Advanced Review

Polymeric nanoparticles for molecular imaging R. Srikar,1 Anandhi Upendran2 and Raghuraman Kannan1,3∗ Conventional imaging technologies (X-ray computed tomography, magnetic resonance, and optical) depend on contrast agents to visualize a target site or organ of interest. The imaging agents currently used in clinics for diagnosis suffer from disadvantages including poor target specificity and in vivo instability. Consequently, delivery of low concentrations of contrast agents to region of interest affects image quality. Therefore, it is important to selectively deliver high payload of contrast agent to obtain clinically useful images. Nanoparticles offer multifunctional capabilities to transport high concentrations of imaging probes selectively to diseased site inside the body. Polymeric nanoparticles, incorporated with contrast agents, have shown significant benefits in molecular imaging applications. These materials possess the ability to encapsulate different contrast agents within a single matrix enabling multimodal imaging possibilities. The materials can be surface conjugated to target-specific biomolecules for controlling the navigation under in vivo conditions. The versatility of this class of nanomaterials makes them an attractive platform for developing highly sensitive molecular imaging agents. The research community’s progress in the area of synthesis of polymeric nanomaterials and their in vivo imaging applications has been noteworthy, but it is still in the pioneer stage of development. The challenges ahead should focus on the design and fabrication of these materials including burst release of contrasts agents, solubility, and stability issues of polymeric nanomaterials. © 2014 Wiley Periodicals, Inc. How to cite this article:

WIREs Nanomed Nanobiotechnol 2014. doi: 10.1002/wnan.1259

INTRODUCTION

M

olecular imaging (MI) can detect functional changes at the cellular level and possesses the ability to detect diseases at an early stage.1–3 In the field of oncology, MI not only detects cancer at an early stage but also aids in the staging of cancer and therapeutic response.4–7 For example, MI holds the potential of detecting cancer lesions less ∗ Correspondence

1 Department

to: [email protected]

of Radiology, University of Missouri, Columbia, MO,

USA 2

Department of Physics, University of Missouri, Columbia, MO, USA 3 Department of Bioengineering, Center for Micro/Nano Systems and Nanotechnology, University of Missouri, Columbia, MO, USA Conflict of interest: The authors declare no competing financial interest.

than 0.5 cm3 in volume. Consequently, detection of disease at its embryonic stage provides a much greater potential for complete recovery. The assistance MI provides toward understanding the molecular changes within the region of interest in body helps physicians develop a highly patient-specific treatment regimen (personalized therapy).4–7 In fact, highly sensitive molecular imaging agents are being developed at a rapid pace to identify the abnormalities within human body.8 These agents will provide future advances in early diagnosis and therapy for several diseases. Nanoparticles are emerging as a new class of MI agents for detecting human diseases.9–15 Advantages for utilizing nanoparticles as MI agents include: (1) smaller size facilitates internalization and probing of cells; (2) payload carrying capacity delivers high concentrations of imaging agents to desired region; (3) multiplexing ability provides opportunity to attach a variety of molecular signaling and receptor

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Monomer Type 1 Nanoconstruct

Type 2 Nanoconstruct

Dye conjugation

Polymerdye solution

NP formation while polymerization

Dye conjugation

Polymerization

NP formation

NP formation while polymerization

Monomerdye solution

NP formation

Preformed polymer

Encapsulation route

Conjugation route

targeting molecules on the surface; (4) multimodal potential offers visualization in more than one imaging modality; and (5) theranostic capability enables detection and treatment of disease using a single nanoconstruct. Polymeric nanoconstructs encapsulated with therapeutic pharmaceuticals have shown excellent success in preclinical and clinical studies.16 Several drugencapsulated polymeric nanoparticles are approved by the US Food and Drug Administration (FDA) for human consumption.16 However, the potential of polymeric nanoparticles have been underutilized in the development of MI agents. The paucity in the data is mainly attributed to challenges associated with poor stability, and rapid release of imaging agent under in vivo conditions.

CLASSIFICATION OF POLYMERIC NANOCONSTRUCTS Polymeric nanoconstructs for imaging applications can be generated by either covalent conjugation (Type I) or physical encapsulations (Type II) of contrast agents to polymeric matrix. In the covalent conjugation method, molecules with imaging characteristics were bound to polymeric backbone to yield Type I nanoconstructs. The grafted polymers were subsequently converted into nanoparticles by conventional techniques. The major advantage of Type I nanoconstructs is that the contrast agent is covalently bound to polymer; thus, the burst release does not occur. However, disadvantages

FIGURE 1 | General synthetic route for Type I and Type II based nanoconstructs.

include poor loading efficiency and nonhomogeneous distribution of contrast agents within polymeric matrix. Type II nanoconstructs were obtained by physical encapsulation of contrast agent within polymeric nanoparticles. These nanoconstructs may have superior advantages over Type I nanoconstructs because of high loading efficiency of contrast agents and homogenous distribution within the polymeric matrix. But, controlling the burst release of contrast agents from nanoconjugates within biological system remains a significant challenge. The general synthetic route for Type I and Type II nanoconstructs are shown in Figure 1. Tables 1 and 2 list polymeric nanoconstructs that were investigated for imaging applications.

