Molecules 2012, 17, 5564-5591; doi:10.3390/molecules17055564
Fluorescent Nanoprobes Dedicated to in Vivo Imaging:
From Preclinical Validations to Clinical Translation
Juliette Mérian, Julien Gravier, Fabrice Navarro and Isabelle Texier *
Département microTechnologies pour la Biologie et la Santé CEA-LETI, Minatec, 17 rue des Martyrs,
38045 Grenoble cedex, France; E-Mail: firstname.lastname@example.org (J.M.)
* Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +33-438-784-670; Fax: +33-438-785-787.
Received: 5 April 2012; in revised form: 6 May 2012 / Accepted: 7 May 2012 /
Published: 10 May 2012
Abstract: With the fast development, in the last ten years, of a large choice of set-ups
dedicated to routine in vivo measurements in rodents, fluorescence imaging techniques are
becoming essential tools in preclinical studies. Human clinical uses for diagnostic and
image-guided surgery are also emerging. In comparison to low-molecular weight organic
dyes, the use of fluorescent nanoprobes can improve both the signal sensitivity (better
in vivo optical properties) and the fluorescence biodistribution (passive “nano” uptake in
tumours for instance). A wide range of fluorescent nanoprobes have been designed and
tested in preclinical studies for the last few years. They will be reviewed and discussed
considering the obstacles that need to be overcome for their potential everyday use in
clinics. The conjugation of fluorescence imaging with the benefits of nanotechnology
should open the way to new medical applications in the near future.
Keywords: fluorescence imaging; nanoprobes; in vivo imaging; nanoparticles; organic
dyes; quantum dots; contrast agents, biodistribution; lymph node mapping
From the first anatomical charts to the most recent development of Magnetic Resonance Imaging
(MRI) or Positron Emission Tomography (PET), setting into images what is normally unseen within
living systems has been a major issue for the understanding of biological processes [1–3]. With the
exception of MRI, which relies on magnetic properties of atom nuclei with half-integer spin, most of
Molecules 2012, 17
the imaging modalities rely on photons with wavelengths ranging from gamma emission to infrared.
Any photon has specific interactions with tissues or bones that will define its uses and drawbacks. On
the one hand, high-energy photons such as gamma emission or X-rays can go through the tissues with
low interactions, making them suitable for deep tissue or whole body imaging, while being potentially
harmful because of the reactions they can trigger within cells. Their use is thus strictly restricted to
dedicated areas and under the supervision of trained personnel. On the other hand, photons in the
visible and infrared wavelengths do not penetrate deeply within organic tissues, but they cause no or
little damage to cells. Moreover, light sources and imaging apparatus are widespread, relatively cheap,
their handling requires moderate levels of training and protection, and does not generate
Contrary to tissue auto-fluorescence recording, near-infrared/visible fluorescence is an optical
imaging modality that relies on the injection of an exogenous probe that will emit light when excited
with suitable wavelength. This method can be used from the macroscopic to the microscopic range,
goes as further as sub-cellular resolution, and is highly sensitive. Targeted fluorescent probes can be
designed to specifically mark and visualize different biological targets, which can be imaged with high
contrast using the appropriate set of optical filters to minimize auto-fluorescence contribution of the
surrounding tissues. Fluorescence is particularly suitable for in vitro and superficial in vivo imaging
applications. However, fluorescence imaging using the near infrared range is also now routinely used
for whole body imaging of small animals in preclinical studies [4–7], and the first human clinical trials
have been performed in 2008/2009 for sentinel lymph node mapping in oncology by different
groups [8–12]. The next challenge to address is to pursue the development of fluorescence imaging
techniques in medical applications. Since the instrumentation tools now exist [13–18], the main
blocking point to overcome for the expansion of clinical trials remains the availability of performing
fluorescent tracers [19,20]. Nanotechnologies could facilitate their design by allowing modular and
flexible constructions. This review will focus on the design of near-infrared fluorescent agents based on
nanostructures, their benefits for in vivo imaging, and the coming challenges for their clinical translation.
2. Clinical Applications of in Vivo Fluorescence Imaging Probes and Associated Constraints
2.1. The Near-Infrared Window
One of the driving issues for the development of new fluorescence imaging probes dedicated to
clinics is the necessity for near-infrared tracers providing high signal-to-background ratio. This
requires both targeting abilities of the probes to accumulate specifically in cells to be labelled, while
being cleared from surrounding tissues, and highly bright fluorophores absorbing and emitting in the
near infrared range. In vivo imaging is indeed limited by the scattering and absorption of light by tissue
components, essentially blood (oxy- and deoxyhemoglobin, respectively HbO2 and Hb) and water. The
spectral properties of these fluids thus define a narrow ‘optical window’, between 650 and 900 nm
(Figure 1), where light is able to penetrate deeper, typically a few centimetres depth. Moreover, tissue
auto-fluorescence is also reduced in the near infrared domain in comparison to the visible range.
Molecules 2012, 17
Figure 1. Definition of the optical window for in vivo imaging. Reprinted from Kobayashi, H.;
Ogawa, M.; Alford, R.; Choyke, P.L.; Urano, Y. New strategies for fluorescent probe
design in medical diagnostic imaging. Chem. Rev. 2010, 110, 2620–2640 . Copyright
(2010) American Chemical Society.
Therefore, contrary to other modalities such as MRI and nuclear imaging for which complete body
scans can be performed, the spectrum of fluorescence imaging applications in clinics could appear
reduced. However, due to the cheap and easy handling of fluorescence tools (absence of radioactivity
for instance), new applications have emerged, which could not be so easily implemented with other
modalities for reasons of cost and apparatus flexibility. This is particularly the case of applications for
which superficial imaging is needed. Up to now, the most investigated clinic applications are in the
field of image-guided surgery . In the future, other applications in the field of diagnostics,
especially thanks to endoscopic instruments, can be envisioned [18,21].
