Nanoscale

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Cite this: DOI: 10.1039/c5nr05264e

Received 4th August 2015,Accepted 17th October 2015

DOI: 10.1039/c5nr05264e

www.rsc.org/nanoscale

Pharmacokinetics, pharmacodynamics andtoxicology of theranostic nanoparticles

Homan Kang,†a Shrutika Mintri,†b Archita Venugopal Menon,b Hea Yeon Lee,c

Hak Soo Choi*a and Jonghan Kim*b

Nanoparticles (NPs) are considered a promising tool in both diagnosis and therapeutics. Theranostic NPs

possess the combined properties of targeted imaging and drug delivery within a single entity. While the

categorization of theranostic NPs is based on their structure and composition, the pharmacokinetics of

NPs are significantly influenced by the physicochemical properties of theranostic NPs as well as the

routes of administration. Consequently, altered pharmacokinetics modify the pharmacodynamic efficacy

and toxicity of NPs. Although theranostic NPs hold great promise in nanomedicine and biomedical appli-

cations, a lack of understanding persists on the mechanisms of the biodistribution and adverse effects of

NPs. To better understand the diagnostic and therapeutic functions of NPs, this review discusses the

factors that influence the pharmacokinetics, pharmacodynamics and toxicology of theranostic NPs, along

with several strategies for developing novel diagnostic and therapeutic modalities.

1. Introduction

Nanoparticles (NPs) possess a relatively small size in the nano-range (1–1000 nm),1 but have a significant advantage overatoms and molecules owing to a larger surface area per unitvolume. NPs also have a greater formulating flexibility forvarious sizes and shapes with different chemical surfacetraits.2 Due to their versatile nature, they have been success-

Homan Kang

Homan Kang is currently a post-doctoral research associate inthe Department of Medicine atHarvard Medical School andBeth Israel Deaconess MedicalCenter, Boston, MA, USA. Hereceived his BS degree (2007) inPolymer Science and Engineeringfrom Dankook University and hisPh.D. degree (2014) in the Inter-disciplinary Program in Nano-Science and Technology fromSeoul National University underthe guidance of Prof. Yoon-Sik

Lee. Dr Kang extended his research into molecular imaging andjoined the Choi Laboratory at BIDMC in 2015. His currentresearch focuses on the development of novel targeted contrastagents and polymer-based nanocarriers for theranostic drugdelivery.

Shrutika Mintri

Shrutika Mintri is a graduatestudent, pursuing her Master’sdegree in Pharmacology at BouveCollege of Health Sciences,Northeastern University, Boston,MA, USA. She completed herBachelor’s degree in Pharmacyfrom Jawaharlal Nehru Techno-logical University, India and iscurrently working at Dr JonghanKim’s laboratory. Her research isfocused on the effects of heavymetals like iron on seleniummetabolism in the brain.

†These authors contributed equally.

aDivision of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess

Medical Center and Harvard Medical School, Boston, MA 02215, USA.

E-mail: hchoi@bidmc.harvard.edu; Fax: +617-667-0214; Tel: +617-667-6024bDepartment of Pharmaceutical Sciences, Northeastern University, Boston, MA

02115, USA. E-mail: j.kim@neu.edu; Fax: +617-373-8886; Tel: +617-373-3214cDepartment of Nanotechnology, Detroit R&D, Inc., Detroit, MI 48201, USA

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fully used as both diagnostic and therapeutic tools.3 “Therano-stics” refers to the development of compounds, whichexhibit the characteristics of diagnostics and therapeutics in asingle entity.1,4 The rapid advancement in nanotechnology hasallowed the emergence of any diagnostic or therapeutic NPs,which have shown advantages of diagnosis and drug delivery aswell as targeting the biomarkers of the disease at the molecularlevel.5 For clinical use, however, the size of a NP has to belimited to 220 nm because a standard 0.22 μm (220 nm) filter isused routinely in the clinic before injecting theranostic agentsinto the body. The National Nanotechnology Initiative (NNI) alsodefines “nanomaterials” as (1) research and technology develop-ment at the atomic, molecular or macromolecular levels, on thelength scale of approximately 1–100 nm range; (2) creating andusing structures, devices and systems that have novel propertiesand functions because of their small and/or intermediate size;and (3) ability to control or manipulate at the atomic scale.2,6

Although theranostic NPs hold great promise in nano-medicine and biomedical applications, a lack of understand-ing persists on the mechanisms of biodistribution and adverseeffects of NPs. An ideal theranostic NP model should possessseveral important properties. For drug delivery, NPs should acton the target tissues and demonstrate appropriate release kine-tics of the drug in optimum concentrations at the site ofaction, illustrating their efficient therapeutic potency. Since italso possesses diagnostic abilities, it should help to determinethe precise location and characteristics of the disease. Alongwith these properties, it is very important that the NP shouldbe non-toxic and easily excretable or eliminated from thebody.4 There have been several reviews providing an in-depthoutlook on the potential of NPs and their applications inseveral aspects, such as their usage as theranostic agents indrug delivery5 and the application of theranostic NPs in cancer

therapy,7,8 which is one of the most rapidly developing thera-pies involving nanosystems. Recognizing that the in vivo avail-ability and efficacy of NPs are mainly determined by theirpharmacokinetics (PK) and potential toxicity, we provide abrief review of these facets of theranostic NPs.