POLYMERIC X-RAY CONTRAST AGENTS X-ray computed tomography (CT) is a common imaging modality that is widely used in clinics for detection, diagnosis, and treatment of tumor, vascular, and other diseases.60,61 It is a noninvasive imaging technology with high spatial resolution. CT imaging utilizes special X-ray instruments to probe inside the body. Significant technological developments in manufacturing of CT instruments have resulted in dual-energy and multislice CT.62–65 In addition, the advent of dual-energy CT imaging modality enables differentiation of contrast materials from biological tissues and bone.66 CT clarity coupled with threedimensional image enables radiologists to predict the

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Polymeric nanoparticles for molecular imaging

TABLE 1 Type I Polymeric Nanoconstructs for Imaging Applications Polymeric Nanoconstruct

Contrast Enhancers

Size (nm)

Modality

Ref

Iohexol

150

CT

17

MAOETIB (2-hydroxyethyl methacrylate)

2,3,5-triiodo benzoic acid

30

CT

18

MAOETIB-GMA (2-hydroxyethyl methacrylate, glycidyl methacrylate)

2,3,5-triiodo benzoic acid

—

CT

19

Cy5

50–100

FL

Polyiohexol (hexamethylene diisocyanate-mPEG-polylactide)

Cyanine-PLA (polylacticacid)

20 21–23

Cyanine-GC (glycol-chitosan)

Cy5.5

231–310, 35–97, 150

FL

Cyanine-PEI-DOCA (polyethylene imine)

Cy5.5

—

FL

24

Cyanine-PEG (polyethylene glycol)

Cy5.5

10.5

FL/MR

25

Cyanine-MMA (poly(PEGethacrylate)-b-oly(triethoxysilyl propylmethacrylate))

Cy7

24

FL

26

Squaraine-PS (polystyrene)

SCEP

100

FL and CL

27

Squaraine-PS (polystyrene)

SREP

100

FL and CL

28

Squaraine-BSA (bovine serum albumin)

NPBT

100

FL

29

Anti-VEGF-PLA-PEG-PLL-Gd (polylactic acid-polyethylene glycol-poly-L-Lys)

Gd-DTPA

82.5 ± 5

MR

30

PHEA-mpeg-ED-DOTA-Gd (polyhydroxyethylaspartamide)

Gd-DOTA

180

MR

31

PCL-b-P(OEGMA-FA) and PCL-b-P(OEGMA-Gd) (polycaprolactone–polyethyleneglycol-monomethyl ether methacrylate)

Gd-DOTA

28

MR

32

Gd-DOTA-alkynyl

67 ± 10

MR

33

Gd-C-DOTA

59

MR

34

Gd-DTPA

97–212

MR

35

Gd-DOTA-alkynyl

68

MR

36

Poly-Gd-Dox (Starch, polymethyl methacrylic acid, polysorbate80) Polysulfide-Gd-DNC (polydisulfide dendrimer) PSI-(mpeg)-GdDTPA (polysuccinimide) P(NIPAM-co-NBA-co-Gd) (poly N -isopropylacrylamide, poly O -nitrobenzylacrylate) CT, computed tomography; FL, fluorescence; MR, magnetic resonance.

presence or absence of a tumor, the tumor’s size, and more importantly, its precise location.67–70 Even metastasis of tumor can be predicted.71 Likewise, the clarity in CT images allows early identification of vascular disease, heart attack, and stroke.72 Indeed, multislice CT is showing high promise in coronary angiography.72 Interestingly, artifacts associated with breathing and others can also be eliminated by faster scan time. Even though the instrumentation technology has been improved, an X-ray contrast agent is still needed to delineate the region of interest and its surroundings. In a typical scan, a contrast agent is administered before imaging is performed. The resultant scans or images aid in visualizing internal organs, bone, soft tissues, or blood vessels with excellent clarity.73,74 Nontoxic molecules containing elements with a high Z-number and excellent X-ray attenuation characteristics are used as contrast agents.75–81 For example, iodinated aromatic

molecules and barium sulfate (for gastrointestinal purposes) are FDA approved for use as contrast agents in humans.75–80 However, much research has been devoted to developing bismuth, and gold containing molecules as X-ray contrast enhancers.75–80 Iodinated small molecules are used as X-ray contrast enhancers to highlight internal organs and blood vessels.81 Even though, iodinated contrast agents have become ‘work horses’ for CT imaging, they have several disadvantages.82,83 Iodinated agents are not suitable for patients with renal diseases and allergic reactions have been reported in many clinical cases.82,83 However, one of the major disadvantages of small molecule iodinated contrast agents is rapid clearance from the body via renal excretion. Repeated injections of iodinated contrast agents could cause adverse reactions in humans. Therefore, CT imaging should be performed immediately after administration (very narrow time window) in the region of interest

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TABLE 2 Type II Polymeric Nanoconstructs for Imaging Applications Polymeric Nanoconstruct

Contrast Enhancers

Size (nm)