2.2. Image-Guided Removal of Sentinel Lymph Node
The first clinical trials in the field of oncology using fluorescence imaging were published in
2008/2009 by Frangioni’s and Sevick-Muraca’s groups using home-made imaging systems [10,12], and
in three Japanese teams using the Photodynamic Eye developed by Hamamatsu [8,9,11] (Figure 2). They
were focused on the use of fluorescence as an image-guiding intraoperative method for sentinel lymph
node resection. Precious insights about the possibility of metastatic progression can be obtained by
removing the sentinel lymph node, first lymph node draining the tumour, and analysing it to determine
the presence or absence of malignant cells . Indocyanine Green (ICG, emission ≈ 800 nm, Figure 3),
presently the only near infrared fluorescent dye with absorbing/emitting wavelengths > 700 nm to be
approved by the FDA (US Food and Drug Administration) for human use, was used as tracer in these
first clinical trials. The dye was locally injected intra- or sub-dermal at the tumour site.
Molecules 2012, 17
Figure 2. Sentinel lymph node mapping in gastric cancer surgery using fluorescence
imaging. Fluorescence (a,b) and video (c,d) images showing fine lymphatic vessels (a,c)
and the succession of lymph nodes (b,d). Reproduced from Miyashiro, I.; Miyoshi, N.;
Hiratsuka, M.; Kishi, K.; Yamada, T.; Ohue, M.; Ohigashi, H.; Yano, M.; Ishikawa, O.;
Imaoka, S. Detection of sentinel node in gastric cancer surgery by indocyanine green
fluorescence imaging: comparison with infrared imaging. Ann. Surg. Oncol. 2008, 15,
1640–1643 , with kind permission from Springer Science+business Media.
This first clinical application of fluorescence imaging certainly emerged for several reasons. From
the instrumentation point of view, the selected applications (breast cancer, gastric cancer surgery)
expose the tissues during intervention, therefore being examples of surface or low-depth image-guided
surgery (typically 1 to 2 cm depth of tissues explored), turning down the challenges of deep
fluorescence imaging. From the tracer point of view, the very short lifetime of ICG in blood (2 to
4 min plasma half-life, followed by a slower clearance rate once bound to plasma proteins ) limits
its use for applications requiring systemic injection and substantial blood concentration of the dye on
the long term, as required for instance for tumour fluorophore uptake. On the contrary, the lipophilic
nature of ICG favours its lymphatic drainage following intradermal or sub-dermal injection at the
tumour site, and its subsequent labelling of the lymph nodes.
This example underlines that fluorescent tracers dedicated to human clinical use should respond to
specific requirements, which may vary according to the applications. In the case of fluorescence-guided
sentinel lymph node resection, the tracer should display no toxicity following local injection at
the imaging doses, be efficiently drained by the lymphatic system, and be retained in the lymph node
to be removed.
The nanometer size (typically less than 50 nm diameter) has been demonstrated to be particularly
suitable for lymph node imaging, since nano-objects, especially lipid-based ones [23,24], are
preferentially retained in lymph nodes (demonstrated in different animal models, from rodents to
pig [19,25–28]), in contrast to low molecular weight fluorescent dyes, more easily washed and
eliminated. The adsorption of ICG on Human Serum Albumin (HSA), forming nanocolloids of ≈7 nm
diameter, has been reported to significantly prolong the fluorescence signal in the sentinel lymph node
of breast cancer patients . Another case study, however, claims that the dye conjugation with the
protein does not bring any significant benefit (time of intervention: 15 minutes following injection) .
Molecules 2012, 17
Yet, sentinel lymph node mapping is one of the most studied applications concerning fluorescent
nanotracers, and will certainly constitute one of the first clinical fluorescence imaging applications
benefiting of improvement brought by nanotechnologies on the design of tracers.
2.2. Other Clinical Applications
Few other applications of fluorescence imaging have been explored in human clinical trials until
now. ICG has been used since the 1970s for retinal angiography, cardiac output and hepatic function
assessment . More recently, it was employed as an intra-operative staining fluorophore in surgery,
to image vascular network [31,32], bile ducts , and the demarcations of liver segments and
sub-segments . Methylene blue (emission ≈ 700 nm, Figure 3), another dye mainly used since now
for its colour staining properties, is also slightly fluorescent in the near infrared domain . It has
been used for years as a tissue staining dye for visible imaging and other clinical applications, and is
FDA approved for some indications . Methylene blue recently allowed the identification of bile
ducts and ureters during fluorescence-guided surgery [33,35]. However, it displays even poorer optical
properties than ICG in terms of extinction coefficient and fluorescence quantum yield, and requires
shorter excitation wavelengths, less favourable to use for deep imaging. The potentialities of ICG for
dynamic fluorescence imaging to assess the lymphatic architecture and transport in healthy and
lymphedema-diagnosed subjects was also explored . ICG was also used to assess the presence of
breast tumours by fluorescence imaging methods associated with pharmacokinetic modelisation of the
dye biodistribution . Another fluorescent dye, Omocianine (Bayer Shering Pharma, emission > 750 nm,
Figure 3), was tested for the detection of malignant breast lesions in women suspected of breast
All these very recent studies (<4 years) demonstrate that clinical fluorescence imaging is
exponentially growing, and will soon be implanted in more surgery rooms, and explored in other
applications. However, they also evidence the poor availability of approved near infrared tracers (only
ICG is validated by the FDA for fluorescence imaging, whereas methylene blue and Omocianine have
been accepted in punctual clinical trials, Figure 3). Hence, there is a large avenue for the development
of more efficient fluorescent probes. In the next paragraph, specific advantages that could be brought
by nanotechnologies on the design of fluorescent tracers for clinical imaging will be discussed,
whereas in Section 4, main nanosystems reported in the literature, currently undergoing preclinical
studies, will be described.