2. Backbone materials of theranosticNPs

NP-based theranostics is considered a promising future ofnanomedicine because NPs can possess several unique fea-tures including targeting, imaging (diagnosis), and therapeuticpotentials within a single nanoplatform. In contrast to smallmolecules, theranostic NPs can be tuned for optical, electrical,magnetic and biological properties and can carry large pay-loads along with contrast agents.3

The backbone materials can be categorized into two classesbased on their compositions: organic vs. inorganic materials,and their key characteristics are summarized in Table 1. Inorganic nanomaterials, synthetic polymers and biopolymersincluding dendrimers, lipoproteins and liposomes have beenoften utilized for targeted drug delivery in the past fewdecades. Organic nanomaterials have biocompatibility thatallows them to functionalize with targeting moieties on theirsurface; however, they usually need a complexation andcovalent conjugation of contrast agents. On the other hand,many inorganic nanomaterials, especially being led from thedevelopment of superparamagnetic iron oxide nanoparticles(SPIONs) and quantum dots (QDs), have been intensivelystudied and already developed as core imaging materials.Consequently, such inorganic nanomaterial-based theranosticNPs can be easily prepared by loading therapeutic drugs onto

Archita Venugopal Menon

Archita Venugopal Menon is cur-rently pursuing her Master’sdegree in Pharmacology atNortheastern University and isworking as a graduate researcherat Dr Jonghan Kim’s laboratory.She received her Bachelor’sdegree in Pharmacy from theUniversity of Mumbai. Hercurrent research interest includesthe elucidation of genetic andnutritional heavy metal toxicitywith a focus on neurobehavioraldysfunction.

Hea Yeon Lee

Hea Yeon Lee is a Director ofNanotechnology in Detroit R&D,Michigan, USA. She received herBS (1987) and MS degrees(1990) in Chemistry fromPukyong National University,South Korea and her Ph.D.degree (1995) in Chemistry fromOsaka University, Japan. Afterfinishing advanced degrees innanofabrication and characteriz-ation technologies, she has beenworking on developing newnanobioelectronic devices and

nanobiosensors. She was a Designated Professor at the Institute ofScientific and Industrial Research, Osaka University, and ResearchAssociate Professor of Mechanical and Industrial Engineering atNortheastern University, Boston, USA. Her research work has beencontributing to accelerating cutting-edge research in the emergingbio-nanoscience area.

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and/or into the NP’s surface.1 However, the inorganic andcarbon-based materials are generally required to modify theirsurface with a biocompatible organic coating due to poorwater solubility, stability, and potential toxicity (Table 1).

Regardless of their compositions, all theranostic NPs mustbe designed to have a reasonable half-life in blood, selectivetargetability, and effective elimination from the body aftercomprehensive delivery to the target site.2,37–39 To acquire

Hak Soo Choi

Hak Soo Choi is Associate Pro-fessor of Medicine at HarvardMedical School, and faculty ofHarvard Medical Faculty Phys-icians (HMFP) at Beth IsraelDeaconess Medical Center(BIDMC) and Dana Farber/Harvard Cancer Center (DF/HCC). He is an expert on engin-eering nanoparticles and bio-imaging, trained in the fields ofmedicinal chemistry and nano-medicine for drug delivery, andhas published over 100 papers in

the field. Since 2008, his laboratory at BIDMC focuses on thedevelopment of novel targeted contrast agents to solve importantproblems in oncology and clinical medicine, with an emphasis onin vivo imaging and tissue-specific contrast agent development.

Jonghan Kim

Jonghan Kim is an Assistant Pro-fessor in the Department ofPharmaceutical Sciences at theNortheastern University, Boston,Massachusetts, USA. He receivedhis BS and MS degrees in Phar-macy from Seoul NationalUniversity, South Korea and hisPh.D. degree in Pharmaceuticsfrom Ohio State University,Columbus, Ohio, with a studyfocused on protein pharmaco-kinetics. During his postdoctraining at the Harvard School

of Public Health, Boston, he investigated the transport mecha-nisms of metals through the olfactory mucosa and the brain. Hisresearch interests center around the characterization of toxico-kinetics and toxicodynamics of drugs and metals in the context ofgenetic susceptibility.

Table 1 Pre-clinically available theranostic NPs2

Class NP type Composition Therapeutic modality Pros Cons Ref.

InorganicNPs

MagneticNPs

Iron oxide Chemotherapy; siRNA;magnetic hyperthermia

Intrinsic MRI contrast;thermal therapeutic agent

Interference in imaging 9–11

QDs Semiconductor Chemotherapy; siRNA;photodynamic therapy

Broadband absorption; smallsize; tunable emission band

High potential toxicity 12

Silica NPs Mesoporous silica Chemotherapy; siRNA Multi functionality; facilesynthesis; solubility

Stability; need contrastagents

13

Carbon NPs Graphene oxide Photothermal therapy;photodynamic therapy;chemotherapy

Large surface area; thermaltherapeutic agent

Size control; difficulty inpurification

14–16

Carbon nanotube Size tunability; mechanicalstrength

High aspect ratio;difficulty inpurification; poorsolubility

17–19

Gold NPs Gold nanoshell Photothermal therapy;chemotherapy

Size tunability; intrinsicthermal therapeutic agent;tunable in NIR region

Potential toxicity 20

Gold nanorod Photothermal therapy High aspect ratio;toxicity; difficulty intherapeutic payload

21,22

Others CuS NPs Photothermal;chemotherapy

Thermal therapeutic agent;tunable in NIR region

Potential toxicity 23,24

MoS2 nano-sheet Photothermal;chemotherapy; siRNA

Large surface area; thermaltherapeutic agent

Need contrast agents;difficulty in size control

25,26

OrganicNPs

BiologicalNPs

Naturally polymersand lipoprotein

Chemotherapy; siRNA Biocompatibility;biodegradability

Need contrast agents;difficulty in size anddegradability control

27,28

PolymerNPs

Linear or branchedpolymer

Photodynamic therapy;chemotherapy

Biodegradability; flexibility;size tunability

Need contrast agents 29–31

Dendrimers Tree-likemacromolecules

Chemotherapy Size tunability; solubility Limited synthesis; needcontrast agents

32,33

Liposomes Phospholipidbilayers

Chemotherapy; siRNA Conventional drug delivery;large payload

Need contrast agents;poor stability

34–36

Abbreviations used are: MRI, magnetic resonance imaging; NIR, near-infrared; NP, nanoparticle; QD, quantum dot; siRNA, small interfering RNA.