Modality

Ref

Iodinated oils

84–101

CT

37

Iodostearic acid, ethyl diiodo stearate

150

CT

38

Gold nanoparticles/Cy7

—

CT/FL

39

PAH-cyanine (poly(allylamine hydrochloride))

ICG

60–100

FL

40

Dextran-cyanine (Dextran)

ICG

500,100

FL/MR

41

Dextran-cyanine (PEG–dextran(−SS–NH2))

ICG

171

Ormosil-PEBBLE-cyanine (phenyltrimethoxysilane, methyltrimethoxysilane)

ICG

200–300

FL

PLGA-cyanine (poly(lactic-co-glycolic) acid))

ICG

137–16744

FL

EUDRAGIT-cyanine (poly(methylmethacrylate)

ICG

100

FL

49

PLGA-porphyrin (poly(lactic-co-glycolic) acid))

meso-tetra(4-hydroxyphenyl)porphyrin

93–157

FL

50

PLA-porphyrin (poly lactic acid)

meso-tetra(4-hydroxyphenyl)porphyrin

118

FL

51

PLGA-pthalocyanine (poly(lactic-co-glycolic) acid))

Zinc(II) phthalocyanine

285

FL

52

Polymersomes-PMAM-Gd-DNC (polyamidoamine)

Gd-DTPA

130

MR

53

Gd-DOTA, Gd-DTPA

241, 244

MR

54

Polymer vesicles (poly(lactic-co-glycolic) acid)

Gd-DTPA

—

MR

55

GC-SPION (glycol, glycidol blocked chitosan)

SPION

34, 36

MR

56

Folate-PEG-PCL-SPION (polyethylene glycol and polycaprolactone)

SPION

40

MR

57

Nanocage polymersomes (lipid containing polymers)

SPION

40

MR

58

Gd-DTPA

80

MR

59

PBD-PEO-Iodine (poly(butadiene)-b-poly(ethylene glycol)) cROMP (poly(styrene)-b-(polyacrylic acid)) Multicomponent (PLGA)

Polymer hydrogels (chitosan and hyaluronic polymers)

PAA-SPION (polyacrylic acid coated iron oxide)

42 43

44–48

CT, computed tomography; FL, fluorescence; MR, magnetic resonance.

before the contrast agent is eliminated. If CT imaging cannot be performed within the narrow time window, incorrect evaluation of disease can occur; hence, recent works by several groups focus on developing contrast agents to retain within specific regions in the body and decrease rate of elimination.82,83 In this context, polymeric nanoparticles encapsulated with X-ray contrast enhancers are expected to provide significant clinical benefit. Current progress in the development of polymeric nanoconstructs (Type I and Type II) as X-ray CT contrast agent is reviewed here.

Type I Several interesting studies have been reported on synthesis and in vivo CT imaging characteristics of Type I nanoconstructs.17–19,84 For example, clinically used iohexol (Omnipaque™) was cross-linked with hexamethylene diisocyanate (HDI) followed by nanoprecipiation using mPEG-polylactide to yield Type I nanoconstruct.17 The hydroxyl groups present in iohexol were O-acylated with HDI to yield a crosslinked product. The resultant hydrophobic product

facilitates formation of nanoparticles by conventional precipitation technique to yield Poly(iohexol) nanoparticles (NPs) of size approximately 150 nm. The CT imaging qualities of poly(iohexol)NPs were compared with iohexol in breast tumor-bearing nude mice.17 Contrast agents were administered intratumorally, and CT attenuation in the tumor region was measured using Hounsfield units (HU) in different time points. Five minutes postadministration, both iohexol and NPs showed similar HU (difference in HU value before and after administration). Interestingly, 4 h postadministration, CT signal of mice treated with NPs were approximately 36 times higher than that of iohexol (Figure 2).17 Intravenous administration of NPs in mice showed longer circulation half-life (15.9 h) compared with iohexol (3.8 h). Consequently, renal and tissue clearance properties of these contrast agents have proven to be strikingly different.17 Above all, the toxicity studies in mice revealed that NPs with dosage up to approximately 300 mg/kg body weight did not show any noticeable side effects. In another study, 2,3,5-triiodobenzoic acid (TBA) was

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Polymeric nanoparticles for molecular imaging

(a) 5 min

1h

4h

Pre

5 min

1h

4h

Poly(iohexol) NP

Iohexol

Pre

(b)

