RSC Advances

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Mini-review: fluo

aCenter for Optical Materials Science and E

Materials Science & Engineering, Clemson

USA. E-mail: foulger@clemson.edu; Tel: +1-bDepartment of Bioengineering, Clemson Un

Cite this: RSC Adv., 2016, 6, 65459

Received 22nd April 2016Accepted 23rd June 2016

DOI: 10.1039/c6ra10473h

www.rsc.org/advances

This journal is © The Royal Society of C

rescence imaging in cancer cellsusing dye-doped nanoparticles

Ragini Jenkins,a Mary K. Burdettea and Stephen H. Foulger*ab

Fluorescence imaging has gained increased attention over the past two decades as a viable means to detect

a variety of cancers. Fluorescence imaging has the potential to provide physicians with high resolution

images with enhanced contrast, which will allow them to be able to better diagnose and treat patients

with cancer. Early detection and treatment are key to eradicating cancer in a patient, and fluorescence

imaging has the ability to identify non-advanced, even pre-cancerous, tumors where imaging based on

white light or radiation overlooked them. Several fluorescent dyes have been identified as possible

fluorophores for enhanced fluorescence imaging, such as cyanine, squaraine, porphyrin, phthalocyanine,

and borondipyrromethane dyes. These dyes have high fluorescence quantum yields, which provides

a high target to background ratio; however, these dyes are often plagued by low water solubility. This

low solubility can be ameliorated by conjugating or covalently attaching these dyes to polymeric

crosslinked micelles, polymersomes, or polymer-core nanoparticles. These particle & dye systems then

can become platforms on which secondary components can be attached to enhance the systems

functionality. For example, dyes attached to these nanocarriers can target tumors through passive

targeting; however, active targeting can be achieved by further modifying these nanocarriers with ligands

that have a binding affinity for receptors overexpressed in tumor cells, cell surface receptors located on

the tumor cell membrane, or endothelium. Fluorescence activation of the probes is another promising

technology for the early detection of cancer. Activation requires that there be a change in fluorescence,

whether it be an emission wavelength change or a fluorescence “on/off” signal when in the presence of

some external stimuli. Activation increases the target to background ratio and enhances the contrast of

the obtained image. This review serves to highlight the recent developments of (1) improved fluorescent

dyes for the detection of cancer, with a specific focus on dyes that are being coupled to nanocarriers;

(2) dye & nanocarrier systems that target, both actively and passively, tumors, and (3) fluorescence

activation of these fluorophore systems for better image quality.

1 Introduction

Cancer, a disease caused by the uncontrollable growth ofabnormal cells, is one of the leading causes of death worldwide,accounting for 13% of all deaths.1 Malignant cancer cells aremore effectively treated when identied early in the disease.Identication of tumors using nanoparticle technologies isbeginning to signicantly impact molecular imaging methods.2

Molecular imaging plays a vital role in the healthcare sector,since abnormal conditions and diseases are oen diagnosedthrough imaging, while therapeutic methods used for thetreatment of the abnormalities are oen guided by imaging. Atpresent, there are various forms of imaging techniquesemployed including tomography, magnetic resonance imaging

ngineering Technologies, Department of

University, Clemson, SC 29634-0971,

864-656-1045

iversity, Clemson, SC 29634-0971, USA

hemistry 2016

(MRI), gamma scintigraphy, ultrasound, and optical imaging(cf. Fig. 1). Computed tomography gives high resolution imagesand has the ability to differentiate between different tissues, butit has several drawbacks, such as high cost and the need ofa contrast agent for an enhanced contrast, as well as exposingthe tissue to radiation.3,4 Magnetic resonance imaging (MRI)does not require ionizing radiation and gives high resolutionimages with better contrast; however, this technique is expen-sive and has low sensitivity.3,5,6 Unfortunately, MRI cannot beused in patients with metal implants or metallic devices, suchas pacemakers.7 Gamma scintigraphy, which includes positronemission tomography (PET) and single-photon emissioncomputed tomography (SPECT), suffers from all three majordrawbacks: ionizing radiation, low resolution, and high cost.7–9

There is a widespread use of ultrasound since its low cost, non-invasive, and an easily performed procedure with no radiation,but the images obtained are low in resolution.3,5,7 Opticalimaging is a highly sensitive and low cost procedure7,10,11 thathas the advantage that multiple probes with different spectral

RSC Adv., 2016, 6, 65459–65474 | 65459

Fig. 1 Standard molecular imaging instruments: (a) magnetic reso-nance imaging (MRI); (b) computed tomography (CT); (c) positronemission tomography (PET); (d) single-photon emission computedtomography (SPECT); and (e) optical imaging (f) ultrasound (reprintedwith permission from ref. 7 Copyright 2010 Elsevier B. V.).

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characteristics can be potentially employed for multichannelimaging.3,12,13 The setbacks for optical imaging are that theimages obtained are generally low in resolution and havea limited tissue penetration7 to light in the UV-vis regime.Tissues exhibit a reduced absorption to light in the far red (i.e.775–925 nm) out to the near-infrared (NIR) range14 and thisreview examines recent developments in the eld of molecularimaging with uorophores that emit in these regions.

The use of NIR uorophores in medical imaging has beengaining popularity. These uorophores overcome some draw-backs present with visible light emitting dyes, such as back-ground noise, and also facilitate deeper imaging in the humanbody.15 Unfortunately, small molecule uorophores have fourmajor limitations: (1) limited aqueous solubility, (2) short invivo circulation lifetimes, (3) low quantum efficiencies, and (4)low signal to noise ratios. These drawbacks can be amelioratedwith varying success by either encapsulating the uorophoreinside a nanoparticle or attaching it on the surface of thenanoparticle. Over the years, uorescent nanocarriers such as

Fig. 2 Schematic structures of (a) polymer cross-linked micelles, (b)polymersomes, and (c) polymer-core nanoparticles (adapted withpermission from ref. 26 Copyright 2012 Multidisciplinary DigitalPublishing Institute AG).

65460 | RSC Adv., 2016, 6, 65459–65474

surface cross-linked micelles,16,17 dendrimers,18 biodegradablepolymeric nanoparticles,19,20 magnetic and other metal parti-cles,21,22 and liposomes21 for tumor diagnosis have been devel-oped. Among these technologies, platforms based on polymericmaterials, especially biodegradable nanoparticles, are ofparticular interest due to the exibility offered by macromo-lecular synthesis methods, high drug loading capacities,improved drug solubility, and their ease of multi-functionalization.23–25 In this review, the developments in uo-rescence imaging with the three polymer based platforms:cross-linked micelles, polymersomes and polymer-core nano-particles (cf. Fig. 2), when coupled with the commonly useduorophores, will be explored.

2 Small molecule imaging

Small molecule organic uorophores have been in demand inthe biomedical community for imaging and image guidedsurgery or therapy.27–34 Imaging with far-red and near-infraredabsorbing and emitting dyes such as cyanine, squaraine, thia-zine, oxazine, porphyrins, and phthalocyanines, is common-place and most of them have been approved for clinicaltrials.35–47 Recently, a new class of dyes, borondipyrromethane(BODIPY) derivatives, have emerged and gained popularity.48–53

However, imaging with small molecules have some challengesin both their optical performance and delivery to the tissue ofinterest, such as water insolubility, aggregation, low quantumyield, insufficient photostability, low tumor to backgroundratio, and short in vivo circulation lifetimes. Researcherscontinue to focus on improving these dyes to enhance theirlight output and localization in specied regions and aredetailed in the following section.