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these desired pharmacokinetic behaviors of NPs for clinicaluse, it is necessary to modulate the hydrodynamic diameter(HD), shape, composition, and surface characteristics of NPsbased on the “Choi Criteria” (Fig. 1).2 For instance, the overall

HD of theranostic NPs is required to be <5.5 nm for renalclearance after complete targeting in order to achieve highsignal-to-background ratio.40 In the following section, wediscuss more details about the physicochemical properties oftheranostic NPs in terms of size, shape, surface, compositionand route of administration.

3. Pharmacokinetics of NPs

The physicochemical properties of theranostic NPs are of sig-nificant importance in modulating PK because they determineimmediate pharmacological response in the body when theNPs are administered. Drugs with low bioavailability can havebetter drug dissolution rates by the technique of “nanosizing”a drug formulation, which would promote increased absorp-tion of the drug.41 Also, NPs can prolong the half-life of drugsin blood circulation, which would otherwise be rapidly clearedor degraded. Since PK plays a major role in determining thetherapeutic efficacy and toxicity of the administered NPs,several key factors influencing the PK of NPs (Fig. 2) are dis-cussed. In this section, we avoid reticuloendothelial system-mediated NP clearance and focus on smaller NPs and theirtheranostic aspects because larger NPs have slim chances of

Fig. 1 Schematic illustration of a theranostic NP and its physico-chemical properties that regulate in vivo pharmacokinetics, biodistribu-tion and toxicity.

Fig. 2 Factors influencing the pharmacokinetics of nanoparticles (NPs). Size, shape, composition, administration route, and surface modificationshould be considered for the enhanced targeted therapy mediated by NPs.

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clinical translation. As previously reported, renal excretion is apreferred and desirable pathway for theranostic NPs comparedwith hepatic clearance because the NPs can be rapidly elimi-nated from the body while little cellular internalization/metab-olism is involved, thus effectively minimizing body exposure tothe NPs.40,42–44

3.1 Size and shape

The ability of the NPs to enter the cell is determined by bothphysicochemical parameters and biological barriers. Due tothe high surface area to volume ratio (small size), they are ableto cross the biological barriers by penetration through the cellmembrane45 and deliver the drug inside the cell. It has beenfound that a general size range of 10–12 nm is ideal and offershigh permeation and minimal accumulation in tissues.46

Choosing a suitable size in designing NP is also essential as itdirects which excretion pathway the drug would follow. Forexample, particles with a smaller HD of <5.5 nm follow theroute of renal excretion,2 whereas larger sized NPs are elimi-nated through the liver.40 Also, choosing an appropriatecarrier is of high significance. NP systems, such as liposomesand organic/inorganic hybrid nanospheres, have been used inprecise targeting of various diseases with intravenous deliveryof theranostic small molecules. The drug-loaded carrier cancontrol the efficiency of drug delivery and also protect the drugfrom inactivation and/or degradation, which can reduce itsadverse effects.47 The cellular uptake is also influenced by theshape of the NPs; for example, elongated NPs are betterabsorbed than spherical ones.48

3.2 Surface properties

A modification in the surface of NPs significantly affects thephysical, chemical and biological nature of the entire moleculein biological systems.

NPs can induce positive/negative charges on the surfacewhere the interactions with the cell membrane change in adifferent manner, which affects their absorption and distri-bution. NPs with a positively-charged surface show greateruptake than negatively-charged NPs due to electrostatic inter-actions.48 Alteration of the NP surface with a neutral non-ionicpolymer imparts stability to the NP by decreasing opsonisationand increasing blood circulation time, as exemplified by NPscoated with polyethylene glycol (PEG) on their surface.48

Surface properties also plays an important role when the NPsare placed in biological fluids (e.g., blood). The surface of NPsis coated with a layer of proteins (protein corona) when incontact with the biological fluid.48 This layer plays a vital rolein determining the attraction of the NP to the cell membrane.Different NPs form different protein coronas and thus, eachtype of NPs has different affinities for a particular protein in abiological fluid and affects the physicochemical character-istics, which would subsequently affect the rate and extent ofbiodistribution.

3.3 Administration route

The PK of a drug from a NP depends upon the route by whichthe drug has been administered, which modifies pharmaco-logical efficacy of the drug. For instance, when bovine insulinwas administered orally by means of a pH-responsive NPsystem of chitosan and poly(γ-glutamic acid) to rats, it showeda greater bioavailability compared with subcutaneously-injected insulin in diabetic patients.49 These oral NPs infiltratethe mucous layer of the intestinal tract and gradually destabi-lize and disintegrate due to their pH sensitivity. The increasedbioavailability may be attributed to the pH-sensitive insulinrelease from the NPs. The difference in biodistribution ofinsulin and prolonged reduction of glucose levels betweensubcutaneous insulin and oral NPs could be because insulin,via the oral route, mimics the physiological pathway of theendogenously secreted insulin, which reaches the liver andhelps to control the glucose levels in the body. In contrast, theinsulin administered by the subcutaneous route fails to mimicthis since it enters the peripheral circulation, which is not thenormal route of insulin production and secretion.