Poly(iohexol) NP Iohexol

ΔHU (HUt – HU0) of tumor

120 100

3.1 x

80 35.8 x 60 40 20 0

5 min

1h

4h

FIGURE 2 | (a) Comparative X-ray computed tomography (CT) imaging study (serial axial images) in mice bearing MCF-7 tumor after injection

with 200 μL of iohexol (upper panel) and poly(iohexol) nanoparticles (lower panel) at 50 mg iohexol/kg. Yellow arrows indicate contrast enhancements in tumor. (b) Change in Hounsfield unit (HU) after administration of poly(iohexol)NPs or iohexol. (Reprinted with permission from Ref 17. Copyright 2013 American Chemical Society)

attached to tissue with 2-hydroxyethyl methacrylate (HEMA) to obtain monomer 2-methacryloyloxyethyl (2,3,5-triiodobenzoate) (MAOETIB) and subjected to emulsion homopolymerization technique to obtain MAOETIB-NPs of hydrodynamic size ∼30 nm. However, the aqueous dispersions were not stable and resulted in agglomeration when the concentration of NPs was greater than approximately 0.3%.18,19 Poor stability of the NPs in high concentrations have resulted in low CT contrast in in vivo animal models.18,19 Modified NPs, P[MAOETIB-GMA]NPs have shown stability with 15 weight% of NPs.17 CT imaging studies in animals have also shown enhanced X-ray contrast in blood, lymph nodes, spleen, and liver.19 Dendrimers conjugated with iodine containing aromatic compounds have been developed for potential utilization as CT imaging agents.84 Type I nanoconjugates did not show any burst release of contrast agents. However, only very low concentrations of contrast agents are covalently attached to the matrix resulting in relatively weak contrast enhancements.

Type II Nanoconstructs of this class can be generated by encapsulating the contrast agent within a polymeric matrix.37–39 There are several pathways to incorporate iodinated contrast agents within the nanoconstruct. deVries et al.37 found that iodine containing aromatic compounds were attached to long chain aliphatic molecules and converted into hydrophobic (oily) material; subsequently, these materials were emulsified in the presence of polymers to obtain nanoparticulate contrast agents. Significant advantages of this method include high encapsulation efficiency, homogenous distribution, and structural Poly(butadiene)-b-poly(ethyleneglycol) integrity.37 (PBD-PEO) polymers were emulsified in presence of hydrophobic iodinated oils to obtain nanoconjugate of size 84 to 101 nm, depending on the molecular size of contrast agents incorporated.37 The in vivo CT imaging efficacy of PBD-PEO-Iodine-NPs, with 520 mg I/kg, as contrast agents have been extensively investigated in mice models.37 After injection, the change in contrast in blood reaches an average value of 190 (HU). In this experiment,

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the blood circulation half-life of these NPs was 3 h. The nanoparticle excreted out within 1 week of injection. It is worth noting that the Gruell and coworkers observed no toxicity in treated mice.37 In another study, Lanza et al.38 showed encapsulation of a mixture of iodinated compounds iodostearic acid and ethyldiiodostearate (37% iodine content) in Poly(styrene)-block-poly(acrylic acid) [PS-b-PAA] polymer.38 In order to prevent the release of iodinated compounds, the encapsulated polymer was immediately cross-linked with diamine containing compounds. These soft colloidal radio-opaque and metal encapsulated (cROMP) nanoparticles (∼150 nm) exhibited very high entrapment efficiency (∼96%).38 Most interestingly, the –COOH groups were available after cross-linking of cROMP, and the carboxylate was subsequently utilized to conjugate with biomolecules (e.g., biotin).38 Detailed analysis showed that each cROMP nanoparticle contains 2,304,841 iodine atoms and 1000 biotin molecules on the surface. Stability of these nanoparticle exceeded 6 months.38 Methodology to entrap iodinated molecule in cROMP can be extended for other CT-active metals such as gold, bismuth, or titanium.38 In vivo CT imaging studies in rat models showed that blood half-life is six times longer (∼56 min) than the clinically used iodinated contrast agents (10 min). All the major organs were visible after intravenous injection of cROMP with a dosage of 6 mL/kg (Figure 3).38 Langer and coworkers39 synthesized highly complex multifunctional nanoparticles with polymer core. In this construct, PLGA polymer is encapsulated with gold nanoparticles and explored as CT agent. In addition to gold nanoparticles, doxorubicin was also loaded. Langer et al. also coated the PLGA core with lipid monolayers.39 Lipid molecules enable subsequent functionalization and also increase biocompatibility. Lipid was coated with polyethylene glycol (PEG) and coencapsulated with sorafenib. PEG coating within the nanoparticle was labeled with cy7 fluorophores to visualize by near-infrared fluorescence (NIRF) imaging.39 Pilot in vivo studies using NIRF confirm the accumulation of these nanoparticles within tumor site.39 This study shows the utility of polymeric nanoparticles to develop multifunctional nanoconjugates for imaging applications.

POLYMERIC OPTICAL CONTRAST AGENTS Noninvasive optical imaging combined with polymeric nanoscience is currently providing an excellent opportunity in the field of cancer diagnostics and directly assists in effective therapy. Currently