2.1 Cyanine dyes

The general structure of cyanine dyes consists of two aromaticnitrogen-containing heterocycles, acting as both electrondonors and acceptors. The two aromatic rings are connected byan odd number of methine groups in which (n + 1) bi-electronsare distributed over n atoms that produce a delocalized cationacross the methine chain (cf. Fig. 3a).54–56 Depending on the

Fig. 3 Structures of (a) cyanine, (b) squaraine, (c) phthalocyanine, and(d) boron dipyrromethane dyes.

This journal is © The Royal Society of Chemistry 2016

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methine chain length, the cyanine dyes absorb in the visibleto infrared regions of the electromagnetic spectrum.57 In1856, C. H. G. Williams synthesized the rst cyanine dye57,58

and since this time many analogs with varying lengths of themethine chain have been developed and employed in variousbiomedical applications ranging from angiography tophotodynamic therapy.54 The majority of commercial uo-rescent probes for in vivo imaging consist of cyanine dyes. Ofall the cyanine dyes, indocyanine green (ICG) is the mostpopular and was approved by the FDA decades ago for eval-uating blood ow and clearance.54,55 ICG andmany other dyesgenerally exhibit high molar extinction coefficients but lowquantum yields, poor photostability, high plasma proteinbinding rate, and undesired aggregation.59 To overcomesome of the limitations, new analogs that have a cyclohexenylin the middle of the methine chain and moieties such ascarboxylic and sulfonate groups were introduced.60,61 Thesealterations result in the improved water solubility of the dye,photostability, and quantum yield.

2.2 Squaraine dyes

Squaraine dyes are 1,3-zwitter ionic donor–acceptor–donor(D–A–D) structures with the central acceptor squaryl ring con-taining donor aromatic or heterocyclic rings on each side(cf. Fig. 3b).62 In 1965, Treibs and colleagues rst reported thesynthesis of squaraine dyes, which have extremely intenseabsorption bands, high molar coefficients, and good photo-conductivity.54,63,64 Despite these molecules having excellentphysical–chemical properties, they gained importance only inthe late 2000s due to their limitations such as low water solu-bility, propensity for aggregation, and poor chemical stabilitywith only a few analogs of the dye emitting at wavelengthshigher than 800 nm.54 Most importantly, the squarate bridge issusceptible to chemical attack by nucleophiles due to its elec-tron deciency and results in loss of uorescence.65,66 In 2005,Gassensmith and colleagues synthesized a rotaxane moleculeencapsulating a squaraine dye. The rotaxane cage protected thesquaraine ring and inhibited aggregation, which improved thechemical and photostability of the dye.67 In order to furtherprotect the squarate bridge from nucleophilic attack, a squarinethat is dicyanovinyl functionalized was synthesized.65 In 2007,Umezawa and colleagues developed another alternative squar-aine derivative dye with improved hydrophilicity due to fourwater-solubilizing sulfonate moieties added to the generalstructure.68 Squaraine dyes can be relatively challenging tosynthesize, but it was shown that a microwave can actually beused to assist in the synthesis of 2,3,3-trimethylindolenine-based squarine dyes exhibiting maximum absorbancebetween 625 nm and 700 nm and emissions between 635 nmand 800 nm. These microwave synthesized dyes exhibiteda better yield and reduced reaction time when comparedsquariane dyes synthesized the conventional way. In addition, itwas shown that both symmetric and nonsymmetric dyes couldbe easily synthesized with this method.69 All these improve-ments to squaraine dyes have made them a very promisingcandidate for protein detection and in vivo imaging.

This journal is © The Royal Society of Chemistry 2016

2.3 Porphyrin and phthalocyanine dyes

Porphyrins are tetrapyrrolic molecules that consist of fourpyrrolic sub-units linked on opposing sides through fourmethine (CH) bridges. Phthalocyanine molecules are an exten-sion of porphyrins where each pyrrolic ring of porphyrin isextended by a benzene ring (cf. Fig. 3c).70 The central cavity of thephthalocyanine contains two hydrogen atoms and these atomscan be replaced by more than 70 different types of metalatoms.71–73 In addition, a variety of substituents can be added tothe periphery of the macrocycle or to the axial positions of thecentral atom.54,74 These blue or green colored dyes absorbstrongly in the red and near-infrared part of the visible spectrum.In 1907, Braun and Tcherniac synthesized the rst metal-free andcopper phthalocyanines.75 In vitro uorescence imaging withphthalocyanines and porphyrins dates back to late the 1980s andearly 1990s.76 In 1993, photofrin, a porphryin derivative, was therst chromophore in this class of dyes to be approved for clinicaluse for the treatment of bladder cancer.70 Since then there havebeen several analogs of the porphyrin and phthalocyaninesynthesized and utilized.77–81 The low water solubility exhibited ofthese dyes has decreased their popularity for imaging.80,82–84

However, a new phthalocyanine chemistry was used to createa series of PEGylated cationic molecules, which has promising invitro behavior against cancer cells.85 Click reactions have beenemployed to create phthalocyanines with different architectures;4-arm star polymers utilizing either polystyrene or poly(tert-butylacrylate) were synthesized with symmetrically tetra terminalalkynyl-substituted phthalocyanines as the core. The centralmetal atom in these systems was either copper or zinc.86 This typeof architecture could be used to improve water solubility andprevent aggregation of the phthalocyanines due to the extensionof the polymeric arms which disrupts the p stacking of thephthalocyanine. In 2015, Bandera and colleagues synthesizednovel silicon phthalocyanine derivatives equipped with azide oralkyne functionality to be able to use the dyes in “click” chem-istry, which allows the dyes to be used in an engineered nano-device for cancer theranostics.87 In 2013, a water solubleporphyrin (THPP), with zinc metal as the central atom wasdeveloped and it showed an increase in in vitro photodynamictherapy activity by 2–3 times when compared to photofrin.88

Photodynamic therapy relies on the ability of the uorophore,when excited at the appropriate wavelength of light, to generatea reactive oxygen species (singlet oxygen) from molecular oxygenthrough an intersystem crossing.89 In the eld of tumor thera-nostics, these dyes have been more successful than the otherclasses of dyes due to their robust uorescence, which yieldsimages with better contrast and signal to noise ratios, as well aslending themselves to superior performance in photodynamictherapy. Yumita and colleagues synthesized a novel porphyrinderivative and demonstrated a mechanism that is related to thegeneration of singlet oxygen to achieve destruction of cancer cellsthrough sound rather than light.90

2.4 Borondipyrromethane dyes

Borondipyrromethanes (BODIPY) have a general structure of4,40-diuoro-4-bora-3a,4a-diaza-s-indacene (cf. Fig. 3d). In 1968,

