents, stimulation ofdrug release, and plasmon-induced thermotherapy indeep tissues [140�142]. Iron oxide nanoparticles,grafted onto mesoporous silica nanoparticles, can beused for platform tracking by MRI as well as localhyperthermia (thermotherapy) and controlled releaseof platform contents via oscillating, external magneticfields. As with their organic-composite hybrid siblings,inorganic-composite hybrid nanoplatforms designedfor theranostic applications frequently employ meso-porous silica as their therapeutic conveyance super-structure due to its larger payload capacity, moreeasily tuned and functionalized pore topology, andgenerally higher biocompatibility than nonporous/crystalline silica constructs. Thus we will focus ourattention on inorganic nanoparticles that have beenencapsulated with either mesoporous silica or MSNsonto which exogenous inorganic nanoparticles havebeen attached.

Encapsulation with silica is an especially simple,benign means of stabilizing and/or surface functiona-lizing inorganic nanoparticles. Qian et al. developeda novel therapeutic nanodevice in which NaYF4upconverting nanocrystals were encapsulated within auniform mesoporous silica shell that contained a zincphthalocyanine photosensitizer [143]. NIR laser irradi-ation of the encased nanocrystals activated the incor-porated photosensitizer generating reactive singletoxygen for cytotoxic effect. Ji et al. synthesizednanotheranostic gold nanoshells in which an internal,amorphous silica layer was used as a dielectric witha superparamagnetic iron oxide-silica core for bothMR imaging and photothemal therapy [144]. For this



work, a superparamagnetic iron oxide nanocrystal wasfirst coated with a layer of amorphous silica via con-ventional sol-gel techniques, then functionalized withamine groups. Gold nanocrystal seeds (2�3 nm) werethen attached to the amorphous silica surface to nucle-ate the growth of a gold shell on the silica. The middlelayer of silica containing hybrid nanoparticles pro-vided a dielectric interface for shifting the plasmonicresonance to the NIR region, enabling both MRI con-trast and photothermal therapy in the same construct.

Chen et al. synthesized gold nanorods withaspect ratios of B4 that, when subsequently encasedwithin amorphous silica, proved to be considerablystrengthened against surface plasmon induced thermaldeformation when optically pumped [142]. With arepetitively pulsed Ti:sapphire laser operating at808 nm and delivering the human maximum permissi-ble exposure (MPE) of 31.12 mJ/cm2 for 90 seconds,the researchers observed 0.7 db and 3.0 db reductionsin the photoacoustic signals of phantoms bearing silicaencased and bare gold nanorods, respectively. TEMstudies, as well as optical absorption spectra of post-irradiated samples, revealed significant reshaping ofbare, unencased nanorods toward a spherical topology,but little change in the shape of the amorphous silicaencased nanorods. As such, amorphous silica coatingcan permit much longer-lived, higher-contrast photoa-coustic imaging than previously was possible. Furtherfunctionalization of the construct, for example, byenveloping the inorganic-composite structure with athermoresponsive copolymer, could enable controlled

delivery and release of small molecules and peptidesembedded within the copolymer encasement.

Mesoporous silica encapsulation of iron oxide nano-particles has been used to both add magnetic proper-ties to mesoporous silica and add targeted, payloadconveyance to iron oxide nanoplatforms, with applica-tions as diverse as enhanced particle separation [145],magnetically triggered drug release [117], and MRItraceability [111,146�148]. Li et al. [145] and Zhanget al. [146] synthesized 40 to 70 nm diameter magneticMSNs that possess 15 nm diameter Fe3O4 inner coreswithin mesoporous silica shells possessing pore-expanded (6.1 nm), radially aligned pores, as depictedin Figure 20.7. Using short salmon DNA, investigatorsadsorbed a maximum of 121.6 mg/g DNA/MSN inchaotropic salt and demonstrated that their magneticMSNs provided an easily manipulated platform forDNA adsorption and desorption processes via externalmagnetic fields. Fe3O4 magnetic MSNs have alsobeen used to convey siRNA. Li et al. developed a mag-netic mesoporous silica nanoparticle (M-MSNs)based, polyelectrolyte (PEI) and fusogenic peptide(KALA) functionalized siRNA delivery system—dubbed M-MSN_siRNA@PEI-KALA—that was shownto be highly effective at initiating target gene silencingboth in vitro and in vivo [149]. M-MSN_siRNA@PEI-KALA construction began with the magneticallyinduced adsorption of siRNA within the mesopores ofM-MSNs, followed by PEI coating the nanoparticle’sexterior. KALA peptides were then grafted onto theouter surfaces of the PEI coating. Cytoplasmic delivery