available optical imaging systems complement the polymer-based fluorescent nanoparticles in achieving molecular level detection of tumor margins.20,85 Fluorescent-based MI predominantly pertains to a specific class of fluorescent dyes known as near infrared dyes (NIRF). It is important to realize that there are other non-NIRF probes that can be brighter and useful in fluorescence-guided surgery. However, the fluorescence of NIRFs has the capability of deep tissue penetration with reduced background noise effects and hence is best suited for clinical diagnostics and imaging.86,87 NIRF can be further classified into several subcategories such as cyanine, squaraine, phthalocyanine, and porphyrin derivatives. In addition to these dyes, inorganic materials such as quantum dots (QDs) exhibit excellent fluorescent properties. However, a detailed understanding of toxicity profile, biodistribution, and cellular kinetics for QD-based technology for human application is still lacking and therefore not covered in this review.88 Also, a detailed review pertaining to the molecular characteristics of these dyes can be found elsewhere.89 This section focuses on the polymeric nanoconstructs containing inherently noncytotoxic fluorescent dyes and their in vivo imaging characteristics. The need for formulation of NIRFs into nanoparticulate systems arises due to the inability of pristine NIRFs to localize within targeted tissues. Indocyanine green (ICG), for example, undergoes rapid clearance from physiological conditions accompanied with fluorescence quenching and has a blood circulation half-life of 2–4 min.90 The rate of plasma clearance of ICG follows exponential decay and is approximately 18.5% per min.91 To overcome the barrier, it is prudent to incorporate ICG within biocompatible nanoconstruct to retain its inherent fluorescent properties for effective biological imaging. As presented in the previous section on CT imaging agents, synthesis of polymeric optical imaging nanoconstructs uses two generic routes (as shown in Figure 1) namely: (1) chemical conjugation of fluorescent agents to polymers and polymeric NPs (Type I) and (2) encapsulation of fluorescent agents within NPs (Type II).39,45,92–94 Type I nanoconstructs can be generated by conjugating selective cyanine dyes to polymer matrix. The conjugated dye molecules are directly exposed to physiological environmental conditions affecting various intrinsic fluorescent properties of the conjugated moiety.95 Type II constructs can be generated by encapsulating NIRF dyes within the desired polymeric nanoconstruct.21,40,96 The encapsulation of NIRF dye within polymeric nanoparticles generally leads to the initial burst release,96 the consequences of which may be ineffective imaging.

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Polymeric nanoparticles for molecular imaging

0 min 1

3

2.5 h

0 min

1h

2h

3h

3.5 h

2 4

2h

3h

5

FIGURE 3 | Serial X-ray computed tomography (CT) images of a rat following tail-vein injection of cROMP (6 mL/kg). Major organs shown above include liver (1), spleen (2), kidney (3, 4), and gastrointestinal track (5). (Reprinted with permission from Ref 38. Copyright 2009 American Chemical Society)

This section presents briefly the synthetic procedure adopted for Type I and Type II nanoconstructs and their in vivo applications. Of particular interests are in vivo imaging studies wherein the effect of fundamental physiochemical properties of the molecular probes is shown interacting with their surrounding environment under physiological conditions.

Type I In this class, polymeric matrix of the nanoconstruct is covalently linked with ‘Cy’ analogs of cyanine

NIRF dye family (Cy5, Cy5.5, and Cy7) due to their amine-reactive properties. Among these dyes, Cy5.5 has been successfully used for targeted optical imaging of subcutaneous U87MG glioblastoma tumorbearing nude mice.97 Examples of Type I nanoconstructs include Cy5-PLA nanoparticles,20 Cy5.5Glycol-Chitosan,21–23 Cy5.5-PEI,24 Cy5.5-PEG,25 and Cy7-polymethylmethacrylate copolymer.26 To study the effect of size and molecular weight of nanoconstructs on in vivo imaging, glycol-chitosan nanoparticles (20 kDa–231 nm, 100 kDa–271 nm, and 250 kDa–310 nm) conjugated with Cy5.5 were

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synthesized.21 The glycol-chitosan nanoparticles were covalently conjugated to Cy5.5 in the presence of 1 wt.% hydroxysuccinimide ester in DMSO at room temperature for 6 h. The unreacted dye molecules were dialysed, and the resulting nanoconstruct was subsequently lyophilized and used for in vivo analysis.21 The nanoconstruct was intravenously administered to Athymic nude mice bearing subcutaneous SCC7 tumors. The results indicated higher blood circulation time for 250 kDa nanoparticles (maintained up to 3 days) when compared with the other two sets of NPs (6 h).21 Tumor-targeting efficiency through enhanced permeability retention (EPR) effect was monitored via fluorescent imaging intensity; the results showed that 250 kDa NPs were 4.1- and 2.4-fold higher than that of 20 kDa and 100 kDa, respectively.21 Biodistribution studies revealed high fluorescence intensities for 20 kDa (231 nm) and 100 kDa (271 nm) nanoconstructs in the kidney 1 h postinjection. Nevertheless, good fluorescent signal was observed throughout the body in the case of a 250-kDa nanoconstruct (Box 1). BOX 1 SIZE VERSUS TUMOR UPTAKE It is important to recognize that 250 kDa is 310 nm and showed higher tumor uptake21 ; this result contradicts several reports which demonstrated that higher tumor targeting efficacy is usually obtained by smaller sized nanoparticles than larger sized particles.98–100 Therefore, NIRFpolymeric modality cannot only be restricted for molecular imaging and diagnostics applications but can also be employed for understanding of fundamental science that underlies the behavior of nanoparticles within physiological conditions.