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Treibs and Kreuzer rst reported the synthesis of BODIPYdyes.91 These molecules typically have the following character-istics: sharp uorescence with high quantum yield, excellentthermal and photochemical stability, and extinction coeffi-cients around 80 000 M�1 cm�1.92 Despite having such attractiveproperties, BODIPYs were not preferred because they emit in theyellow to deep-red region, and the extinction coefficients wereconsidered to be relatively low.54 To overcome these disadvan-tages, two approachs have been undertaken. First, polymeric andcopolymeric BODIPY dyes were synthesized to shi emission tothe near-infrared region.93 Second, modication of the pyrrolecore shis the emission to the red end of the visible spectrum.The most signicant modication is to include aryl groups to theBODIPY. Using thismodication, BODIPYs withmolar extinctioncoefficients of 253 000 M�1 cm�1 and emission in the near-infrared region can be obtained.94,95 In 2014, BODIPY deriva-tives were synthesized via a Suzuki reaction where a hetero arylsubstituent on the 3- or 3,5-position resulted in approximatelya 150 nm bathochromic shi in both maximum absorbance andmaximum emission. This series of BODIPYs resulted in animproved production of singlet oxygen.96 An increased uores-cence quantum yield (approximately 0.82) was achieved throughthe modication of the phenyl group with 2 methyl substituentsin themeso position. Thesemethyl groups provided rigidity to themolecule and resulted in a red shi in maximum emission to644 nm (in chloroform) or 649 (in DMSO). These BODIPYs werefurther modied by a terminal bromo group in order to attachthe dye onto carbon nano onions.97 This modication showedthat BODIPY could be modied in a variety of ways in order toattach the dye to a scaffold, which could serve to incorporatethem into a system for possible biomedical applications. In 2016,Kim and colleagues synthesized new BODIPY uorophores thatwere capable of 1 or 2 photon uorescence imaging. Further, theyshowed that the free uorophores accumulate in the lysosomesof cells. The uorophores have low cytotoxicity, which makesthem ideal for imaging agents.98

3 Imaging with polymer basednanoparticles

It is well established that nanosized objects accumulate moreefficiently in tumors and increase the target to background ratiodue to the enhanced permeability and retention (EPR) effect.99

The EPR effect results in passive targeting of nanoparticles totumor sites. Blood vessels supplying tumor tissues have largerpore sizes compared to those in healthy tissue, and tumortissues have poor lymphatic drainage. These factors combinedallow for a preferential tumor accumulation of nano-particles.100–103 Small animal studies indicate that a 50 foldincrease in accumulation of nanoparticles within tumor tissuesare due to the EPR effect, coupled with an increase in in vivocirculation lifetimes.103 Apart from the increased accumulationand lifetimes, the nanoparticles have another advantage: theyprovide a platform for an increased surface area per volume forenhanced loading of imaging and therapeutic moieties.102 Tothis end, conjugating a small molecule uorophore to the

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surface of the nanoparticle is preferred over using the uo-rophore by itself for optical imaging. In addition to the EPReffect, the particle characteristics such as size distribution,surface charge, biocompatibility, biodegradation behavior, andavailability of functional groups for conjugation play animportant role in determining the circulation lifetimes andaccumulation behavior in tumor tissues.26,102 It is establishedthat biodegradable microparticles made of starch, albumin, orpoly lactic acid, are rapidly cleared by the reticuloendothelialsystem (RES) and are unable to enter capillaries, making themless attractive for imaging applications.102 As mentioned previ-ously, polymeric nanocarriers are of particular interest due tothe exibility offered by macromolecular synthesis methods,high drug loading capacities, improved drug solubility, andease of multifunctionalization.23–25 The rst preparation andcharacterization of polymeric nanoparticles was reported in1976; since, the research in this area has grown exponen-tially.102,104 It has been established that neutrally chargedparticles, with an average diameter of 10–100 nm, and molec-ular weights up to 800 kDa were required to obtain longercirculation lifetimes.102,105 In addition, the choice of the parentpolymer is important in designing such systems because thepolymer tends to determine the nal behavior of the system invarious environments.102 Based on the above mentionedrequirements for the nanocarrier, some of the successfulorganic uorescent carriers used in uorescence imaging willbe discussed in detail in the following sections (cf. Fig. 2).

3.1 Crosslinked micelles

A crosslinked micelle (CM) is a nanocarrier that is based ona self-assembled aggregation of amphiphilic molecules orsurfactants with the uorescent probes conjugated to it.Micelles have a dynamic structure in equilibrium and, ata critical micellar concentration (CMC), they exist as freemonomers, so care should be taken to ensure that theconcentration is below the CMC to avoid a fast and undesirablerelease of the probe upon administration.26 Typically, CMs arein the 5–150 nm size range,106 which allows extravasation andpermeation into tissues and avoids clearance by the RES.107 TheCMs are simple aqueous based preparations with highlytunable emission wavelengths and an expanded Stokes shi ofthe dye from 20 nm to about 160 nm,108 which made them anattractive carrier for uorescence imaging.109 In addition, mostCMs are not toxic and are biologically compatible. The excep-tion is surfactant based micelles as the large amounts ofsurfactant used in their formation might lyse the cellmembrane and denature proteins, so they are not a preferreddesign.107 In polymer based micelles, the hydrophobic units ofthe block copolymers form the hydrophobic core of the micelleand hydrophilic units surround the core forming a hydrophilicshell, thus making the micelle sterically stable and protecting itfrom EPS uptake.106,107 Using this strategy, a number of blockco-polymer micelles that encapsulate near infrared emittingchromophores have been synthesized over the years andutilized in imaging applications.26,110,111 Recently, in 2013, Chenand colleagues synthesized PEGylated tripropargyl amine

This journal is © The Royal Society of Chemistry 2016

Fig. 5 Temperature induced self-assembly of ELPBC to form sphericalmicelles with multivalency. An N-terminal ELP[V1A8G7�n] gene(hydrophilic, high Tt) and C-terminal ELP[V5�n] gene (hydrophobic, lowTt) are fused together to create a gene that encodes an ELPBC. Whenthe size and ratio of the blocks are optimal, the ELPBC self-assemblesinto a spherical micelle at approximately 40 �C. In the schematic, uponself-assembly the spherical micelles present several copies of anaffinity targeting moiety (green triangle) and capture a drug or imagingagent (lightning bolt) within the core of the micelle (reprinted withpermission from ref. 113 Copyright 2008 American Chemical Society).

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micelles conjugated to BODIPY uorophores, which had excel-lent water solubility and membrane permeability. Thesecompounds exhibited an increased Stokes shi without anycovalent structure modication of the uorophore.108,112 Inaddition, these BODIPY conjugated PEGylated tripropargylamine micelles were observed to readily penetrate cellmembranes and preferentially accumulate in the cytoplasmrather than the nucleus of the cell (cf. Fig. 4).112 In most of themicelles, poly(ethylene glycol) (PEG) remained as the choice ofthe hydrophilic unit as this polymer is widely accepted asa biocompatible material and is easily available.106 The recentadvances in crosslinked micelles have come in the form of“smart” micelles that respond to various biological stimuli,including the pH of the environment, and can be designed totarget specic tissues (cf. Fig. 5).106,113 Apart from synthesizingstimuli responsive micelles, there has been development ofmicelles based on recombinant proteins. Kim and colleagues114

developed a 50 nm recombinant protein micelle for targeted invivo imaging. In these micelles, multiple uorescent moietieswere covalently conjugated to surface amines of crosslinkedamphiphilic elastic-mimetic protein micelles utilizing commonprotein chemistry, namely N-hydroxysuccinimide ester chem-istry. These micelles offered enhanced biocompatibility and canbe further modied in order to tailor the micelle nanocarrier forspecic applications. Self assembled micelles consisting ofpoly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate) (PEG-PCC) covalently attached to amine modiedindocyanine green (NH2-ICG) were successfully produced andshowed efficient photodynamic therapy behavior (i.e. signi-cant singlet oxygen generation).115

3.2 Polymersomes

Polymersomes are articial vesicles or small hollow spheresthat enclose a solution and have radii ranging from 50 nm to50 mm.116 They have a large hydrophilic reservoir and a thickhydrophobic lamellar membrane that supports the storage ofa large quantity (i.e. more than 10 mol/wt%) of hydrophobicuorescent dyes and can protect the dyes from quenching anddegrading.117,118 The polymer chains dictate the average uo-rophore–uorophore interspatial separation along with the

Fig. 4 (a) Fluorescence, (b) transmission, and (c) overlapping fluo-rescence & transmission images of HeLa cells observed with confocallaser scanning microscopy. The cells were incubated for 30 min with 1mM of BODIPY conjugated surface crosslinked micelles (ca. 10 nm) at37 �C in an atmosphere of 95% air and 5% CO2. Fluorescence signalswere detected between 570 nm and 620 nmwith a constant excitationof 561 nm (reprinted with permission from ref. 108 Copyright 2013 TheRoyal Society of Chemistry).