There are several publications concerning the effect ofinjection routes on the biodistribution and elimination ofNPs.50–52 Very recently, Huang et al. reported the biodistribu-tion, clearance and tumor uptake of renally clearable carbondots with three different injection routes, including intra-venous, intramuscular and subcutaneous administrations.53 Theblood clearance and urinary accumulation rate of administeredNPs followed the order of intravenous > intramuscular > sub-cutaneous injections. In addition, tumor uptake of carbondots by subcutaneous and intravenous injections was higherthan that by intramuscular injection. Such examples areindicative of the route-dependent therapeutic potential andclinical benefits of NP-based theranostic systems. Absorption,biodistribution, elimination and pharmacologic and toxiceffects of NPs following different routes of administration aresummarized in Fig. 3.

3.4 Composition

Many therapeutic NPs are composed of several differentelements with specific geometry/conformation such as core–shell, core–satellite, linear, and hyper-branched structures.The small differences in geometry or conformation can con-tribute to their in vivo performance such as absorption, bio-distribution, elimination as well as targeting ability.2 In addition,the geometry/conformation changes of NPs and decompositionin in vivo environments can significantly affect toxicity.

The biodegradability of theranostic NPs relies on theirchemical compositions. Polymeric NPs containing hydrolys-able linkages, such as ester, ortho-ester and anhydride, in theirbackbones are biodegradable in the body.54 The use of bio-degradable polymers can significantly increase the eliminationof NPs from the body and reduce long-term toxicity. On thecontrary, most inorganic NPs are not biodegradable. Such in-organic NPs remain for a relatively long period of time in thebody due to their larger size and greater hydrophobicity com-

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pared with small molecules, therefore, concerns have beenraised about the potential long-term toxicity of these NPs.

Taken together, both physicochemical properties (i.e.surface charge, chemistry and size of the NP) and exposureroutes are critical factors that determine the PK of NPs,41 andthese factors can be modified to control (enhance or decrease)the blood circulation and tissue permeation of the drug. Onthe other hand, poorly designed NPs can promote anenhanced delivery of the drug molecules to certain non-targettissues non-specifically and cause undesirable side effects,which warrant the appropriate assessment of toxicity for theuse of NPs.

4. Imaging and therapeuticmodalities

The selection of imaging modality is another important com-ponent for theranostic NPs. The use of minimal or non-inva-sive imaging modality is beneficial to characterize the PK andbiodistribution as well as therapeutic efficacy of theranosticNPs.55 Current clinically available imaging modalities includenuclear imaging (positron emission tomography; PET, andsingle photon emission computed tomography; SPECT), mag-netic resonance imaging (MRI), computed tomography (CT),ultrasound (US), optical imaging, and photoacoustic (PA)imaging (Table 2). However, for efficient molecular imaging,the surface or core of NPs should be modified with variousradioisotopes, paramagnetic ion chelates, or fluorophores,except in the case of using inherent contrast NPs such as iron

oxide NPs, QDs, and dye-doped silica NPs. Based on the intrin-sic sensitivity and tissue penetration ability of imaging modal-ities, the theranostic NPs could be visualized via an non-invasive (more desired) or minimally invasive (less desired)manner in diagnostic procedures. The pros and cons of eachimaging modality are summarized in Table 2. In this section,we describe therapeutic modalities of NPs.

4.1 Chemotherapy

Since cancer is one of the leading causes of death worldwide,NP-based cancer therapy has great potential for overcomingbiological barriers and selective targeting to desired sites.56

Furthermore, NPs are relatively small and have greater affinityfor the cell membrane, thus can easily enter the cancer cellsafter binding to the cell surface specifically through targetingligands, which decreases non-specific biodistribution and tox-icity in non-target organs.57

Polymeric NPs like liposomes and micelles have been usedto solubilize hydrophobic drugs so that a higher percentage ofinjected dose (%ID) can be achieved at the target site. Doxil,for example, is a PEGylated liposome coated on doxorubicin(DOX), where the PEG coating prevents the degradation ofdrugs by the immune system and controls the release of drugsinto the blood, resulting in a prolonged terminal half-life andhigher drug efficacy.57,60 Another example is a QD–aptamer–DOX conjugate [QD–Apt(DOX)] for prostate cancer therapy.The QD–Apt(DOX) conjugate can perceive and render DOX atthe target site by using the fluorescence resonance energytransfer (FRET) effect between DOX and QDs. The conjugate iscomposed of the following 3 parts; (1) therapeutic DOX, (2) tar-

Fig. 3 The pharmacokinetics, pharmacodynamics and toxicity of theranostic NPs. Shown are absorption, biodistribution, elimination and pharma-cologic and toxic effects of NPs following different routes of administration. The fate of the theranostic NPs depends on physicochemical propertiesof NPs and the route of administration as well as altered body functions (e.g., nutrition status and disease conditions).

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geted RNA aptamers, which are covalently attached onto thesurface of QDs, and (3) diagnostic QDs for fluorescenceimaging. This activatable system works by turning “on” thefluorescence by releasing DOX in the tumor cells, while theDOX-loaded QD–Apt is “off” in the normal cells.61

4.2 Gene therapy

Gene therapy implies the replacement of a faulty gene in thecell with a proficient gene or by overexpression or silencing ofa gene by introducing foreign DNA and modifying the cellularsignalling.62 NPs have a capability to replace viral vectors asthey are small in size and therefore can communicate withmany biological moieties like cytokines and proteins. Althoughthey possess some drawbacks, such as inefficient transfectingefficiency, these can be overcome by chemical modification ofthe functional groups.63 Magnetic NPs have been used in genetherapy by intercalation of the functional gene with the SPIONand its effective transfection into the desired cell by highgradient magnets. Morishita et al. demonstrated that magneticNPs, with cell fusion vectors hemagglutinating virus of Japanenvelope with protamine sulfate magnetic NPs, showed asignificantly improved transfection efficiency in the presence of amagnetic source along with reduced toxicity in BHK21 cells.64,65

4.3 Thermal therapy

NPs can be used in thermal therapies such as photothermalablation and magnetic hyperthermia, due to unique surfaceplasmon resonance or magnetic susceptibility of NPs.66 Forinstance, the magnetic NPs continuously emit heat via Néeland Brownian relaxation pathways upon exposure to the alter-nating external magnetic field.9 Noble metal nanostructuressuch as nanorods,21,67,68 multi-branched particles,69,70 nano-shells,71,72 and hollow-shells73,74 have been used for photo-thermal therapeutic applications with non-invasive therapy.