FIGURE 20.7 Alternate approach to conveyance of DNA using MSNs. (a) 40 to 70 nm diameter spherical mesoporous silica shells weresynthesized around 15 nm diameter iron oxide (Fe3O4) nanocrystal cores. MSN pores were expanded up to 6 nm in diameter. Through the useof an externally applied magnetic field, up to 89.5% of DNA (salmon) double-strands were adsorbed within the confines of the MSN’s meso-pores. (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.



of the nanoparticle M-MSN_siRNA@PEI-KALA wasobserved to initiate via electrostatic membrane asso-ciation and subsequent endosomal internalization.Once MSN-bearing endosomes matured to becomeendolysosomes, the lower pH (B5) of the latter trig-gered a conformational change in the nanoparticles’KALA peptides—from α-helix to random coil—thatsimultaneously destroyed the endolysosome mem-brane and enabled the release of loaded siRNA as freeentities within the cytoplasm (nanoparticles not labeledwith KALA peptides were observed to remain withinintact mature endolysosomes, with no siRNA release).In vitro knockdown studies of enhanced green fluores-cent protein (EGFP) and vascular endothelial growthfactor (VEGF) in tumor cells demonstrated high effi-ciency, while in vivo experiments employing intratu-morally injected M-MSN-VEGF siRNA@PEI-KALArevealed significant inhibition of tumor growth, likelythe result of the suppression of neovascularization.

Ma et al. developed highly versatile, inorganic-composite hybrid nanotheranostics for multimodalimaging and multimodal therapy [150]. In this con-struct, ellipsoidal Fe3O4 cores were encased withinmesoporous silica shells via conventional sol-gel chem-istry. Post-synthesis, the IO-MSNs were decoratedwith PEGylated gold nanorods and the mesoporoussilica nanochannels loaded with dox. As such, thenanotheranostic enabled, in a single particle, chemo-therapy, photothermotherapy, T2-weighted MR imag-ing, and dark-field optical and infrared thermalimaging. Considerable in vivo MRI contrast enhance-ment was observed, while infrared thermal imagingrevealed in vivo heating efficacy of these nanomaterialsto be substantial; following 5 mm diameter 808 nmlaser irradiation of tumors previously injected withnanoellipsoids, tissue temperature increases of 3�C at1 W/cm2 and 18�C at 2 W/cm2 irradiation wererecorded, the latter causing irreparable tumor damage.Wu et al. developed similar tumbler-like multimodalMSNs that employed both T2-weighted MRI and opti-cal reporters—termed Mag-Dye@MSNs—by combiningamorphous silica coated Fe3O4 cores and organic fluo-rescent dyes with an ammonia solution that containeddilute TEOS and low concentration of C16TAB sur-factant [151,152]. In a somewhat similar approach,Liong et al. fabricated dye-doped MSNs that incorpo-rated magnetite nanoparticles and folic acid (FA) tar-geting ligands to obtain an inorganic-composite hybridnanoplatform for simultaneous drug delivery, MR andfluorescence imaging, and magnetic manipulation[153]. These nanoparticles underwent greater thantwofold increased cellular uptake in pancreatic cancercells than did comparable, untargeted nanoparticles.

Loading of these magnetic dye-doped FA-targetingMSNs with the hydrophobic anticancer drug camp-tothecin demonstrated greatly increased cytotoxicitywith pancreatic cancer cells but not with fibroblast(control) cells.