This journal is © The Royal Society of Chemistry 2016

uorophore-localized electronic environment.116 Greater uo-rescence was obtained for more hydrophobic uorophoreswhen relatively apolar membranes, such as poly(g-methyl-3-caprolactone), were used, and the more amphiphilic uo-rophores were better dissolved when bilayers of poly(3-capro-lactone) were employed.118 For most polymersomes, the outershell is a layer of dense polyethylene oxide (PEO), which confersthe “stealth” like character and results in increased biocom-patibility, structural integrity in plasma, and circulation life-times.116,117,119 In addition, the surface has terminal functionalgroups, such as alcohols, which can be used to covalentlyconjugate targeting molecules, drugs, and biomolecules tocreate a multifunctional unit.117 There has been extensive workand investigation into developing polymersomes into stimuliresponsive multimodality agents,120–123 but for this review, wewill focus on the polymersomes that contributed signicantlyfor uorescence imaging.118,119,124 Initially, Hammer andcolleagues synthesized poly(ethylene oxide)-block-poly(ethyl-ethylene) (PEO-b-PEE) diblock copolymers based polymersomesand expanded that technique to generate a number ofbiocompatible PEO-based amphiphilic block copolymers, suchas poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD).119

They loaded these polymersomes with hydrophobic dyes, suchas Nile Red, and hydrophilic dyes, such as calcein, anddemonstrated its use in deep-tissue uorescence basedimaging.119,125 PEO-b-PBD polymersomes encapsulatingporphyrin based uorophores generated a uorescence signalthat was penetrable through 1 cm of solid tumor.119 However,these systems were not biodegradable and were not fullybiocompatible. To this end, Ghoroghchian and colleaguesdeveloped the rst set of self-assembled polymersomes thatwere composed entirely of United States Food and DrugAdministration (FDA) approved biodegradable diblock

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copolymers. They were fully bioresorbable diblock copolymersof poly(ethylene oxide)-block-poly(3-caprolactone) (PEO-b-PCL)and the diblock copolymer poly(ethylene oxide)-block-poly(g-methyl 3-caprolactone) (PEO-b-PMCL) (cf. Fig. 6).118 In 2012,Massignani and colleagues developed poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-diisopropylaminoethyl meth-acrylate) (PMPC-PDPA) diblock copolymers based polymer-somes and loaded it with rhodamine, which had enhancedemission compared to free rhodamine and CellTracker dye.126

This increase was attributed to the effective dye delivery whenthe polymersome was used. In 2014, Quan and colleaguessynthesized a uorescent polymersome that could encapsulatehydrophobic or hydrophilic drugs instead of the traditionalpolymersome encapsulating uorophores. It was prepared byself-assembly of block copolymer hydrophilic poly(ethyleneglycol) borondipyrromethenes (MPEG-BODIPY) in aqueous

Fig. 6 Cartoon representing various means of incorporating oligo(-porphyrin)-based NIRFs within polymersomes. (a) The NIRFs aredifferent as they have various numbers of porphyrin subunits (N),various linkage topologies between porphyrin molecules, and variouspositions of ancillary aryl-group substituents (R), and the nature ofeach R group is different based on its chemistry. (b) Diblock copolymerat a variety of compositions have been used to generate polymer-somes that have emission in the NIR. (c) Interfacial interactionsbetween polymers and ancillary aryl group substituents, and the varietyof conformational populations in which the NIRF can arrange itself, arevaluable tools when manipulating and/or tuning the emission of theNIRF. (d) Control over the chemical composition and polymersomemembrane thickness forces individual NIRFs into dielectric environ-ments of matching polarity. (e) A family of nanoscale polymersomespossessing emission wavelengths corresponding to the NIR region ofthe visible spectrum (reprinted with permission from ref. 118 Copyright2009 John Wiley & Sons Inc.).

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solution. These ca. 57 nm particles exhibited low toxicity andhigh accumulation in tumor site through passive targeting.127

Polymersomes are also being used to encapsulate super-paramagnetic iron oxide nanoparticles (SPIONs), an ubiquitousimaging agent in MRI. These polymersomes consist of eitherzwitterionic or cationic block copolymers (poly(2-methylacryloyloxy)ethyl phosphorylcholine)-block-((3-(meth-acryloylamino(propyl)trimethylammonium chloride)); PMPC20-b-PMAPTAC190) or zwitterionic and anionic block copolymers(poly(2-methylacryloyloxy)ethyl phosphorylcholine)-block-poly(sodium 2-(acrylamido)-2-methylpropanesulfonate; PMPC20-b-PAMPS196). In order to better encapsulate SPION, the SPIONswere coated with a cationic chitosan derivative that had beenfurther modied with the uorophore Alexa Fluor® 647. Theuorophore provides additional imaging capabilities andtracking. These polymersomes have a semipermeablemembrane that is capable of encapsulating a large amount ofhighly charged nanoparticles; the chitosan coated SPIONsreside in the interior and in the membrane of the polymersome.Through laser scanning confocal imaging of the Alexa uo-rophore, it was seen that the particles are uptaken by theendothelium.128 Polymersomes have started to be producedfrom chemically modied proteins in conjunction with poly-mers. Polymersomes of a poly ion complex based on chemicallyaltered chitosan were used in conjunction with uorescentquantum dots (QDs), where the hydrophobic QDs were encap-sulated by the polymersome. The in vivo study showed thatthere was a longer retention time and greater uorescencesignal in the tumor for the polymersomes conjugated with QDswhen compared to the isolated QDs. The uorescence signal ofthe free QDs was quickly extinguished within 1 hour aertreatment with a strong uorescence signal in the liver sug-gesting that the free QDs are rapidly cleared from the tumor.129

In another study, poly(N-isopropylacrylamide) (PNIPAM),a thermally responsive polymer, along with a protein, namelya derivative of green uorescent protein (amilFP497), wasdeveloped. The polymersome is a block copolymer with PNI-PAM-b-amilFP497. These polymersomes exploit the conforma-tion changes that occur when PNIPAM is subjected totemperatures in the lower critical solution temperature (LCST)of 37 �C. At temperatures below 37 �C, the polymersomes selfassemble and are capable of encapsulating drugs, such asdoxorubicin (DOX), and uorophores during the self assemblyprocess. The uorophore phycoerythrin 545 was used in thisstudy and was found to be in the core of the polymersome, whileDOX was found in both the core and the membrane of thepolymersome. At temperatures above the LCST, the polymer-some unfolds and releases its load.130

3.3 Polymer-core and uorophore nanoparticles

The polymer-core nanoparticles can be divided into two cate-gories: natural and synthetic. As the name of the categorysuggests, particles made from naturally occurring polymerssuch as dextran, albumin, gelatin, chitosan, heparin, bacterio-phages, or lipoproteins fall under the natural category, and theparticles synthesized from articially produced polymers such

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Fig. 7 In vivo biodistribution of intravenously administered Cy5-PLAnanoparticles. Lateral tail vein injection using the LI-COR Odysseyscanner was performed and the biodistribution of Cy5-PLA nano-particles was obtained. More than a 10-fold increase in accumulationwas seen in Cy5-PLA nanoparticles contained in visceral organs whencompared to background autofluorescence. Cy5-PLA nanoparticlesshowed the most accumulation in the spleen when compared to theother organs sampled (i.e. spleen, liver, kidney, lung, and heart); barscale is 5.5 mm (reprinted with permission from ref. 145 Copyright2010 WILEY-LISS INC.).