Stern et al. have recently demonstrated an NIR activated goldnanoshell as a preclinical treatment modality-which comple-tely enabled photothermal destruction of human prostatecancer in a xenograft model.75 These NPs can be utilized aspromising drug carriers for thermally triggering drugs as wellas thermal therapy.

4.4 Photodynamic therapy

Photodynamic therapy is used for selective destruction ofcancer cells and tissues by utilization of photosensitizers.When external light excites the photosensitizers in the pres-ence of oxygen molecules, the photosensitizers produce toxicsinglet oxygen species, which lead to cell death.76 Tsay et al.designed peptide-coated QD–photosensitizer conjugates usingrose bengal and chlorin e6 photosensitizers.77 These photo-sensitizers were covalently bound to the peptide-overcoatedgreen and red CdSe/CdS/ZnS QDs. The production of singletoxygen was enhanced by direct or indirect activation of thephotosensitizers. Upon activation, these photosensitizerscould perform both imaging and therapeutic activities.

5. Theranostic applications andpharmacodynamics of NPs

The application of theranostic NPs has probably been mostsuccessfully implemented in cancer research. For theranosticand clinical applications, however, most NPs should have inertsurface coatings with organic polymeric and/or biologicalmaterials as discussed above. In addition, selective targeting isan essential property to overcome one of the limitations ofconventional therapy and to minimize potential side effects.There are two major approaches for efficient tumor targeting:in passive targeting, therapeutic NPs reach the tumor site

Table 2 Minimal- or non-invasive imaging modalities for theranostic NPs5,58,59

Modality Probe(s) Pros Cons

Nuclear imaging:PET, SPECT

Radionuclides (e.g. F-18,In-111, Cu-64)

Quantitative analysis Radioisotope exposureHigh sensitivity Expensive procedures

MRI Paramagnetic atoms(e.g. Gd, Mn)

Able to image physiologicaland anatomical details

Limited acquisition time

Superparamagnetic NPs(e.g. SPION)

Soft tissue contrast High cost

CT Heavy elements(e.g. iodine)

Quantitative anatomicalinformation

Radiation exposure

High spatial resolution Limited to morphologicalinformation

Ultrasound imaging Gas filled microbubbles Ease of procedure Low resolutionLow cost Low sensitivity

NIR imaging Fluorophores(e.g. fluorescence dye, QD)

High sensitivity andspatial resolution

Limited penetration(<5 mm)

Low autofluorescenceCost efficiency and simplicity

Photoacoustic imaging Light absorbates(e.g. fluorophore, quencher)

High spatial resolution Limited penetration(<5 cm)Functional information

Abbreviations used are: PET, positron emission tomography; SPECT, single photon emission computed tomography; MRI, magnetic resonanceimaging; NP, nanoparticle; SPION, superparamagnetic iron oxide nanoparticle; QD, quantum dot.

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through leaky endothelium surrounding tumor tissues(enhanced permeability and retention (EPR) effect). In con-trast, active targeting is based on targeting ligands, such asantibody, aptamer and peptide, on the NP surface which allowNPs to bind to the receptors overexpressed on cancer cells. Inthis section, we introduce several advanced examples oftheranostic nanoplatforms.

5.1 Synthetic polymer NPs

Photosensitizer-conjugated amine functionalized polyacryl-amide NPs synthesized by an oil-in-water microemulsion tech-nique have been reported by Kopelman and co-wokers.54 Fortumor-specific targeting, the surface of NPs was modified withcell-permeable peptide and biologically inert PEG. Oncefluorophore-embedded NPs enter the tumor, the fluorescencedye lights up the tumor cells and the drug is photosensitizedby irradiation, which specifically kills the cancer cells. Inaddition, Liu et al. reported polyelectrolyte-based polyprodrugswhich possess imaging, chemotherapeutic and photodynamicproperties.31 The NPs were covalently conjugated to doxo-rubicin through a reactive oxygen species (ROS) cleavablelinker. PEGylated polyelectrolytes efficiently produce ROS

under light irradiation, which then not only kill the cancerouscells by photosensitization but can also release doxorubicinfor chemotherapy. Light-triggered chemotherapy and photo-dynamic therapy have been combined to produce betterresults to cure cancers with synergistic advantages such asovercoming multiple drug resistance and improved therapeuticefficacy.31

5.2 Biological NPs (naturally derived polymers)

The self-assembled micellar nanocomplex (MNC) has beendeveloped for delivery of protein drugs. Chung and Kurisawa’sgroup reported that simple sequential self-assembly of the epi-gallocatechin-3-O-gallate (EGCG) derivative, a major ingredientof green tea, with anticancer proteins leads to the formation ofa stable micellar nanocomplex.78 The anticancer effect of theHerceptin-loaded micellar nanocomplex (Herceptin–MNC) wasinvestigated in vitro and in vivo and compared with those ofbovine serum albumin (BSA)–MNC and free Herceptin. Her-ceptin–MNC exhibited a 2.3-fold greater accumulation in thetumor site, 29-fold longer blood half-life, and significantlyhigher anticancer effect in the tumor in comparison with freeHerceptin (Fig. 4).