Figure 3. Near infrared fluorescent dyes that have been used in clinical trials until now.
Molecules 2012, 17
3. The Benefits of Nanotechnologies
3.1. Limitations of Low Molecular Weight Fluorophores for in Vivo Fluorescence Imaging
Low molecular weight fluorescent dyes (typically molecular weight up to ≈1,500 Da for near
infrared fluorophores) are currently the most widespread type of tracers. This is mainly due to their
well-defined chemical structure, which can be tailored according to the desired optical and chemical
properties, and the very large commercial offer. The most common organic fluorophore families
include rhodamines, BODIPY, indocyanines, porphyrines and phthalocyanines, but this list is far from
comprehensive . Each of the basic structures presented in Figure 4 has been modified by functional
groups in order to adjust emission wavelength, enhance photostability, change hydrophobic/hydrophilic
balance or to enable conjugation with targeting moieties. In the near-infrared domain (600–900 nm),
mainly cyanine, bodipy, porphyrine and phtalocyanine structures are explored .
Figure 4. Basic chemical structures of (a) fluoresceins, (b) rhodamines, (c) bodipys,
(d) indocyanines, (e) porphyrines and (f) phthalocyanines.
a.X1, X2= OH, O
X1, X2= NH2, NH2+
However, small organic fluorescent dyes present drawbacks that arise especially when it comes to
in vivo applications. Firstly, due to their aromatic structure, these molecules are generally poorly
soluble in aqueous medium, thus poorly bioavailable. This limited water solubility issue is particularly
stressed for near infrared dyes, for which the addition of pi-conjugated bounds increase the molecule
hydrophobicity. Chemical modifications to improve water solubility by adding polar groups, as for
instance sulfonate [42,43] or saccharide [44,45] functions, have been proposed. Another drawback is:
the more red-shifted the emission of a dye is, the lower its fluorescence quantum yield, which
generally results in reduced brightness. Indeed, due to the increasing number of bounds involved in the
pi-conjugated system, infrared emitting dyes possess a higher degree of vibrations, leading to an
increasing number of non-radiative decay pathways.
Molecules 2012, 17
As already illustrated with the ICG dye, a second driving issue in the development of potent
fluorophore for in vivo imaging applications, is their very fast body clearance, by kidneys and urine
excretion for highly hydrophilic compounds (such as fluorescein), by liver and bile excretion for more
lipophilic molecules (such as ICG). If this fast clearance can be advantageous to limit dye toxicity, or
for specific applications such as for instance repetitive vascular imaging, it also limits the temporal
window in which imaging can be performed, and the list of organs in which the fluorescent tracer can
be distributed. A “targeting” moiety can be conjugated to the fluorescent tracer to improve the
localization and binding of the dye in the area to image, and to modify its pharmacokinetics. The
targeting moiety can be for instance an antibody, protein or peptide, an oligonucleotide, a saccharide,
or any other molecular template known for its specific affinity for a cellular compartment, cellular
receptor, biological fluid or tissue. The use of nanotemplates, where the fluorescent dye can be
encapsulated, adsorbed, or grafted on the surface, constitutes another strategy to modify low molecular
weight fluorophore biodistribution. In this strategy however, caution should be paid on the potential
toxicity issues raised by the new biodistribution, especially uptake and prolonged retention in liver and
spleen, as further discussed below.
3.2. What Can Nanotechnologies Bring in the Design of Efficient Fluorescent Probes
Nanotechnologies can benefit in three ways to the design of efficient fluorescent probes dedicated
to in vivo imaging: (1) by taking benefit of the nanometer-size governed biodistribution of the probe;
(2) by designing, in an easy and versatile manner, complex and modular tracers associating different
functionalities; (3) by obtaining brighter fluorescent tracers.
It is now well established that nanosized objects can passively accumulate in tumours more
efficiently than in healthy tissues. This process, named the “enhanced permeability and retention“
(EPR) effect is linked to the presence of fenestrations (up to a few hundreds of nanometers) in the
blood angiogenic vessels, which are built to supply fast-growing tumour cells, associated with the
inefficient lymphatic drainage of cancer tissues  (Figure 5). This process is not observed for low
molecular weight fluorophores (<5 nm), for which fast extravasation from the blood vessels to the
tissues is counterbalanced by the inverse diffusion process. Low molecular weight dyes can however
be designed to display stronger affinity for tumour than healthy tissues . The passive EPR targeting
effect, demonstrated in tumours for a wide range of nanoobjects, could also exist in inflamed tissues
and be used for the diagnostics of different pathologies. Passive tumour uptake (generally leading to
signal-to-background ratio of about 2:1 to 4:1) can also be enhanced by the decoration of the
nanoparticle surface with targeting ligands, able to specifically bind to receptors overexpressed by
tumour cells (Figure 5). Different receptors and associated ligands are identified and widely used in
animal models, such as folate or vascular endothelial growth factor (VEGF) receptors, and the v3
integrins. If targeted tracers generally yield improved signal-to-background ratios (5:1 or higher) in
comparison to EPR effect, as well as molecular information, their signal is intimally linked to the
molecular nature of the tumour tissues, and a cocktail of several tracers might be necessary to establish
diagnostics. On the contrary, nanoparticles based on EPR effect could constitute “universal” first
intention diagnostic contrast agents.