In case of larger animals or humans, it would always be preferred if the fluorescence was observed only in the targeted location. An interesting approach for such modality would be activation of fluorescence ONLY at the targeted sites enabling improved imaging and high contrast. In this direction, a self-assembled chitosan nanoparticle conjugated with fluorogenic Cy5.5-peptide-BHQ-3 was developed.23 BHQ-3 is a NIRF dark quencher.101 The nanoconstruct was synthesized using self-assembly of glycol chitosan in water and subsequent conjugation of activatable fluorogenic peptide, Cy5.5-Gly-Pro-Leu-Gly-Val-ArgGly-Lys(BHQ3)-Gly-Gly in the presence of catalyst. Nanoconstruct upon contact with the specific matrix metalloproteinase (MMP), cleaves the substrate and the NIRF dye. The release of Cy5.5 dye leads to

Active MMPs

Polymer NPs

Multi-Nirf quenched

Activated

FIGURE 4 | Matrix metalloproteinase (MMP) enzyme-activated fluorescent polymeric nanoconstructs for imaging applications. (Reprinted with permission from Ref 23. Copyright 2009 American Chemical Society)

fluorescence as a result of dequenching as shown in Figure 4.23 To investigate the in vivo efficacy, the nanoconstruct was intravenously injected to MMP2/9-positive SCC7 tumor-bearing mice under two different conditions: (1) without pretreatment (direct) and (2) with pretreatment using a MMP-2/9 inhibitor. The results indicated increased fluorescence in case of direct modality while the pretreated model showed considerably reduced fluorescent intensity as shown in Figure 5.23 Few studies have covered the synthesis of squaraine-dye-incorporated polymeric nanoparticles. The primary focus of this synthesis is based on staining or labeling of dyes onto the nanoparticles. Examples of squaraine-polymeric NPs include Squaraine Catenane Endoperoxide (SCEP). This dye is a bicomponent, comprised of chemiluminescent macrocyclic endoperoxide and fluorescent squaraine chromophore. SCEP conjugated on carboxyl functional polystyrene (PS) nanoparticles via particle swelling technique was used to analyze the potential of the nanoconstruct for whole-body fluorescence and chemiluminescence imaging independently in normal mice.27 The results of the whole body imaging showed a striking difference between fluorescent and chemiluminescent imaging.27 It is worthy to note that the fluorescent intensity is highly ‘surface weighted’, that is, organs closer to the scan area would result in ‘high florescent intensity’ while organs and tissues located in larger depths would generate relatively lesser intensity. In the study,27 the florescence imaging showed lesser accumulation of nanoparticles in the lungs and higher uptake in the liver. In contrast, the chemiluminescence imaging indicated lesser accumulation of the nanoparticles in liver compared to lungs. Presumably, the sensitivity of the imaging modalities plays a role in showing accumulation status.27 Squaraine rotaxane endoperoxide (SREP) was another squaraine derivative optical contrast agent, which was conjugated on PS nanoparticles via a similar swelling technique.28 The results of the study confirmed the superiority

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Polymeric nanoparticles for molecular imaging

(a) 1h

2h

1h

Intensity (NC)

2h

1.47e+005

1.1e+005

7.38e+004

3.74e+004

1e+003

+ Nanosensor/ − inhibitor

SCC7 tumor

(c)

(d) Z (mm)

20

Tu

Liv

Lu

H

Sp

A

Kid

15

A

3

10

B

4

C

5

0

− Inhibitor

(e) A

C

B

5

+ Inhibitor

(g)

(f) B

D

1 cm

Fluorescence intensity (a.u.)

Tumor ROI and T/N ratio (a.u.)

(b)

+ Nanosensor/ +Inhibitor

5000 4000 3000 2000 1000 0 0

3

6

9

12

15

Relative MMP 2/9 activity

FIGURE 5 | (a) In vivo near-infrared fluorescence NIRF tomographic images of subcutaneous SCC7 tumor-bearing mice after intravenous injection of the nanosensor (NS) with or without the inhibitor. (b) Tumor region of interest (ROI) and T/N ratio. (c) NIRF images of excised SCC7 tumor and organs. (d) Two-dimensional slices of the tumor images. (e) NIRF microscopy of SCC7 tumors injected with nanoconstruct. (f) NIRF images of excised NS-treated SCC7 tumors from mice with different size tumors. (g) Correlation between tumor grades (total fluorescence intensity) and the relative MMP-2/9 activity of excised SCC7 tumors. (Reprinted with permission from Ref 23. Copyright 2009 American Chemical Society)

of deep tissue imaging by chemiluminescence compared to fluorescence. Several other squaraine dyes namely N-propanesulfonate-benzothiazolium (NPBT) squaraine and N-propanesulfonatebenzoindolium (NPBI) squaraine labeled to bovine serum albumin (BSA) nanoparticles synthesized via desolvation method have also been explored for optical imaging.29,102

Type II ICG is a tricarbo cyanine, NIRF dye utilized for optimal imaging in humans.103,104 It is approved by US-FDA for various clinical applications.103,104 Examples of Type II nanoconstructs with encapsulated