Fig. 4 (a) Schematic diagram of the self-assembly process used to form the micellar nanocomplexes, which are formed via two sequential self-assemblies in an aqueous solution: complexation of OEGCG with proteins to form the core, followed by complexation of PEG–EGCG surroundingthe pre-formed core to form the shell. (b) Anticancer effect on BT-474-xenografted nude mouse model. PBS (vehicle control, open circles), BSA–MNC (open triangles), Herceptin (2.5 mg kg−1, open squares), sequential injection of BSA–MNC and Herceptin (filled inverted triangles) and Hercep-tin–MNC (filled circles). (c) Real-time intraoperative tumor detection and NIR fluorescence image-guided resection at 24 h post-injection. Arrowsindicate nonspecific uptake (liver, kidneys, intestine). The red dashed circle delineates the region of interest. Abbreviations used are: BSA, bovineserum albumin; EGCG, Epigallocatechin-3-O-gallate; MNC, micellar nanocomplex; OEGCG, oligomerized EGCG; PEG, polyethylene glycol; T (+), posi-tive tumor. Reprinted with permission from ref. 78. Copyright 2014 Nature Publishing Group.

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5.3 Mesoporous silica NPs

Mesoporous silica NPs have been used successfully in cancertherapy, mainly because of a large surface area and porevolume, and ease of surface modification.13,79 Recently, mag-netic NPs or gold NPs were embedded into mesoporous silicaNPs for thermally triggered drug release. An anticancer drugwas loaded into porous cavities of mesoporous silica NPs andporous structures were capped with thermally releasable mole-cules. When external stimuli, such as magnetic field and NIRlaser, are applied to these mesoporous silica nanoplatforms,drug release can be controlled precisely. This controlledrelease behavior is a very important feature in target specifictherapy as it can overcome the side effects of a conventionaldrug delivery system.

5.4 Magnetic NPs

Another example of theranostic NPs in cancer therapy is theuse of magnetic NPs (MNPs). MNP-based theranostics can bedivided into three ways in terms of therapeutic methods: (1)hydrophobic drug or gene delivery, (2) thermal therapy in amagnetic field, and (3) magnetic/mechanical controlling incell signalling. Theranostic MNPs normally contain a super-paramagnetic iron oxide core, which is used for MRI to detectthe tumor, covered by a hydrophilic surface coat on theoutside, and have been linked with an anticancer drug orsiRNA to treat the tumor.80 As one of the key examples, Mooreand co-workers have reported dextran-coated SPIONs forin vivo siRNA delivery.11 The amine-dextran coated SPIONs were

labelled with Cy5.5 dye for simultaneous optical imaging, andcovalently linked to thiolated siRNA duplex and myristoylatedpolyarginine peptides, which are membrane translocationmodules, for intracellular delivery. This study showed advance-ment of siRNA delivery and silencing with imaging strategies.MNPs can be also developed by conjugating chemotherapeuticdrugs on the surface of NPs to target and treat cancers. Leeet al.81 have also developed a nanocarrier containing MNP con-jugated to the anticancer drug gemcitabine. These NPs deliverthe drug by receptor-mediated endocytosis to its target, uro-kinase plasminogen activator receptor and also allows in vivoMRI of the tumor.81

Magnetic thermal therapy utilizes heat induced from MNPsin an external high frequency alternating magnetic field,which allowed the control of heat generation after specific tar-geting to the tumor region of interest.82 Although external trig-gering is one of the advantages in magnetic field inducedthermal therapy, the efficacy is limited even with high concen-trations of therapeutic MNPs. Very recently, to control cell sig-nalling, a magnetic switch method has been developed byusing zinc-doped iron oxide MNPs.83 The thiolated MNPs wereconjugated with antibody for targeting death receptor 4 (DR4)of DLD-1 colon cancer cells. When a magnetic field is appliedto MNP bound DR4s on DLD-1 cells, clustering of DR4s wasformed and apoptosis signalling pathways were induced. Forin vivo apoptosis experiments, the magnetic switch methodwas applied to zebrafish and apoptotic morphology changes ofzebrafish could be observed in the magnetically activatedgroup (Fig. 5).

Fig. 5 A schematic representation of the magnetic switch for apoptosis signalling in in vitro cells and zebrafish. MNPs first bind to the death recep-tors, and subsequent aggregation on the application of a focused magnetic field triggers extrinsic apoptosis signalling. Magnetic switching of deathreceptor clustering results in the death of cells and also causes morphological changes in zebrafish. Reprinted with permission from ref. 83. Copy-right 2012 Nature Publishing Group.

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6. Toxicity of theranostic NPs

Although the development of NPs has advanced the field ofdrug delivery and nanomedicine, it is relatively new to otherforms of pharmaceutical formulations. Moreover, certainmechanisms by which nanosystems can lead to toxicity havenot been fully characterized in a proper manner,84 likelybecause there are only a small number of nanomaterial drugsapproved by the FDA.2 The toxicity of NPs depends on variousconditions, including not only physicochemical properties ofNP (e.g. HD size, shape, surface charge, and chemical compo-sition), but also the physiological status (e.g. genetics, diseaseconditions). For example, patients with coronary artery dis-eases are more prone to heart attack when they are subjectedto NP therapy.85 Although small size and large surface area aretwo of the unique properties making NPs popular, these pro-perties also significantly affect toxicity.40 Since NPs are smallerin size than cells and cell organelles86 and they possess thepotential to penetrate into these cellular structures in severalorgans by circulatory, nervous and lymphatic systems, they candisrupt physiological functions and promote tissue inflam-mation, abnormal cell functioning or even cell death.46