Molecules 2012, 17
Figure 5. Passive and active tumour targeting of nanoparticles.
As mentioned earlier, the use of nanoparticle templates also allows the easy design of complex
objects. For instance, a nanoparticle surface can be decorated by a controlled number of targeting
moieties, promoting cell recognition by the multimerised presentation of the ligands. Drugs or
contrast agents for different modalities, including fluorescence imaging,can be associated to design
multifunctional probes [47,48] or for theranostic strategies [49,50]. Such objects have been reviewed
recently and will not be discussed in the present document [47–50].
The use of nanostructures is also expected to lead to improved optical properties of the tracer, and
ultimately, higher image contrast. Indeed, as will be detailed in the next paragraph, the construction of
fluorescent nanotracers can rely either on two different strategies: the use of intrinsically fluorescent
semi-conducting nanocrystals (quantum dots); the encapsulation or binding of low molecular weight
fluorophores to nanoscale templates (silica or organic nanoparticles for instance). Quantum dots
display outstanding optical properties, such as high absorption coefficient and fluorescence quantum
yields, but raise cytotoxicity issues. The inclusion of several dyes in a single nanostructure increases
their local concentration on the targeted site. Moreover, in the latter dye-inclusion strategy, the
nano-matrix in which the fluorophores are encapsulated plays a protecting role, preventing direct
chemical contact between the dye and the biological fluids. This notably prevents dye water solubility
issues and aggregation, often responsible for loss of optical properties (increased sensitivity to
photo-bleaching, protein quenching effects). Therefore, the use of nanotechnologies can lead to the
design of more efficient and targeted nanoprobes for high contrast fluorescence imaging.
4. Fluorescent Nanoprobes
Fluorescent nanoprobes can be distinguished in two main categories: those based on organic
nanoparticles and those constructed with inorganic nanocrystals, such as semi-conducting quantum
dots or silica nanoparticles (Figure 6). In this paragraph, the main in vivo preclinical studies carried out
using fluorescent nanoprobes will be reviewed.
Molecules 2012, 17
Figure 6. Schematic structures of fluorescent nanoprobes for in vivo imaging. Inorganic
nanoprobes are quantum dots (a) or dye-loaded silica, calcium phosphate, gold or oxide
nanoparticles, for which the organic dye can be included in the inorganic matrix (b), or
grafted on the nanoparticle surface (c). Organic nanoprobes can be divided in two main
families: dye-loaded polymer-based and dye-loaded lipid-based nanoparticles. In each
family, different architectures can be found: polymer- or lipid- core particles (polymer
nanospheres (d), proteins (e), lipid nanoparticles (f), lipoproteins (g)), self-assembled
constructions (polymer (h) or lipid (i) micelles), nanocapsules (polymersomes (j) or
liposomes (k)). The fluorescent organic dye can be either included in the hydrophobic core
or shell of the structure, or grafted on the nanoparticle surface (hydrophilic organic dye).
Molecules 2012, 17
4.1. Quantum Dots
Quantum dots are nanocrystals of semi-conducting materials . They are made of elements from
the II and VI periodic groups (i.e., ZnS, CdSe or CdTe compositions for instance), or more recently III
and V periodic groups (InP) and IV and VI periodic groups (PbS, PbSe, PbTe). They are chemically
grown and their emission properties, being related to the confinement effect of an electron-hole pair
(exciton) within the crystal, mainly depend on their shape and size. The inorganic semi-conducting
core (typically 2–12 nm diameter) is generally coated by an inorganic shell (typicall ZnS), which
reinforces the optical properties of the crystal. The outer shell can further be functionalized by an
organic layer- typically thiol bi-functional molecules or amphiphilic polymers-, or entrapped in
phospholipid micelles (Figure 6a).
Thanks to their optical properties - tunable, narrow and symmetric emission band, long
luminescence lifetime, and good photostability -, these objects have been very intensively studied for
bio-imaging since late 1990s [52–56]. Cadmium-free nano-crystals emitting in the near-infrared more
specifically dedicated to in vivo applications have been developed [57–64]. A decisive step towards
their potential use in clinics has been made in 2005 with the evidence that very small neutral
nanocrystals (diameter < 5–6 nm) could be eliminated by kidney clearance . This was further
confirmed by other studies . Indeed, larger quantum dots are known to accumulate for very long
time in reticulo-endothelial system (RES) organs, especially in liver [63,66,67], even if no major acute
or prolonged toxicity effects seem to have been observed in rodents [68,69]. The coating of the
nanoparticles with poly(ethyleneglycol) (PEG), a well-known polymer to prevent surface protein
adsorption and prolong blood lifetime of drugs or nanoparticles , does not hinder their RES
uptake . Associated to the fact that many quantum dot core compositions include toxic elements
such as cadmium, arsenide or lead, it clearly constitutes an obstacle for their translation to clinics.
Therefore, even if studies demonstrated the possibility to passively or actively address the particles to
tumour sites following their intravenous injections [60,64,72,73], their future use in tumour diagnostics
in clinics seems compromised. The application of quantum dots for sentinel lymph node fluorescence
mapping [26,61,64,74,75] could be more promising. This application should be less restricting
concerning toxicology issues, since a local injection and therefore a reduced dose are required with
further removal of the tracer included in the resected node.
4.2. Dye-Loaded Inorganic Nanocarriers
Other inorganic probes have been proposed as fluorescent nanotracers, relying on the encapsulation
(Figure 6b) or surface binding (Figure 6c) of organic fluorescent dyes to inorganic nanoparticles,
mainly silica, calcium phosphate oxide or gold nanoparticles.