Although a variety of inorganic nanoconstructs canbe encapsulated in silica shells to fabricate multifunc-tional silica nanoparticles, such encapsulation is notalways straightforward or without consequence, suchas the morphological disruption of neighboring nano-pore structure. Such difficulties can, however, occa-sionally be circumvented via the decoration ofinorganic nanoconstructs onto the surface of previ-ously synthesized silica nanoparticles to yield yetanother inorganic-composite hybrid class. Chelebaevaet al. designed hybrid structures that combined two-photon fluorescent dye-doped MSNs with coordina-tion polymer nanoparticles (CPNs)—the latter a formof magnetic inorganic nanoparticle—for multimodalimaging [154]. Lee et al. synthesized highly versatile,composite nanoparticles via decorating the outermostsurfaces of mesoporous dye-doped silica nanoparticleswith multiple superparamagnetic magnetite nanocrys-tals [112,155]. Integration of numerous magnetite nano-crystals on the silica nanoparticle’s surface yieldedhighly synergistic magnetic effects (e.g., specificnuclear relaxivity (r2) of 80 mM21s21) that resulted inremarkable 2.8-fold enhancement of T2-weighted MRsignals. In addition to serving as fluorescence and MRIcontrast agents, these nanoplatforms also incorporatedhydrazone-linked dox for time-dependent/pH-sensi-tive drug release following nanoparticle endocytosis,revealing only 4% dox release at pH 7.4 but 78% doxrelease at pH 4.0, at 56 hours exposure. Cellular uptakeof dox hydrazone-MSN-FITC-Fe3O4-PEG was verifiedby confocal laser scanning microscopy.

Another function of exogenous inorganic nano-constructs associated with the exteriors of poroussilica nanoparticles is that of gatekeepers in stimuli-responsive delivery systems. By capping nanochannelentrances with environmental (e.g., pH, osmolarity,photoactivated) sensing inorganic nanoentities thatprevent the premature release of conveyed cargo,MSNs have been designed to operate as “smart” drugdelivery platforms [131,156]. Gan et al. developedmagnetic (Fe3O4) nanoparticle-capped MSNs by usingreversible acid-labile 1,3,5-triazaadamantane units forpH-sensitive endosomal delivery and release [157]. Inthis design the functionalized Fe3O4 nanoparticlesserved directly as nanogates, to regulate dosing.In vitro payload release studies revealed that the pH-sensitive MSN ensembles quickly release the nano-channel contents between pH values of 5.0 and 6.0,



but gave no detectable release whatsoever at physio-logical pH 7.4. Sun et al. developed gold nanoparticle-capped MSNs (Au-MSNs) for intracellular codeliveryof enzymes and substrates, with no apparent loss ofbioactivity [158]. In this construct, Au-MSN nano-channels were loaded with bioluminescent substrateluciferin and their PEGylated exteriors embeddedwith the bioluminescent enzyme luciferase. Whenexposed to disulfide-reducing antioxidants, the goldnanoparticle caps dissociated from the MSNs’ pores,enabling the release of the MSN interior’s luciferin.After proximal dispersal, enzyme and substrate link-age arose, as evidenced by the chemiluminescent sig-nal observed. Photosensitive nanogated MSNs fordrug delivery have also been developed by Knezevicand Lin. [159]. In these constructs, magnetite nano-particle incorporated MSNs were loaded with antican-cer drug camptothecin. Mesopore entrances werethen capped with CdS quantum dots through photo-cleavable o-nitrobenzyl based carbamate linkages.Camptothecin release was successfully triggeredby UV photoirradiation, while the nanoplatform’sposition was manipulated via a variable, external,magnetic field.

Lai et al. recently created a redox-controlled drugdelivery system based on cadmium sulfide (CdS)nanoparticles capping of MSNs [160]. In this system, adisulfide linker was used to chemically attach CdSsto MSNs, cleavable with various disulfide reducingagents such as dithiothreitol and mercaptoethanol.Giri et al. developed an inorganic-composite redox-controlled drug delivery system that could be posi-tioned via externally applied magnetic fields [64]. Thisdesign employed 3-(propyldisulfanyl)propionic acid-functionalized MSNs whose mesopores were cappedby 3-aminopropyltriethoxysilyl-functionalized super-paramagnetic iron oxide nanoparticles (Fe3O4).Incorporation of surface ferrite nanoparticles has alsobeen used to enhance the operation of organic nano-valves situated at mesopore entrances. Thomas et al.demonstrated such enhanced nanovalve performanceby encapsulating zinc-doped iron oxide nanoparticleswithin mesoporous silica and capping the pores withcyclic cucurbit[6]uril nanovalve to derive a magneti-cally activated release system [161]. Nanochannelrelease of particle payload in this system was achievedby application of an external oscillating magnetic fieldwhose interaction with the magnetic nanoparticlesresulted in thermal energy generation sufficientenough to break the electrostatically bound nanovalvemolecules. The introduction of zinc doping to ironoxide nanocrystals provided a fourfold increase in

hyperthermic effects compared to undoped iron oxidenanocrystals.