ICG include poly(allylamine hydrochloride), dextran, and PLCL-Poloxomer-based nanoparticles synthesized through electrostatic assembly, poly(lactideco-glycolide (PLGA)) nanoparticles synthesized via spontaneous emulsification solvent diffusion (SESD) or double emulsion method, ormosil PEBBLES using sol–gel combined emulsion polymerization and Eudragit-based nanoparticles synthesized via precipitation method.40–49,105,106 Kim and coworkers synthesized ICG-encapsulated pluronic nanoparticles via solvent evaporation method and intravenously administered the nanoconstruct in mice bearing CT-26 colon carcinoma.107 The studies showed both ICG and ICG-nanoconstruct accumulated more in liver and kidney compared with

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(a)

36 h postinjection Free ICG

5 d postinjection

ICG/PF-127

Free ICG

ICG/PF-127 10.0 9.0 8.0 ×107

7.0

Tumor

6.0 5.0 Radiant efficiency p/s/cm2/sr μW/cm2

(b)

(c) Spleen

Kidney Heart

Lung

Tumor

7×107

1.2

Free ICG

1.0 0.8 ×108 0.6

ICG/PF-127

0.4

Fluorescence efficiency [(p/s/cm2/sr)/(μW/cm2)]

Liver

Radiant efficiency p/s/cm2/sr μW/cm2

Free ICG ICG/PF-127 micelle

6×107 5×107 4×107 3×107 2×107 Liver

Spleen Kidney Heart

Lung

Tumor

FIGURE 6 | In vivo whole-body imaging of CT-26 tumor-bearing mice injected with Indocyanine green ICG and ICG-encapsulated nanoconstruct. (Reprinted with permission from Ref 107. Copyright 2010 Springer)

tumor 1.5 h postinjection period. However, the fluorescent images recorded 36 h postinjection period showed higher accumulation of nanoconstruct in tumor compared to liver as shown in Figure 6.107 Free ICG, on the other hand, showed relatively lesser fluorescence than nanoconstruct and was found to be evenly distributed throughout the body.107 The results validate high retention efficiency of nanoconstruct within tumor sites, confirms retarded fluorescence quenching and lower clearance in physiological conditions. In addition to ICG, porphyrin, and its derivatives have been encapsulated within polymeric nanoparticles for optical imaging. Most porphyrin derivatives have an inherent hydrophobic characteristic responsible for its affinity toward neoplastic tissues.108 Phthalocyanine, a second generation photosensitizer, exhibits enhanced photoluminescence relative to porphyrins.109 Examples of porphyrin and phthalocyanine derivative incorporated polymeric nanoparticles include meso-tetra(4-hydroxyphenyl)porphyrin within PLGA and PLA nanoparticles synthesized via SESD method,50,51 Hexadecafluoro zinc phthalocyanine encapsulated in PLA nanoparticles was

synthesized via salting-out technique,110 and zinc(II) phthalocyanine encapsulated in PLGA nanoparticles was synthesized via SESD method.52 A detailed study of formulation for synthesizing stable polymeric nanoformulation with porphyrin or phthalocyanine having high blood circulation time could lead to a promising optical imaging agent. Although most of the porphyrin and phthalocyanine have been applied to photodynamic therapy in reported literature, the fact remains the compounds exhibit NIRF properties. The interest in noninvasive optical imaging is experiencing exponential growth with the emergence of robust new NIRF imaging instruments. The Fluorescence-Assisted Resection and Exploration (FLARETM ) intraoperative NIRF imaging system was developed and applied in the first-ever human clinical trials for mapping sentinel lymph nodes (SLN) in breast cancer.111 In this work, ICG–HSA solution was injected intratumorally and subcutaneously in six human subjects and the FLARETM imaging system was used to optically image the SLN. The successful results, which conformed to that of lymphoscintigraphy, are expected to bring a revolution in optical imaging. Nevertheless, the technology is also a tremendous

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Polymeric nanoparticles for molecular imaging

leap forward toward MI in humans using fluorescent agents. The authors also mentioned in their study that future applications of FLARETM technology depend upon the development of specific contrast agents. Thus, the need of the hour for exploiting the maximum potential of such imaging technologies is to develop suitable and specific optical contrast agents. Development of such probes would not only complement current technology but would also be responsible for developing highly sophisticated NIRF imaging systems for comprehensive bioimaging applications.