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as poly(amino acids), poly(alkyl-cyano acrylates), poly(esters),poly(orthoesters), poly(urethanes), and poly(acrylamides) areconsidered synthetic.26,102 In this current review, the focus is onsynthetic polymer-core nanoparticles. The polymer-core nano-particles have an inner core made of a cross-linked polymerwhile the uorophore is either encapsulated within the core orcovalently conjugated to the polymer. Encapsulation protectsthe uorophore from direct interactions that could decrease itsemission, but, at the same time, the uorophore can neitherform benecial host/guest assemblies nor complex with bio-macromolecules. Physical encapsulation of the uorophorefurther suffers from leakage and non-specic release of theuorophore. These drawbacks are overcome easily when theuorophore is covalently conjugated to the outside of theparticle.26,131 For the inner core, hydrophobic polymers such aspoly(lactic-co-glycolic acid) (PLGA), poly(3-caprolactone) (PCL)and poly(methyl methacrylate) (PMMA) are preferred, as thehydrophobic matrix provides a strong affinity for the entrap-ment of poorly water solubilized uorophores and drugs.132,133

Typical nanoparticle size varies from 50–500 nm, and theoptimum nanoparticle size for tumor accumulation is between70 and 200 nm as the nanoparticle uptake by the RES is in thesize range between 150 and 300 nm.134,135 In addition, it hasbeen well established that a coating of PEG on the hydrophobicparticle is necessary to render the nanoparticles biocompatible,avoid rapid clearance by the RES, and increase the circulationlifetime, which increases accumulation and allows distributionin different tissues.26,131,136–138

As mentioned previously poly(lactic acid) (PLA) basedsystems are the most popular choice for nanoparticles. PLA andits copolymers are one of the most extensively investigatedmatrix materials for nanoparticle-based diagnostic and drugdelivery applications as they offer excellent biocompatibility,biodegradability into metabolizable moieties, and manu-facturability.132,139 In addition, PLGA is approved by the FDA andis a component of many biodegradable market products forparenteral application.133,140 Initially, in 2004, Saxena andcolleagues prepared 300–410 nm PLGA nanoparticles entrap-ping ICG by a modied spontaneous emulsication solventdiffusion method and were able to achieve over 74% loading ofthe uorophore in the particles.141,142 Later in 2006, theydemonstrated the use of these particles for tumor diagnosis andphotodynamic therapy and also determined the biodistributionof ICG loaded PLGA nanoparticles in healthy C57BL/6 mice(female, 10 week old).143 In 2011, Schadlich and colleaguesencapsulated different Nile Red and DiR (a carbocyanine baseduorophore) in PEGylated PLA nanoparticles and performed invivo and ex vivo imaging studies to investigate the impact ofparticle size in tumor accumulation, distribution, and elimi-nation.133,144 In addition to encapsulated systems, there arecovalently conjugated uorophore–PLA or PLGA nanoparticlesystems being investigated. In 2010, Tong and colleaguesdeveloped Cy5-conjugated polylactide (Cy5-PLA) nanoparticles.They found these particles to have excellent signals with hightumor to background ratio uorescence in various organs whenadministered intravenously to balb/c mice (cf. Fig. 7).145 In 2012,Reul and colleagues, evaluated the concept of covalently

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attaching a NIR dye to poly(lactide-co-glycolide) (PLGA) to createstable NIR uorescent nanoparticles. For their studies, theycoupled PLGA with DY-700 (a near infrared uorophore) andinjected the system intravenously into mice. According to theirresults, these particles had very good stability and preferentiallyaccumulated in the liver where the free uorophore would notenter. This system could be developed further into a nelytraceable PLGA nanosystem for uorescence imaging.140

Apart from PLA based nanosystems, there have been parti-cles based on other polymers such as poly(propargyl acrylate)(PA), poly(styrene-co-methacrylic acid), and poly(acrylic acid)(PAA). In 2011 Rungta and colleagues prepared sub-100 nm PAnanoparticles which had azide-terminated indocyanine green(azICG) covalently conjugated to the surface. The azICG wasattached to the particles via azide/alkyne Huisgen cycloadditionusing a copper catalyst.59 Since the inception of the “click”cycloaddition reaction in the eld of drug discovery in 2001 bySharpless and coworkers, it has been used by numerousresearchers to prepare nanosized particles including polymericmicelles and nanoparticles, liposomes and polymersomes,capsules, metal and silica nanoparticles, carbon nanotubes andfullerenes, and bionanoparticles for tumor diagnostic and drugdelivery applications.146–160 In addition, signicant growth andinterest is seen in developing new processes for the “click”reaction to achieve high reaction kinetics, excellent biocom-patibility, selective labeling of specic targets, easily accessiblereactive tags, and long shelf-life.146 In the future, it can be ex-pected that a majority of the nanosystems for tumor thera-nostics will utilize the “click” reaction.161,162 These PA-azICGnanoparticles particles demonstrated great potential forphotodynamic therapy (PDT), but their brightness was too low

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for imaging applications. Later in 2013, the same groupsynthesized particles that were surface modied with an azideterminated squaraine dye (azSQ) instead of azICG; thesePA-azSQ particles performed better than the PA-azICG particlesfor uorescence imaging. Co-localization studies were per-formed to establish that the uptake of the PA-azSQ particles wasthrough endocytosis and in vitro uorescence imaging wascarried out in UMSCC22A (head and neck) and A549 (lung)cancer cells.131 In 2011, Palma and colleagues, prepared BF2chelated tetraarylazadipyrromethene (near infrared dye)conjugated poly(styrene-co-methacrylic acid) particles. The dyewas attached to the nanoparticles via a standard N-hydrox-ysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) coupling reaction. Using those particles,they were able to obtain promising results for real time in vitroimaging in MDAMB-231 (breast cancer), HEK293T (kidney), andCAKI-1 (renal cancer) cell lines (cf. Fig. 8).163 In 2013, Tao andcolleagues reported the synthesis of the rst biocompatibleNIR-II agent nanoparticles, for in vivo imaging. These particleshad IR-1061, a commercially available water insoluble poly-methine dye, embedded in a poly(acrylic acid) (PAA), anamphiphilic polymer, matrix. These nanoparticles also hada coating of the surfactant DSPE-mPEG (polyethylene glycol-conjugated phospholipid, ca. 5 kDa) for improved biocompati-bility and longer circulation lifetimes. Using these IR-1061nanoparticles, the inner organs and blood vessels of micewere imaged and the results matched the images previouslyobtained with inorganic carbon nanotubes and quantumdots.164 Most of the recent literature published on nanoparticlesystems not only are biocompatible, fully biodegradable, andexhibit high signals but are also designed to target particularcells or tumors and avoid healthy cells. Specic details of thesesystems and examples will be discussed in the next section.Passive targeting of nanoparticles has also been recently usedwith self assembled D-a-tocopheryl polyethylene glycol 1000succinate conjugated to cis-aconitic anhydride modied doxo-rubicin, a common chemotherapy drug. Aer the self assembly

Fig. 8 Real-time fluorescence imaging of BF2 chelated tetraar-ylazadipyrromethenes (near infrared dye) conjugated poly(styrene-co-methacrylic acid) particles uptake into HEK293T cells (scale bar 10 mm)(reprinted with permission from ref. 163 Copyright 2011 AmericanChemical Society).