To increase the payload capacity of mesoporous silicananotheranostics further, and produce new topologicaldomains for additional functionalization, silica nanor-attles have recently been developed by a number ofinvestigators. These hybrid constructs are generallyinorganic-composite silica structures that consist of aporous silica shell whose hollow interior is filled witha suspension of therapeutics (e.g., hydrophobic andhydrophilic drugs, siRNA, proteins, peptides) and/orcontrast agents (e.g., quantum dots, iron oxide nano-particles, dye-doped silica nanosphere) [162�164].Liu et al. fabricated B150 nm diameter multifunctionalsilica nanorattles that had been encapsulated withinPEGylated, thin, gold nanoshells for a combination ofremotely controlled photothermal therapy (via plasmo-nic heating) and chemotherapy (via docetaxel), asshown in Figure 20.8 [165]. In vitro cell (HepG2) studiesand in vivo (hepatoma H22) small animal studies dem-onstrated the dual therapeutic moiety to possessmarkedly higher therapeutic efficacy and lower sys-temic toxicity than free docetaxel chemotherapy,although not with the hyperthermia-enhanced cytotoxic-ity characteristic of conventional, combined docetaxel-thermotherapy. Zhang et al. developed mesoporousmultifunctional upconverting luminescent and magneticnanorattles for targeted optical imaging and chemother-apy, as described in Figure 20.9 [166]. In this system,nanorattles comprised of porous hydrophilic, rare-earthdoped (Yb,Er) NaYF4 shells, each containing aloose magnetic nanoparticle, were synthesized via anion-exchange process. The region between the magneticcore and the NaYF4:Yb,Er shell was void, making it pos-sible to load the nanorattle with significant quantities oftherapeutic agents. To preclude the leakage of iron inbiologically acidic environments such as endosomes andlysosomes, mesoporous silica was used to encase theconstituent magnetic Fe3O4 nanoparticle cores. After2-photon NIR excitation, the resulting visible lumines-cence enabled nanoplatform localization within tumors,while external magnetic fields were used to position thenanorattles to regions of interest. Incubation of nanorat-tles with human hepatoma cells (QGY-7703), followedby MTT (3�4,5�dimethylthiazol�2�yl)-2,5-diphenylte-trazolium bromide) assay and fluorescence microscopy,showed the nanorattles to have low cytotoxicity andexcellent cell imaging properties. In vivo experimentsyielded substantial tumor shrinkage when nanorattlesvoid spaces were loaded the antitumor drug dox, andthe nanoparticles magnetically navigated toward thepathology.



FIGURE 20.8 Combined nanoplatform for photothermal therapy and chemotherapy. (A) Cross-sectional structure of core and shell inter-face 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 sil-ica nanorattle, and (f) PEGylated gold shell-encased silica nanorattle. (B) Thermal imaging of excised hepatoma 22 (H22) solid tumors underlaser irradiation, showing temperature rise (�C) as a function of exposure time. Source: Reproduced from [165] with kind permission of Wiley-VCHVerlag GmbH & Co. Copyright 2011. All rights reserved.