Nanomaterials, involving dendrimers, cationic polymers,QDs, magnetic and metallic NPs, and carbon based materials,have been widely used as biosensors, contrast agents, anddrug carriers.87 However, the toxicity of these NPs is still aproblem for in vivo applications. Metallic NPs (e.g. silver, gold)can enter the cells either by endocytosis or diffusion due totheir relatively small size. Upon their entry into the cell, theyreach mitochondria and can impair the mitochondrial func-

tion by disturbing the electron transport chain, resulting inoxidative stress. Moreover, these metallic NPs can generateROS, which enter the nucleus and cause oxidative stress,leading to DNA damage by cross-linking or formation of DNAadducts. These ultimately promote cell death. The ROS canalso cause protein oxidation and lipid peroxidation, whichgenerally inhibit cell adhesion and proliferation. Silica NPsreduce levels of antioxidant glutathione as well as cause oxi-dative stress via ROS production, resulting in DNA damage. Inaddition, silica NPs can induce the formation of proteinagglomerates and inhibit the cell growth. QDs can causetoxicity by disruption of mitochondrial function, which leadsto DNA damage.88 SPIONs can release iron, which can betaken up by cells through iron transporters or SPIONs them-selves can be endocytosed into the cell and then release ironby lysosomal degradation of the SPION. The intracellular ironcan mediate the Fenton reaction to produce ROS, which entersthe nucleus and damages DNA.89 Furthermore, iron canaccumulate in tissues and cause iron-related toxicity.90–93

Carbon-based nanomaterials (e.g. carbon nanotubes and gra-phene) are known to interfere with the cell signaling andcause cytotoxicity.94 Toxicity mechanisms of different types ofNPs are summarized in Fig. 6. In this section, we describemore examples of NP toxicities and discuss the relationshipbetween toxicity and physicochemical properties of NPs.

6.1 Toxicity of cationic lipid and polymer NPs

Cationic lipids and polymers as non-viral vectors have beendeveloped for gene delivery because of their relative safety,capacity to transfer large genes, site-specificity and their low

Fig. 6 Toxicity mechanisms of different types of NPs.

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immune response.95 However, toxicities of cationic lipids andpolymers in gene vectors are still an obstacle to the applicationof non-viral vectors to gene therapy. The cytotoxic effect isassociated with the cationic nature that can interact with criticalenzymes such as protein kinase C.96 To overcome toxicity pro-blems, degradable polymers in a low pH environment havebeen developed. For example, the toxicity of low molecularweight polyethylenimine polymers is found to be reducedbecause of the degradation of acid-labile linkage.96,97 Inaddition, the PEGylated polycomplex shows a marked decreasein toxicity compared with the non-PEGylated cationic polymer.98

6.2 Toxicity of metallic NPs

The in vivo use of heavy metals is debatable with regard tosafety concerns. Divalent metal ions even in small concen-trations are toxic as they accumulate in kidneys andbecome nephrotoxic. Cadmium and selenium are two metalschiefly used in making the core of QDs and these metals arerelated to moderate toxicities in vertebrates. It has beenreported that, under oxidative stress, cadmium ions arereleased and bind to the sulfhydryl group in the mitochondriaand cause a loss in their function, leading to cell poisoning.99

Metallic NPs (e.g. gold NPs) have been frequently used for diag-nosis and therapy. They offer several advantages, such as lowtoxicity, better photo-stability, and surface-enhanced and dis-tance-dependent spectroscopic properties.100 Although goldNPs are thought to possess low toxicity, certain forms like thecationic gold nanospheres could exhibit moderate cellular tox-icity, whereas their anionic counterparts are non-toxic. The tox-icity of the cationic form could be explained by the interactionwith the negatively charged membrane, leading to distortionof the cell membrane.101 Silver NPs have also shown to causeoxidative stress in the brain94 and a few other tissues likeliver102 by reducing the respiratory chain complexes I, II, IIand IV of mitochondria.94 These particles can also inducenecrosis and apoptosis.103 SPIONs are composed of a hema-tite, magnetite or maghemite core which is coated withorganic or inorganic polymers. And, SPIONs have a uniqueproperty called superparamagnetism in which each magneticparticle has a distinct magnetic moment and acts like a super-paramagnetic atom and spontaneously responds to externalmagnetic fields.104 These NPs have been actively used asimaging tools in MRI, in targeted drug delivery, and inducedhyperthermia cancer therapy. However, these particles also haveseveral concerns about toxicity because they can generate a hugeamount of free radicals due to redox cycling at the surface of par-ticles. The ROS can promote oxidative stress and interfere withthe cellular functioning. Furthermore, since SPIONs containiron, they can lead to excessive accumulation of iron at the targetsite when the SPIONs are administered in excess, which can leadto metal toxicity related to iron overload in the target tissue.105

6.3 Toxicity of carbon based nanomaterials

Carbon nanotubes (CNTs) are tubular cylinders of carbonatoms and can either be single-walled CNT (SWCNT) or multi-walled CNT (MWCNT). Although CNTs have attracted great

interest due to their remarkable tunability, and ability to in-corporate multiple functionalities, it is necessary to disperseentangled/bundled CNTs uniformly in solution without impu-rities.106 Studies have shown that the aggregation of MWCNTscauses toxicity in tissues due to deposition in the presence oflarger particles when exposed for long time, whereas theSWCNTs that are composed of smaller particles undergophagocytosis and are non-toxic. Also, cationic carbon nanotubescould be more toxic than the neutral or negatively chargedCNTs since they can cause platelet aggregation.107 Grapheneoxide (GO) has been found to be one of the promising candi-dates as nanocarriers due to its unique 2D shape.108,109