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of the nanoparticles, the uorophore chlorin e6 was loaded intothe nanoparticles via encapsulation. The nanoparticle system asa whole is nonuorescent. However, cis-aconitic anhydridemodied doxorubicin is acid sensitive, and upon cellularinternalization and subsequent transport to the acidic lyso-somes, the amide linkage is hydrolyzed and the uorophoreand drug are released. Successful photodynamic therapy wasconrmed with this system.165 Self-assembly of nanoparticleswas also utilized to yield tetraphenylethene uorophore nano-particles. Tetraphenylethene exhibits aggregation inducedemission, making it an ideal candidate for uorescenceimaging. Further, the tetraphenylethene self-assembled nano-particles exhibited very good singlet oxygen generation as wellas emission in the NIR region. Tetraphenylethene nanoparticlesshowed a maximum absorption of 450 nm with a maximumemission of approximately 700 nm.166 Another study investi-gated PEGylated graphene oxide nanoparticles with encapsu-lated sinoporphyrin sodium (DVDMS). These nanoparticlesexhibited increased accumulation in cancer cells than DVDMSalone. Further, the nanoparticles showed more efficientapoptosis/necrosis via photodynamic therapy when comparedto that induced by DVDMS alone.167 Nanoparticles made fromderivative of anthraquinone, a uorophore exhibiting aggrega-tion induced emission, via a reprecipitation method weresynthesized. These particles showed low cytotoxicity and goodstability in cells (A549 cell line) for 15 days over six generations.This system is a good candidate for performing uorescenceimaging of cancer over an extended period of time.168

4 Targeting

Up to this point examples of passive targeting of tumors inuorescence imaging have only been discussed. Encapsulatingor conjugating small molecule uorophores to nanoparticlesallows the particles to preferentially accumulate in tumors dueto the EPR effect (cf. Fig. 9).100,101 In this case, the size is used topassively target tumors. Apart from size, modifying the surfacecharge of the nanoparticles is an additional way to passivelytarget the tumors. The surface charge of tumors is highlynegative when compared to normal cells,169 so if the surface ofthe nanoparticles is modied to be more cationic, then thenanoparticles bind electrostatically to the negatively chargedphospholipid head groups expressed on tumors and in turnpreferentially accumulate in tumors.170–173 However, passivetargeting is limited; therefore, it is necessary to add an activetargeting component to the nanoparticles so that they affecttumors and avoid healthy cells (cf. Fig. 9). An ideal target shouldbe universally and uniquely expressed by tumors. In most casesthe target has an overexpression of a particular cell surfacemarker in tumors.170 The overexpression of the target groupfacilitates increased binding of the targeting agent, whichincreases the cellular uptake of the targeting agent.106

In ligand-based targeting, the ligand binds specically toa receptor that is overexpressed by tumor cells. The ligands usedvary from small molecules, vitamins, carbohydrates, peptides,antibodies, proteins, and nucleic acids.100,170,174,175 A system wasdeveloped to detect nitroreductase (NTR), which is overexpressed

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Fig. 9 Cartoon depicting different mechanisms of drug delivery tocancer cells with nanocarriers. Polymeric nanoparticles are shown asrepresentative nanocarriers (circles). Passive targeting is achieved byextravasation of nanoparticles through the enhanced permeation andretention (EPR) effect. Active cellular targeting (inset) can be achievedby tagging the nanoparticle surface with targeting ligands thatspecifically bind with a receptor on the cell surface. The nanoparticlescan either (i) release their cargo when in proximity to the target cells;(ii) attach to the cell membrane and function as an extracellular sus-tained-release drug reservoir; or (iii) internalize into the cell (reprintedwith permission from ref. 100 Copyright 2007 Nature PublishingGroup).

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in tumors experiencing hypoxia. Hypoxia occurs when tumors aredeoxygenitated; in the tumor, the oxygen level is 0–4%, whichinhibits many therapies that require oxygen in order to beeffective. The system for the detection of NTR consists of para-nitro benzoate functionalized Cy7 cyanine dye. The nitro-benzoate linker performs the detection of NTR, while thecyanine dye provides the uorescence signal. The aromaticnitrogen group binds to NTR through strong hydrogen bonds.Once the probe is bound to the NTR, the system, which wasoriginally nonuorescent, becomes uorescent due to thechanging strength of the electron withdrawing group uponbinding, serving as an optical indicator of NTR.176 Another recentdevelopment in ligand-based targeting is the use of a GE-137peptide that binds to human tyrosine kinase c-Met; humantyrosine kinase is prominent in pre-cancerous colorectal polyps.In this targeting system, GE-137 is conjugated to a cyanine dye,which uoresces allowing for an optical cue for physicians per-forming uorescence colonoscopies that there is a pre-cancerouspolyp in the colon. Fluorescence colonoscopies led to 18% morepolyps being detected by physicians than when only white lightcolonoscopies were performed on the same patients.177 Anotherstudy used a phthalocyanine dye that had been modied viastreptavidin–biotin chemistry to have an avb6 integrin targetingligand. It was found through in vitro, in vivo, and ex vivo uo-rescence and optical imaging and ex vivo PET scans that thetargeted phthalocyanine system did successfully bind to avb6

integrin, which is widely upregulated in cancers, especiallypancreatic cancer.178 In another investigation, silica nano-particles with encapsulated salicylaldehyde were surface modi-ed with a DNA aptamer that selectively binds to nucleolin.

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MCF-7 cancer cells, which overexpress nucleolin, in comparisonwith normal MCF-10A cells were used to evaluate the targetingefficiency of the functionalized nanoparticles. In vitro studiesshowed that the nanoparticles preferentially accumulated in thecancer cells and effective uorescence imaging was achieved withthe nanoparticles via aggregation induced emission.179

Commonly targeted surface cell receptors are transferrin,folate, epidermal growth factor receptors (EGFRs), and glyco-proteins.170,180–185 A study was performed where a targetingligand was conjugated to a sulfur substituted BODIPY deriva-tive. The target was g-glutamyltranspeptidase (GGT), which isoverexpressed on the plasma membrane of tumor cells andserves to cleave glutamate. The probe, when in the presence ofGGT, causes the enzymically triggered conversion of the sulfursubstituted BODIPY derivative to the amino-substitutedBODIPY, which causes a large shi in the maximum emissionof the BODIPY. In this way, GGT levels can be monitored andtumors can be imaged.186 Early 2016 saw the development ofa NIR-II targeted PEGylated uorophore (CH1055) probe thatwas 90% cleared by the kidneys within 24 h. The probe wassurface functionalized with an anti-EGFR Affibody. The uo-rophore exhibits a desirable large Stokes shi of 175 nm witha maximum absorption of 750 nm and a maximum emission of1055 nm. In immunodecient nude mice, successful imaging,targeting, and cancer ablation was achieved.187 Silica nano-particles were used to encapsulate palladium(II) tetraphe-nyltetrabenzoporphyrin (donor) and perylene (acceptor) or toencapsulate palladium(II) tetraphenyltetrabenzoporphyrin(donor) and 9,10-bis-(phenylethynyl)anthracene (BPEA)(acceptor) to yield two distinct triplet–triplet annihilationupconversion uorophore pairs, which emit either green orblue. Further, the nanoparticle surface was covalently modi-ed with various peptides or antibodies for active targeting. Itshould be noted that upconversion is an anti-Stokesphenomenon in which at least 2 photons exhibiting lowfrequency are combined and converted into one photon witha high frequency.188