CONCLUSIONS

Nanotheranostics seek to enhance both diagnosticinsight and treatment outcome through the use of mul-tifunctional, nanometer-sized, organic/inorganic con-structs. Ideally nanotheranostics also enable in vivomonitoring of agent biodistribution and fate, and pro-vide feedback as to disease progression/regression.The easily manipulated, size-dependent physicochem-istries of these nanoplatforms often give rise tounusual physical, chemical, and biological propertiesquite unlike those of bulk material having the samecomposition, with bioactivities that depend heavilyon the environment in which the nanoparticles findthemselves—some therapeutically beneficial, otherspotentially toxic. Silica nanostructures, however, donot acquire any additional or unusual physicochemicalcharacteristics as a result of their diminutive size otherthan a corresponding increase in surface area. Rather,much of the utility of silica nanoplatforms in theranos-tic applications originates from their ease of surfacefunctionalization and ability to form solid, hollow, andporous structures—the last two of which provide evengreater, topologically distinct surface areas for furtherfunctionalization. Silica nanoparticles are also effec-tively “transparent” at UV, visible, NIR, and RF wave-lengths and unaffected by presence of internal orexternal electric or magnetic fields. Moreover, silicananoparticles are easily synthesized, chemically inert,inexpensive, biocompatible and excretable, and hydro-philic, making them especially well suited to biomedi-cal applications, as is evident from the currentliterature [131,167�173].

Despite the enormous number and variety of silicananotheranostics developed to date, a number of funda-mental challenges remain to be addressed prior to theirwidespread clinical use. First, and perhaps foremostamong them, are improvements in nanoplatform target-ing specificity. PEGylation generally reduces reticulo-endothelial system (RES) recognition of nanoplatformssignificantly, and can increase circulation times 2 to10 times that over identical particles that are notPEGylated. However, the paucity of suitable biomarkersfor pathology-specific targeting—the bane of all currentforms of molecular imaging—inevitably results in signif-icant nonspecific binding and/or uptake by the RES,frequently followed by entrapment in the liver, spleen,and bone marrow. Indeed, a significant source of

FIGURE 20.9 Mesoporous upconversion luminescent and mag-netic nanorattles for targeted chemotherapy. (A) Construct synthesisbegan with 20 nm diameter Fe3O4 nanocrystal cores onto which wasgrown a SiO2 shell until B90 nm in diameter to prevent iron dissolu-tion in acidic biological conditions. Particle was then coated with alayer of amorphous Y/Yb,Er(OH)CO3-H2O and heated to derive 20 nmthick Y2O3 cubic phase polycrystalline shell, termed magnetic upcon-verting oxide nanospheres (MUC-O-NS). Ion exchange in the presenceof HF and NaF resulted in a 115 nm diameter, porous α-NaYF4:Yb,Eroptically upconverting shell (20 nm thick, 4.8 nm diameter pores)whose hollow interior retained the iron oxide core encased in a now10 nm thick silica shell, termed magnetic upconverting fluoride nanor-attle (MUC-F-NR). (B) (a) TEM and SEM (inset) of Fe3O4@SiO2 nano-spheres; (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

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



nanoplatform-induced toxicity, aside from that poten-tially inflicted by components of the nanoplatform itself,arises simply from RES accumulation of nanoparticles.In vivo aggregation of nanoplatforms often leads notonly to more probable RES sequestration but also to lossof nanotheranostic functionality and occlusion ofcapillaries in the liver, lungs, kidneys, and spleen.

Dosing is also problematic in the clinical translationof nanotheranostics as the dose typically required fortherapeutic efficacy is generally much higher than thatrequired for a diagnostic effect. For example, a freedrug may need to be delivered at mg/kg of bodyweight levels whereas a radioactive tracer for a PET/SPECT scan requires much less than 1 μg/kg bodyweight. Lastly, and unique to theranostic compounds,is the difference in agent dwell times needed for diag-nosis versus treatment. Although diagnostics andtherapeutics each require highly specific targeting,imaging mandates that the region of interest(i.e., pathology) have markedly higher signal thanthat of surrounding tissue during image acquisition(i.e., contrast). Consequently most conventional con-trast agents for imaging are designed to clear from theblood as quickly as possible, usually within minutes tohours. Rapid clearance of contrast agents also enablesperfusion (dynamic) study of the pathology andpermits earlier “follow-up” imaging. A therapeuticapproach, however, demands that the nanotheranosticshave longer circulation times, to permit not only accu-mulation at the pathology but for drug release as well.

Notwithstanding such seemingly formidable impedi-ments, silica-based nanotheranostics, with their intrinsi-cally high biocompatibility and tunability of both formand function, are especially well positioned amongnanomaterials to succeed in accommodating the numer-ous platform-pathology specific optimizations required.

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cancer theranostics || silica nanoparticle platform

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