One of the most described silica nanoprobes are certainly the C-dots developed at Cornell
University , which entered clinical trials in Fall 2011 for lymph node diagnosis and staging in
advanced melanoma . Other groups also develop dye-loaded silica nanoparticles for bioimaging
applications [78–82]. Silica is an interesting material for the design of functional nano-objects: active
molecules can be encapsulated inside the pores of the matrix, or the use of organic modified silanes
can allow their covalent attachment in the core or the shell of the particles. Synthesis protocols such as
Molecules 2012, 17
the Stöber ‘s process are well mastered, and easy to implement . The optical properties of the dyes
such as cyanines are well preserved and even enhanced in the silica matrix [84–86]. Similarly to
quantum dots, very small nanocrystals (<10 nm) seem to undergo urinary excretion [81,87], whereas
larger nanoparticles (>20 nm) rather distribute in liver, spleen, stomach and undergo hepatobiliary
Calcium phosphate (also named apatite) or calcium phosphosilicate have also been proposed as
interesting matrixes for dye encapsulation and in vivo vectorization [88–91], but very few preclinical
experiments have been performed in rodents [88,89]. 20 nm diameter gold nanoparticles have been
used as templates for the covalent grafting of cyanine fluorophores via metalloprotease-cleavable
spacers . The cyanine emission is quenched by the surface-energy transfer properties of the
nanoparticles, and further activated in protease-rich tissues, such as in SSC7 carcinoma, after breaking
of the bound by which they are linked to the gold surface . This design of “activatable probe”,
possible thanks to the environment-sensitivity of organic dye fluorescence, allows imaging with high
signal-to-background ratio, and underlines one of the benefit that can be brought by fluorescence
imaging in comparison to other modalities to get molecular information in vivo. Very small gold
nanoclusters (2 nm) have also been shown to display luminescent properties by their own, however
with emission wavelength below 600 nm, limiting in vivo depth imaging .
The biodistribution of different oxide based nanocrystals loaded with or encapsulating organic
fluorophores have been studied, and is governed, as for other inorganic nanoparticles, by their size,
charge, and nature of the polymer coating [94,95]. Yttrium oxide (Y2O3) [96,97] or sodium yttrium
fluoride (NaYF4) [75,98,99] nanoparticles can also be loaded with erbium and terbium cations to
achieve up-converting nanomaterials. Up-converting nanoparticles are excited in the infrared range
(typically 980 nm) and display up-converted emission in the green or near infrared depending on
doping concentrations [75,96–99]. Due to the up-conversion process, tissue autofluorescence is
considerably lowered, achieving very high-to-background ratio in biological samples. However, the
multiphotonic process involved in up-conversion requires the use of imaging systems with high power
excitation light, and imaging is restricted to surface (a very few mm of penetration depth) [75,96,98].
4.3. Dye-Loaded Organic Nanocarriers
Organic nanocarriers have aroused much interest for the last 30 years for in vivo drug vectorisation,
and several nanosized drug formulations are presently used in clinic [100,101]. Therefore, several set
of data are already available on the biodistribution and in vivo fate of most of these nanocarriers
[102–109], even if each nanoparticle displays unique properties. It has well been evidenced that a
stealth coating (PEG essentially) is necessary to limit rapid nanoparticle uptake in the RES and allow
their distribution in different tissues [110–112]. Targeting strategies using different set of ligands such
as antibodies, peptides and saccharides have also be explored in different animal models, especially for
tumour targeting. In the present review, we will mainly focus on near-infrared dye loading in the
organic nanocarriers, its impact on fluorophore properties, and the potential clinical applications that
can be envisioned with these systems.
The loading of nanoparticles with organic fluorophores to design dedicated in vivo fluorescent
nanoprobes has been explored quite recently, mainly in the last 10 years, with the arrival of small
Molecules 2012, 17
animal imaging devices [4–7]. The fluorescent dyes, mainly near infrared cyanines, porphyrines or
phtalocyanines as far as in vivo imaging is concerned, can be included in the nanoparticle core, can be
intercalated in or be part of the nanoparticle shell, or linked to the particle surface by either adsorption
or a covalent bond (conjugation) (Figure 6d–k). Encapsulation, either by the entrapment of the dye
using molecular affinity (i.e., a lipophilic dye will present a strong affinity with a lipophilic matrix) or
by covalent conjugation to the material constituting the particle core (a hydrophobic polymer chain for
instance), presents the advantage of protecting dyes from direct interactions with biological fluids,
which can alter their optical properties. Chemical conjugation of the dye to the particle core or shell
limits its leakage while in buffered media.
We will divide the organic nanocarriers in two main families, polymer-based and lipid-based
nanoparticles (Figure 6). In both categories, “natural” and “synthetic” template nanoparticles are
found. For instance natural template nanoparticles include proteins (such as serum albumin, Figure 6e),
natural polymers (dextran, chitosan…), bacteriophages or lipoproteins (Figure 6g), whereas synthetic
template nanoparticles include polymer nanospheres (Figure 6d), polymer micelles (Figure 6h),
dendrimers, liposomes (Figure 6k), solid lipid nanoparticles (Figure 6f).
4.3.1. Polymer-Based Nanoparticles
Polymer-based nanoparticles can present different architectures (Figures 6 and 7): linear chains
arranged in extended (Figure 7a) or coiled conformation (Figure 7b), according to polymer
hydrophobicity, cross-linked core nanospheres (Figure 7c), dendrimers (Figure 7d), which were
grouped in Figure 6 as “polymer-core nanoparticles”, self-assembled amphiphilic micelles (Figure 6h),
polymersome capsules (i.e., “polymeric” liposomes, Figure 6j) . Polyesters, as well as poly(lactic
acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA), which hydrophilic/hydrophobic balance
can be finely tuned, are extensively used as synthetic nano-materials for their biocompatibility and
biodegradability. “Nature-based” polymers include proteins (human serum albumin for instance),
polypeptide constructions (poly(lysine), poly(glutamic acid) for example), saccharides (dextran, chitosan).