In tumor endothelium targeting, the endothelial cells aretargeted to prevent angiogenesis (production of new bloodvessels), as it plays an important role in regulating cancergrowth. Targeting vascular endothelial growth factors (VEGF)receptor, avb3 integrin, vascular cell adhesion molecule-1(VCAM-1), and matrix metalloproteinases (MMP) are someexamples of tumor endothelium targeting.170,189–193 One study intumor endothelium targeting employed poly(lactic acid) (PLA)nanoparticles that had encapsulated Endostar, a recombinanthuman endostain that is known to inhibit angiogenesis.Further, the PLA nanoparticles had been surface modied witha uorophore, namely NIR IRdye 800CW, and a GX1 peptide,which has been shown to bind to the endothelium. It was foundthat the complex did bind to the endothelium through the invivo uorescence molecular imaging of mice with colorectaltumors. It should also be noted that functionalized PLA nano-particles accumulated better and resulted in a higher uores-cence signal than free uorophore, IRdye 800CW, alone.194 Itwas found that the BODIPY uorophores synthesized by Kimand colleagues could be successfully encapsulated in silica

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Fig. 10 (a) Design of lactoferrin-conjugated biodegradable polymer-some for glioma targeting. Polymersomes are loaded with doxorubicin(Dox), an antitumor drug, and tetrandrine (Tet), a multi drug resistance(MDR) inhibitor, at the same time. Lactoferrin (Lf) was conjugated onthe surface of polymersomes for two reasons: (1) to act as a gliomatargeting ligand and (2) to help overcome the obstruction of theblood–brain-barrier (BBB). (b) Accumulation of targeted polymer-somes in glioma (EPR effect and overcoming BBB) was shown throughfluorescence imaging. (c) The specific interaction of Lf-conjugatedpolymersomes with glioma cells was shown by an improvement in thesurvival rate of rats treated with the system. The improved survival rateis a direct result of the Lf-conjugated polymersomes improving thetherapeutic effect from chemotherapy (reprinted with permissionfrom ref. 124 Copyright 2012 Wiley Periodicals Inc.).

Fig. 11 Internalization of rhodamine B-tagged NPs. Fluorescencemicroscopy images of MCF7 cells show the cellular uptake of non-functionalized (N0) and biotin-functionalized (N1) NPs (red) after 5 h ofincubation. The nuclei were stained with DAPI (blue) and phalloidin-fluorescein isothiocyanate (Ph-FITC, green) which was used to label F-actin. The last column of images is an overlay of all the staining(reprinted with permission from ref. 196 Copyright 2012 American

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(SiO2) nanoparticles. Then the surface of the silica nano-particles could be modied to have an arginine-glycine-aspartic(RGD) peptide that would selectively target avb3 integrin, whichlead to enhanced uorescence imaging with higher signal tobackground ratios when compared to the free uorophoresalone.98

In the majority of the applications, the targeting agent is notused by itself but is attached to a uorophore or a nanocarrier toimprove the accumulation. Most of these targeting agents areattached via a simple covalent conjugation technique orthrough electrostatic interactions. Attaching the targeting agentdirectly to the small molecule uorophore compromises theemission and other properties of the probe, so attaching thetargeting agents to the actual nanocarriers, which does not alterthe properties of the uorophore, is much preferred.107 Inaddition, conjugating the targeting agent to the polymericnanocarrier overcomes weak reproducibility and poor control oftuning the number of targeting agents attached to theparticle.132 Once the polymeric particle is conjugated with thedesired amount of the targeting agent, they can be utilized toaccumulate in a specic tumor for imaging applications.

Zheng and colleagues developed a novel ICG containingphospholipid-polyethylene glycol (ICG-PL-PEG) based micelle.They attached two targeting agents, a small molecule, namelyfolic acid (FA), and a large protein called integrin avb3 mono-clonal antibody (mAb), to the ICG-PL-PEG micelles and dis-played their target specicity using three different cell lines. Thethree cell lines used were EMT6 (murine mammary tumor cells),U87-MG (human glioblastoma cancer cells) and MCF-7 (humanbreast cancer cells). Laser scanning confocal microscopy, owcytometry, and other in vitro experiments were employed toconrm that the targeting probe was mainly internalized intocells via ligand–receptor or antigen–antibody mediated endo-cytosis pathway. In addition, according to their data the amountof targeting agent conjugated to the ICG-PL-PEG micelle doesnot affect the integrity and properties of the nanoprobe.107

Pang and colleagues developed a 1,10-dioctadecyl-3,3,30,30-tetramethylindotricarbocyanine iodide (DiR) (uorophore)loaded methoxy poly(ethylene glycol)-poly(s-caprolactone)based polymersome. Apart from the uorophore the polymer-some was loaded with drugs, doxorubicin (Dox) and tetrandrine(Tet), to give a therapeutic functionality to the system. Thesemultifunctional polymersomes were conjugated with a target-ing agent called Lactoferrin (Lf) and this polymersome wasreferred to as Lf-PO-Dox/Tet-DiR. Lf is a novel brain targetingligand, which enables drug-loaded nanocarriers to transportacross the blood–brain barrier (BBB) with higher efficiencywhen compared to transferrin, another targeting agent. In vitrostudies in C6 cells (rat glial tumor cells) were utilized to showthat Lf-PO-Dox/Tet-DiR polymersomes were uptaken throughreceptor-mediated endocytosis, while the free Dox accumulatedmainly through a diffusion mechanism. Moreover, in vivostudies performed in glioma model rats further conrmed thatthe Lf-PO-DiR crossed the BBB and had an increased accumu-lated at the tumor site, which is indicated by the strong increasein uorescence at the site of the tumor (cf. Fig. 10). Overall, they

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constructed a promising targeted nanoprobe for diagnosis andtherapy of gliomas.124,195

Le Droumaguet and colleagues developed a rhodamine B (u-orophore) functionalized poly(alkyl cyanoacrylate) (RhB-PACA)nanoparticles for uorescence imaging. The RhB-PACA nano-particles were surface functionalized with biologically active tar-geting ligands, such as biotin, curcumin derivatives, and a variety

Chemical Society).

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Fig. 12 (a) Increase in the emission intensity ratio of PA-azICG-azPEG1K particles dispersed in a phosphate buffered solution (PBS)and after the addition of 0.014 mM BSA; the graph depicts the timeevolution of the intensity at 819 nm relative to the initial intensity. Theinset presents emission of particles after 2 min (B), 37 min (C), and1174 min (V). Excitation was at 710 nm; particle density was 1.259 �1012 cm�3. (b) Optical image of emission intensity of PA-azICG-azPEG1K particles in deionized water (far left), PBS (center), and 2 hafter the addition of 0.014 mM BSA to the PBS solution (far right);images were taken with a Caliper Xenogen IVIS Lumina II XR Instru-ment with excitation at 745 nm and emission was observed with anICG emission filter; particle density of 1.259 � 1012 cm�3 (reprintedwith permission from ref. 59 Copyright 2011WILEY-VCH Verlag GmbH& Co. KGaA).