POLYMERIC MR CONTRAST AGENTS Magnetic resonance imaging (MRI) is one of the prevalent noninvasive diagnostic techniques utilized for disease detection. It is a preferred and safe mode of imaging specifically for the brain, central nervous system, and for patients with complications as it does not involve the use of radiotracers or ionizing radiations. MRI provides 3D images at high spatial and sequential resolution of sections in soft tissue samples by showing differences in proton density, enabling delineation separating diseased lesions from normal tissues. Contrast agents are used to overcome the inherent sensitivity problems that hamper the visualization of small changes in tissues at cellular and molecular levels. Contrast agents induce proton density changes in the longitudinal (T1) and transverse (T2) relaxation processes, subsequently aiding in identification of lesions. Based on the type of relaxation produced, MR contrast agents can be classified into two types: T1 contrast agents (bright images, shortening T1 relaxation time) and T2 (negative contrast agents, i.e., dark images, shortening T2 relaxation time).112,113 The widely used T1 contrast agents are mainly gadolinium based and some of them (ProHance®, Magnevist®, and Doterm®) have been approved for clinical use.114 Gd is paramagnetic in nature and, therefore, increases contrast in T1-weighted images. However, super paramagnetic materials such as iron oxide (SPIO) and ultrasmall paramagnetic iron oxide (USPIO) materials alter the transverse relaxation to produce dark T2weighted images.114 These negative contrast agents have the ability to alter T2* relaxation (a process that accounts for both basic molecular interactions and external magnetic field in homogeneities); therefore, T2/T2* weighted sequences can be used for data analysis.114,115 In general, SPIO nanoconstructs are anticipated to have greater sensitivity compared to Gd-based T1 contrast agents. However, it is important to mention that T2-based contrast agents

result in dark images, and this may potentially lead to visualization challenges in some tissues. At present, only two iron-based contrast agents (Endorem®/Ferridex® and Lumiren®) have been FDA approved for clinical use. The sensitivity of MR images are improved by combining both dynamic and static imaging techniques using T1 and T2 weighted images to provide more useful patterns for image enhancement. Both T1 and T2 agents have been explored for diagnosis of cancer and vascular diseases.114,115 However, T2 or negative contrast agents can specifically be used as diagnostic tool to locate pathological lesions precisely and other neurological disorders.115 There has been a burgeoning interest in developing sensitive and targetspecific MR agents to selectively identify regions of interest, increase retention time, minimize toxicity, and monitor disease progression. In this aspect, polymeric and metallic nanoparticles have gained wide attention due to their inherent advantages over small molecules or metal complexes.116 Currently used T1 contrast agents are Gdbased small molecule chelates such as Gd-DTPA, Gd-DOTA, and Gd-DTPA-BMA. If Gd agents exceed recommended dosage limits, it can cause nephrotoxicity112 due to release of free Gd ions. Macrocyclic forms of Gd chelates are preferred to those of linear chelates as the macrocyclic chelates will tightly bind the Gd ions and prevent them from leaching out in vivo. There are disadvantages associated with macrycylic chelates as they lack in vivo stability and target specificity. Also the smaller size enables distribution throughout intravascular and interstitial space resulting in rapid renal clearance.112 Therefore, to eliminate toxicity issues, it is important to design the polymer nanoconstruct for optimal circulation time and concurrently obtain images with high signal intensity. SPIO, the commonly used T2 agents, on the other hand, suffer from solubility and magnetic saturation causing signal loss issues.114 One possible way to circumvent these issues is to use biocompatible polymeric nanoconstruct for modulating solubility issues, prevent magnetic saturation, improve in vivo circulation time, and target specificity for increasing signal intensity ratio in the region of interest. Indeed, polymeric nanomaterials are generally used as coatings on both Gd and SPION agents.112,115,117,118 The coatings are generally achieved through covalent bonding or electrostatic attractions. Custom-made polymeric nanoconstructs were used for selective transportation of T1 (Gd agents) or T2 (SPION or USPION) type contrast agents. As classified in previous sections, polymeric nanoconstructs incorporated with MR contrast agents

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FIGURE 7 | In vivo magnetic resonance imaging MRI performed in hepato cellular carcinoma (HCC) mice models at different postinjection time ®

points for Magnevist (a: 0 min, b: 1 min, c: 10 min, and d: 1 h); PLA-PEG-GdNPs (e: 0 min, f: 1 h, g: 3 h, and h: 12 h); and anti-VEGF-PLA-PEG-GdNPs (i: 0 min, j: 2 h, k: 12 h, and l: 24 h). (Reprinted with permission from Ref 30. Copyright 2013 Elsevier)

can also be categorized into two types: MR Contrast agents covalently conjugated with polymeric nanomaterial (Type I) or MR agents encapsulated inside the polymeric core (Type II). Tables 1 and 2 list polymeric nanomaterials that were developed for imaging applications.

Type I In this class, Gd-based MR agents are covalently conjugated to polymeric matrix and subsequently converted into nanoparticles for MRI applications. These nanoconstructs have been used as targeted vehicles for delivery of contrast agents and drugs selectively to cancerous region. The targeted delivery is achieved by attaching either PEG for EPRbased targeting or receptor-specific biomolecules. The targeted detection of hepatocellular carcinoma (HCC) using MR has been achieved using Gd-DTPAconjugated polymeric nanoconstructs.30 Vascular endothelial growth factor (VEGF) receptors are overexpressed in HCC.30 Polylactic acid-polyethylene glycol-poly-l-Lys was conjugated with anti-VEGF and Gd-DTPA to obtain the polymer nanoconstruct antiVEGF-PLA-PEG-PLL-Gd. The nanoconstruct was targeted to VEGF expressed HCC in mice models and MRI were compared with Magnevist®. The nanoconstruct aid in prolonging the imaging time to 12 h compared to that of Magnevist® (