Figure 7. Different architectures of “polymer-core nanoparticles”.
Linear poly(lysine) chains bearing peptide-fluorophore moieties on pending amino groups were one
of the first architecture proposed for the design of fluorescent nanoprobes for in vivo imaging, and
certainly remains one of the most elegant (Figure 8) [113–116]. The peptide-fluorophore ratio on
the polymer backbone was optimized so that the spatial proximity of the fluorophores induced
self-quenching of their emission properties. Therefore, the nanoprobe was non-emissive upon injection
in the animal tail vein. The peptide sequence linking the fluorophore and the polymer was selected
Molecules 2012, 17
according to the desired proteolytic activity to image. Enzymatic cleavage occured specifically in
tissues where the proteases were expressed, freeing the fluorophores, which recovered their emission
properties as they diffused in the tissues.
Figure 8. Design of activatable probes for protease activity imaging. (a) Architecture of
the probe, based on linear poly(lysine) with peptide-fluorophore moieties on pending
amino groups. In this example, the peptide sequence can be cleaved by cathepsin D (CaD).
(b) In vivo fluorescence images obtained 24 h after i.v. injection of the reporter probe in
Nude mice implanted with a CaD+ (red arrow) and CaD− (blue arrow) tumours. Adapted
from Tung, C.-H.; Mahmood, U.; Bredow, S.; Weissleder, R. In vivo imaging of
proteolytic enzyme activity using a novel molecular reporter. Cancer Res. 2000, 60,
4953–4958  by permission from the American Association for Cancer Research.
The encapsulation of ICG in different polymer or co-polymer matrixes (typically particle diameter
of 100–500 nm) was shown to improve the dye optical properties and enhance its chemical stability
[117–120]. The encapsulation of different dyes in PEG-PLA nanospheres has been studied . The
experiments showed that the amphiphilic Nile Red dye (logP = 3.8) was quickly released from the
polymer matrix after injection resulting in its rapid elimination from the blood stream, whereas the
hydrophobic near infrared DiR dye (logP = 17.4) lead to a constant fluorescent signal in blood up to
6 hours post injection. DiR-loaded particles subsequently allowed tumour labelling . The covalent
conjugation of the Cy5 fluorescent dye to the polymer matrix prevented the burst released effect .
Such polymer nanoparticles, largely studied as drug delivery systems, are known to be efficiently
up taken by the reticulo-endothelial system if not functionalized by a stealth coating such as
Near infrared fluorescence dyes were also encapsulated in poly(ethylene imine) or biodegradable
aliphatic polyester dendrimers (typically 10–30 nm diameter). The rather small sizes of these polymer
structures favour their renal clearance . The dendrimers can be surrounded by a PEG shell to
improve their water solubility. The fluorophores thus display enhanced stability, resistance to
enzymatic oxidation and prolonged in vivo residence time . These structures can also be used to
transport both hydrophilic and hydrophobic molecules .
The adsorption of ICG on plasma proteins to prolong the otherwise very short blood lifetime of the
dye has already been mentioned (see part 2.2) since this constitutes the only clinical trials made with
Molecules 2012, 17
nanosized probes in the field of fluorescence imaging until now . Block co-polymer micelles have
also been used to encapsulate ICG [126,127] or other dyes . Polymeric nanomicelles with a PEG
shell were functionalized with different targeting ligands for in vivo sentinel lymph node mapping or
tumour follow-up . Because micelles are dynamic structures in equilibrium with the free
monomers at the critical micelle concentration (cmc), the polymer design must be optimized to lower
the cmc and avoids fast burst release of the fluorescent dye upon in vivo administration.
Bacteriophage particles were functionalized conjointly by pH-sensitive (HCy-646) and
pH-insensitive (Cy5) cyanine dyes . This double labelling was used to evaluate the stability of the
nanocarrier after injection by double checked in fluorescence signal, and to probe the disruption in
acid/base homeostasis often found in tumour hypoxia.
Polymersomes are polymer vesicles generated through cooperative self-assembly of amphiphilic
diblock copolymers (similar structure than liposomes but with a polymer instead of a phospholipid
bilayer). Their near-infrared properties can be generated including in the polymer shell monomeric or
oligomeric dyes (Figure 9) [131,132]. These polymersomes were designed in order to uniformly
distribute numerous large hydrophobic porphyrins exclusively in their lamellar membranes. Within
these sequestrated compartments, long polymer chains regulated the mean fluorophore-fluorophore
interspatial separation as well as the fluorophore-localized electronic environment to optimize optical
Figure 9. NIR-emissive polymersomes. In aqueous solution, amphiphilic poly(ethylene
oxide)/poly(butadiene) polymers (PEO30-PBD46) self-assemble into polymer vesicles
(polymersomes) with the hydrophobic PBD tails oriented end-to-end to form a bilayer
membrane, in which (porphynato)zinc(II) oligomers can be encapsulated. Adapted from
Duncan, T.V.; Ghoroghchian, P.; Rubtsov, I.V.; Hammer, D.; Therien, M. Ultrafast
excited-state dynamics of nanoscale near-infrared emissive polymersomes. J. Am. Chem.
Soc. 2008, 130, 9773–9784 . Copyright (2008) American Chemical Society.