However, uncoated GO exhibits in vivo toxicity such as indu-cing blood clots or pulmonary edema and granuloma throughaccumulation in lungs after intravenous administration.110,111

Yang et al. reviewed the behavior and the toxic effects of gra-phene and its derivatives in different biological organisms andsuggested that their physicochemical properties mainly fromthe functional groups on the surface could determine thein vivo behavior and toxicity.109

6.4 Toxicity of mesoporous silica NPs

Mesoporous silica NPs have been used as theranostics incancer therapy since they can target the tumor and release thedrug in a controlled manner. However, it was found that,despite reduced toxicity compared with colloidal silica, meso-porous silica NPs could still induce cytotoxicity associated withoxidative stress by increased ROS production and decreasedGSH levels, which can ultimately lead to cell death.112,113 Ithas been known that oxidative stress and apoptosis are respon-sible for the dysfunction of endothelial cells and several cardi-ovascular disorders like bradycardia and pericardial toxicity.114

Silica NPs can also inhibit gene expression by forming proteinagglomerates and can interfere with processes involved in thecell growth and replication.94,115 Although NPs have a hugepotential for diagnosis and therapy, they also possess thecapacity to cause severe toxic effects in the body and thus,their formulation should be carefully designed keeping theirtoxic properties in mind.

7. Future directions

Many different types of theranostic NPs have been successfullyimplemented in targeting and therapy of various diseases.Anti-cancer agents may be enclosed within or embedded ondifferent NP structures for targeted imaging and therapy. Infact, most therapeutic NPs boast unique size-dependentoptical or magnetic properties, multifunctionalities, strongEPR effect and long-term blood circulation. However, NPs areinsufficient to meet some important properties for clinicaltranslation such as high physiological stability, efficient clear-ance, minimum accumulation in non-targeted tissues andorgans, and rapid distribution to various organs and tissues.44

Also, clinical applications of most NPs have been hampered bylack of comprehensive knowledge of toxicities of individual

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NPs. More studies in the context of preclinical and predictivetoxicology are warranted to address this question.

In terms of therapeutic method, the majority of appli-cations have been related to curing cancers by killing cancer-ous cells by drugs, heat, and gene transfections. Very recently,a promising theranostic application of multifunctional NPshas been proposed for diagnosis and treatment of chronic neu-rodegenerative disorders such as Alzheimer’s disease, Parkin-son’s disease and stroke.116,117 For instance, multifunctionalNPs can be applied for the amelioration of brain disordersthat are associated with iron overload. Oxidative stress causedby metals like iron, which accumulate in the brain in excess, isconsidered one of the major causes of many neurodegenerativediseases, including Alzheimer’s disease.118 Iron chelators havebeen extensively used with the aim to remove excess iron fromthe brain to improve these disease conditions. An ideal chela-tor should demonstrate increased specificity to the targettissues with minimal distribution into other tissues andorgans. Unfortunately, this is the major drawback of many che-lators because they do not act via a specific targeting mechan-ism and bind to several tissues which can cause differentadverse effects and toxicities.119 Liu et al.120,121 proposed apossible mechanism using NPs in iron chelation therapy fortreating Alzheimer’s disease based on the fact that NPs arecapable of crossing the blood–brain barrier through apolipo-protein E (Apo E) or low density lipoprotein (LDL) receptor inthe brain by imitating LDL. This mechanism allows NPs toenter the brain where they chelate the excess iron and exit thebrain with the chelated complex. This would reduce the toxicityof chelators since they would be directed to the brain and thetherapy would prove to be more efficient since a low amount ofchelators would be needed for therapeutic effects with reducedside effects.120 This mechanism seems plausible and, if develo-ped properly, the formulations could be a major breakthroughin treating not only Alzheimer’s disease, but many other neuro-degenerative disorders. However, other tissues could exertreceptor-mediated uptake of these NPs and deplete iron that isrequired for essential physiological functions. While rigorousassessments of toxicity are needed, NPs with imaging modalitycould track the real-time PK/pharmacodynamics and help todesign better formulations to avoid the distribution into non-target tissues. In addition, nanobiosensors including nanowellarrays and microfluidic chips with ultra-high signalling accuracycan be utilized for ex vivo molecular diagnosis, since it is impor-tant to validate the in vivo functionality of NPs by analysingnanoliters of biosamples (i.e., blood, urine or cerebrospinalfluid) with the detection of isolating single cells, toxic moietiesor secreted proteins.122 This will enhance clinical benefits oftheranostic NPs and further contribute to the development ofadvanced, multifunctional NPs.

Abbreviations

BSA Bovine serum albuminCNT Carbon nanotube

CT Computed tomographyEPR Enhanced permeability and retentionHD Hydrodynamic diameterLDL Low density lipoproteinMNC Micellar nanocomplexMRI Magnetic resonance imagingNNI National Nanotechnology InitiativeNIR Near infraredNP NanoparticlePA PhotoacousticPEG Polyethylene glycolPET Positron emission tomographyPK PharmacokineticsQD Quantum dotRNA Ribonucleic acidROS Reactive oxygen speciesSPECT Single photon emission computed tomographySPION Superparamagnetic iron oxide nanoparticleUS Ultrasound.

Acknowledgements

This study was supported by the following grants from NIH/NIBIB grant #R01-EB-011523 (H.S.C.), NIH/NIEHS grant #R00-ES-017781 (J.K.) and Basic Science Research Program throughthe National Research Foundation (NRF) of Korea funded bythe Ministry of Science, ICT & Future Planning (NRF-2014-R1A6A3A03057790); the contents of this paper are solely theresponsibility of the authors and do not necessarily representthe official views of the NIH.

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