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of antibodies. In vitro studies were performed in two cancer celllines, MCF7 (human breast adenocarcinoma cells) and M109(murine lung cancer cells), which both overexpress biotin recep-tors on their surfaces (cf. Fig. 11). The results obtained conrm thatthe biotin conjugated RhB-PACA nanoparticles were uptaken bythe cells through a specic receptor mediated endocytic pathwayas the uorescence seen was mainly around the vesiclessurrounding the nuclei. Moreover, the uorescence signal ofcovalently conjugated rhodamine B to PACA nanoparticles wassharp and strong in comparison to the typical diffuse signal ob-tained when hydrophobic dyes are encapsulated. The specicity ofbiotin targeting strategy was further supported by in vitro studiesperformed in L1210 cells that do not overexpress biotin. Thebiotin-RhB-PACA nanoparticles did not show the same stronguorescence in L1210 cells as shown in MCF7 and M109 cells.196

5 Activation

One of the issues noticed with polymer-core nanoparticles wasthat the emission of the probe was oen initially quenchedwhen dispersed in aqueous solutions due to the aggregation ofthe uorophore on the surface of the particles.59 This drawbackactually turned into an advantage for nanoparticulate systemsbecause the probes are in a “turned off” uorescence stateunder normal conditions and a “turned on” uorescence stateonly under specic diseased conditions, which results in anenhanced tumor to background ratio.197 To this end, identifyingdifferent activation techniques for nanoprobes is necessary todevelop activatable NIR nanoprobes. To date, various generalactivatable small molecule probes have been developed to imagetumors and in most of these systems, the emission is “turnedon” upon binding to a specic protein, enzyme, or receptor.197

Pham and colleagues reported a probe that is activated uponbinding to matrix metalloproteinase 7 (MMP), a protease over-expressed in tumors.198 Tung and colleagues developed a NIRprobe that activates when interacted with cathepsin D, which isanother protease overexpressed in tumors.199 Urano andcolleagues developed pH-activatable probes based on BODIPYuorophore that had a cancer-targeting monoclonal antibodyconjugated to it.200 There are only a few reported activatablenanoparticle systems. In 2011, Rungta and colleagues, used PAparticles modied with an azide terminated ICG to demonstratethe activation of uorescence when mixed with bovine serumalbumin (BSA) (cf. Fig. 12).59 It is well established in literaturethat albumins can “turn on” the emission of aggregated uo-rophores. Albumins bind to the hydrophobically aggregateduorophore via a combination of hydrophobic, hydrogenbonding, and electrostatic interactions, which deaggregates theuorophores and activates the emission.201 Later in 2011, Palmaand colleagues demonstrated that the emission of BF2 chelatedtetraarylazadipyrromethenes (near infrared dye) conjugatedpoly(styrene-co-methacrylic acid) particles can be activated bysodium dodecyl sulfate (SDS), a surfactant. In addition, theydemonstrated the activation in vitro in MDAMB-231 (breastcancer), HEK293T (kidney), and CAKI-1 (renal cancer) cell lines.In this latter system, the phospholipids in the cell membrane,specically lecithin is responsible for the activation and the

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mechanism is similar to that of SDS.163 These two strategies ofactivation are viable but are not specic. There has beenadvancement in the development of nanoparticle systemswhich respond to various stimuli and environments to activateemission (cf. Fig. 5),106,113,202 but less advancement has been seenfor particle-based systems which activate emission uponbinding to specic proteins or receptors that are overexpressedin tumors. It is expected that future endeavors in the eld oftumor nanotheranostics will be in the direction of developing“smart” activatable NIR nanoparticles. For example, the wellknown uorescence quenching effects of gold nanoparticleswas employed by the Mirkin group203 to develop “nanoares”which are designed to provide an intracellular emission signalthat directly correlates with the concentration of a specicnucleic acid or other molecular target. These particles areoligonucleotide-functionalized gold nanoparticles that are tag-ged (i.e. hybridized) to short, uorophore-labeled probes.Without a target, the uorophore is close to the surface of thegold particle and the uorescence is quenched, while binding tothe target releases the uorophore, resulting in a signal.Similarly, activation was achieved for a BODIPY probe cova-lently conjugated to N-benzyl-4-hydroxyaniline. N-Benzyl-4-hydroxyaniline is sensitive to nitric oxide, which is respon-sible for regulating a wide variety of physiological processes;however, when the amount of nitric oxide becomes unbalanced,it can be a cause or indicator of cancer. Therefore, detectingirregular levels of nitric oxide is desirable. Originally, theBODIPY/N-benzyl-4-hydroxyaniline conjugate is nonuorescentdue to N-benzyl-4-hydroxyaniline quenching the uorescence ofBODIPY. However, upon interaction with nitric oxide, the N-benzyl-4-hydroxyaniline undergoes a nitrosation reaction,which effectively activates the uorescence of BODIPY andserves as a visual indicator of nitric oxide. Further, the amountof nitric oxide can be detected by monitoring the emission seenfrom the BODIPY uorophore.204 Yuan and colleagues createda novel nanoprobe with a derivatized tetraphenylethylenecovalently attached to 2,4-dinitrobenzenesulfonyl. It should be

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noted that tetraphenylethylene is a commonly used uorophoreexhibiting aggregation induced emission. However, this absorp-tion of the uorophore is in the UV range of the electromagneticspectrum, which decreases the effectiveness of the uorophore inuorescence imaging. Therefore, tetraphenylethylene was modi-ed to have a dicyanovinyl group andmethoxy group, which act asa donor/acceptor pair which redshis the absorption of the uo-rophore. Upon reaction with 2,4-dinitrobenzenesulfonyl, the uo-rescence of tetraphenylethylene is quenched. However, uponcellular uptake and interaction with biothiols, the quencher iscleaved from the uorophore, which activates the uorescence oftetraphenylethylene, and a high signal to noise ratio was observed.Tetraphenylethylene is a singlet oxygen generator and with MDA-MB-231 cells, cell death was observed. The nanoprobe wasfurther functionalized with a cyclic RGD probe, providing activetargeting for cells overexpressing avb3 integrin.205 Nanoparticlesmade from curcumin (Cur), a chemotherapy drug, with encapsu-lated perylene and 5,10,15,20-tetra(4-pyridyl) porphyrin (H2TPyP)(a donor–acceptor pair) were synthesized. The intact system, uponinitial cellular internalization, have no therapeutic effect, and theyhave a red emission due to FRET between the uorophores, whichenhances the effect of photodynamic therapy due to the increaseduorescence emission cause by FRET. Aer some time, Curnanoparticles begin to dissociate, treating cancer via chemo-therapy, while the uorescence of the system shis from red togreen. Cur emits green, and, due to the disintegration of the Curparticles, FRET is no longer achievable between perylene andH2TPyP. However, any part of the intact Cur particles will continueto emit red; therefore, this system can be used to estimate real timedrug dosage.206

6 Perspectives and conclusions

Strides have been made in ensuring that uorescence imaging isan enhanced way to detect cancer in its early stages without thedrawbacks of traditional methods. Great care has been taken inimproving the uorophores used for imaging, such as couplingthem with nanocarriers, which has ameliorated many of thedownfalls of the currently FDA approved uorophores, such asICG and photofrin. Further advancements have beenmade in thedelivery of these uorophores to the intended cancerous or pre-cancerous site to provide physicians with as much informationabout the tumor as possible while limiting the side effects for thepatient (i.e. radiation exposure). Little doubt remains that theseuorophores and their nanocarriers will eventually revolutionizethe way cancer is diagnosed and treated.

Acknowledgements

The authors thank the Gregg-Graniteville Foundation and theNational Science Foundation (DMR-1507266) fornancial support.

References

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