As a conclusion on polymer-based fluorescent nanoprobes, the choice of the matrix polymer and the
chemical process (often employing organic solvents whenever synthetic polymers are concerned) is
crucial to achieve low-cytotoxicity dye-loaded nanocarriers with good optical properties and suitable
biodistribution. Thanks to the well mastered synthetic chemical process, the wide range of materials
Molecules 2012, 17
available, polymer-based probes with finely tuned properties and sizes ranging from 10 nm
(dendrimer, micelles) to micrometer (polymer spheres or capsules) can be designed and precisely
characterized. Very different biodistribution patterns can be achieved, according to the particle size,
charge, and flexibility [123,134], opening the way to design well-tailored specific fluorescent probes
for each envisioned medical applications.
4.3.2. Lipid-Based Nanoparticles
Lipid-based nanoparticles also present a wide variety of architectures, constructed with lipophilic
lipids such as mono-, di- or triglycerides, and/or amphiphilic lipids such as phospholipids, which
present surfactant properties. The lipids and phospholipids used can be extracted and purified from
natural products such as animal or vegetal oils, or synthetically produced. Polymeric moieties, can also
be included in the structures: for instance hydrophilic poly(ethyleneglycol) moieties are often grafted
on the external bilayer of liposomes to prevent their rapid in vivo RES uptake and subsequent
degradation. The great advantage of lipid nanocarriers is their intrinsic very low cytotoxicity, which
make them outstanding nanocargos concerning potential cytotoxicity issues encountered in clinical
applications. For instance, IC50 values as high as 0.1 to 1 mg/mL of lipids have been reported for
liposomes or solid lipid nanoparticles .
Lipid nanoparticles [105,108,109], nanocapsules  or nanoemulsions  (particle diameter
typically of 50–300 nm) have been studied for a few years for the delivery of lipophilic drugs. It was
shown that ICG formulated in soybean emulsion displays improved optical properties and slower
plasmatic clearance . Oil nanodroplets encapsulating iron oxide crystals were stabilized by
dye-functionalized phospholipids for multimodality imaging , whereas perfluorocarbon
nanoparticles were loaded with a hydrophobic near-infrared dye for sentinel lymph node mapping
using photoacoustic imaging . Our group developed a new technology for the encapsulation of
lipophilic molecules, including near infrared dyes, based on oil-in-water nanoemulsions processed by
ultrasonication (Figure 10) [139,140]. The main drawback of nanoemulsions -namely intrinsic poor
colloidal stability- has been overcome by the use of a complex mixture of core lipids (mixture of long-chain
mono-, di- and triglycerides) and surfactants (phospholipids and PEG-stearate), bringing entropy
mixing stabilization to the physico-chemical system . These dye-loaded lipid nanoparticles,
termed “Lipidots™” display very low cytotoxicity (IC50 ≈ 300 µg/mL of lipids ), high in vivo
tolerance dose (>150 mg lipids/kg), and distribute in liver in 30 minutes following their intravenous
injection in healthy animals . Their liver uptake seems to be mainly due to the accumulation of
the particles in hepatocytes rather than macrophages, since very low levels are detected in spleen and
lungs, other organs known for macrophage homing. Lipid nanoparticles can be loaded with different
lipophilic dyes , including ICG , to obtain highly bright fluorescent nanoprobes (Figure 10b).
Passive tumour uptake of 50 nm-diameter DiD-loaded Lipidots™ has been demonstrated in a variety
of tumour models implanted sub-cutaneous in Nude mice using fluorescence imaging [143,144]. The
grafting on the nanoparticle surface of the cRGD peptide, exhibiting specific adhesion to αvβ3 integrins
overexpressed in 25% of tumour cell lines, can improve tumour accumulation and cell internalization
of the functionalized lipid nanoparticles (Figure 10d) . ICG-loaded lipidots also constitute
promising nanotracers for sentinel lymph node mapping (Figure 10c) [24,142]. For this later
Molecules 2012, 17
application, we demonstrated that 10 times less DiD-loaded lipidots (2 pmol) could lead to the same
image contrast than commercial QTracker™ quantum dots (20 pmol), while displaying decreased
cytotoxicity on NIH-3T3 fibroblasts (IC50 < 30 nM for quantum dots, >70 nm for DiD-loaded lipidots) .
Figure 10. Dye-loaded lipid nanoparticles (“lipidots™”) for in vivo imaging. (a) Structure
of the particles. (b) Photograph of 50 nm diameter lipidots loaded with different organic
dyes, covering the visible and near-infrared range. (c) Lymph node imaging 4 hours
after sub-dermal injection of ICG-lipidots in the right paw. (d) Specific uptake of
cRGD-functionalized lipidots in comparison to non-functionalized nanoparticles in HEK3
xenografted tumours in Nude mouse. This tumour model is known for its poor EPR effect
and over-expression of v3 integrins, for which the cRGD ligand presents a strong affinity
(right images: microscopy photographs of HEK3 cells (nuclei stained in blue) after 24 h
incubation in the presence of DiD-loaded lipidots (in red)). Adapted from [24,143].
Another strategy is to use as fluorescent dye nanocarriers natural lipoproteins present in the blood
stream, especially high density lipoproteins (HDLs) and low density lipoprotein (LDLs) [145,146].
Lipoproteins (10 to 30 nm diameter) are formed of a glyceride and cholesterol core, coated by a
phospholipid and apolipoprotein shell (Figure 6g). HDLs [147–149] and LDLs [150,151] were
modified by the inclusion of fluorescent phospholipids, or lipophilic fluorophores such as DiR or
DiR-bis-oleate. The surface of the lipoproteins can be modified to target epidermal growth factor
(EGF) or folate receptors, overexpressed in different tumour cells. Moreover, HDLs and LDLs are
Molecules 2012, 17
involved in cholesterol transport and linked to cardio-vascular affections. Dye-loaded apolipoproteins
could therefore also constitute interesting nanocarriers for atherosclerosis imaging.
Phospholipid-PEG micelles were shown to improve ICG optical properties and prolong up to a few
weeks its stability in aqueous buffer [127,152]. The PEG extremity can also be functionalized to
design targeted nanomicelles directed against folate receptors and v3 integrins . However, in a
similar way than polymer micelles, lipid micelles must be optimized to lower the cmc and avoids fast
burst release of the fluorescent dye upon in vivo administration.
Liposomes labelled with the DY-676-C18 ester dye were used for near infrared optical imaging of
cultured macrophages and of inflammatory process like oedema in an in vivo mouse model . ICG
was included in different liposomal formulations (typical diameter from 70 to 150 nm) to image
tumours [154,155], arthritic tissues  or lymphatic vessels . Porphysomes, nanovesicles
formed from porphyrine-modified phospholipids self-assembled in bilayers, allowed imaging of the
lymphatic system using photoacoustic tomography and fluorescence .
To conclude on lipid-based fluorescent nanoprobes, specific features seem to be associated to these
nanostructures. Indeed, lipid-based nanocarriers have been reported to display strong affinity for lymphatic
channels and lymph nodes [23,24,138,142], more importantly than polymeric particles . Their
lipid nature could also favor their affinity for numerous tumors over-expressing lipoprotein receptors
(breast and prostate cancer especially) [149,150] or atherosclerotic lesions . Another point to
underline is their intrinsic very low cytotoxicity, with for instance IC50 values as high as 0.1 to
1 mg/mL of lipids for liposomes or solid lipid nanoparticles [135,141]. Taking together, these
properties should favor the emergence of dye-loaded lipid-based nanostructures to dedicated imaging
applications in clinic. On the contrary, lipid-based nanostructures can appear more difficult to tailor
than polymer constructions, which can be very finely tuned for each specific application.
5. Conclusions and Perspectives: Transfer to the Clinic
During the last decades, advances in technology (lasers, cameras) have enabled the development of
high performance instrumentation for near-infrared fluorescence imaging. This imaging modality has
rapidly been propelled toward clinical applications, since the near-infrared region is the best suitable
optical window for body in-depth scanning. The use of the currently available near-infrared dyes,
especially FDA-approved ICG, has been fully revisited by extending the field of their clinical
applications. Due to the depth issue (photon penetration into tissues), near-infrared fluorescence
imaging applications have been restricted to superficial tissues, or deep tissues in intraoperative
context. For instance, clinical trials are presently ongoing on the accurate identification and resection
of the sentinel lymph node, using fluorescence guided surgery.
In the meantime, nanotechnologies have opened exciting new avenues in the quest of more
photostable and highly bright near-infrared tracers, more performing than classical organic dyes in
terms of optical properties. The use of nanostructures in comparison to small organic dyes is mainly
driven by their different and tunable biodistribution that can be tuned according to article size, shape,
charge and flexibility. The wide range of nanostructures explored for the design of efficient
nanoprobes is impressive. Quantum dots present several strong physicochemical advantages for this
purpose, but their short term translation toward clinics seems impeded by the toxicity issue still in
Molecules 2012, 17
debate for their use in patients. Another encouraging way relies on the entrapment of organic near-
infrared dyes into toxic element-free nanoparticles, especially those developed in pharmaceutical
science for drug delivery. These safe nanoparticles, especially polymer- or lipid- based carriers, allow
the protection of the near-infrared dyes against chemical and/or biological degradation, improve their
photophysical properties, and can be targeted for reaching specific diseased cells, increasing thus the
fluorescence signal-over-noise ratio. In fine, the choice of a fluorescent nanoprobe dedicated to a
specific medical application will be discussed on the basis of toxicity/adverse effects at the dose to be
injected for good image contrast. The same criterion will dictate the choice between small organic dye
and nanometer size tracer. Certainly, several nanoprobes of different chemical nature will come to
market for different applications in the near future.
The development of such interesting smart nanoprobes usually relies on interdisciplinary teams in
basic sciences in fields ranging from physics to biology, in close environment to clinicians. Their
future translation to clinics depends on the hardiness of their production processes in Good
Manufacturing Practice (GMP) conditions, their compelling safety, and the benefit they may offer for
human health in indicated clinical applications (early diagnosis, functional imaging guided surgery…).
Taking all together the aforementioned elements, there is no doubt that new clinical uses of fluorescent
nanoprobes will emerge in the next few years.
This work is supported by the Commissariat à l’Energie Atomique et aux Energies Alternatives
Conflicts of Interests
The authors declare no conflict of interest.
1. Massoud, T.F.; Gambhir, S.S. Molecular imaging in living subjects: Seeing fundamental
biological processes in a new light. Genes Develop. 2003, 17, 545–580.
Massoud, T.F.; Gambhir, S.S. Integrating noninvasive molecular imaging into molecular
medicine: An evolving paradigm. Trends Mol. Med. 2007, 13, 183–191.
Frangioni, J.V. New technologies for human cancer imaging. J. Clin. Oncol. 2008, 26, 4012–4021.
Leblond, F.; Davis, S.C.; Valdès, P.A.; Pogue, B.W. Pre-clinical whole-body fluorescence
imaging: Review of instruments, methods and applications. J. Photochem. Photobiol. B: Biol.
2010, 98, 77–94.
Hassan, M.; Klaunberg, B.A. Biomedical applications of fluorescence imaging in vivo.
Comp. Med. 2004, 54, 635–644.
Koo, V.; Hamilton, P.W.; Williamson, K. Non invasive in vivo imaging in small animal research.
Cellular Oncol. 2006, 28, 127–139.
Licha, K.; Olbrich, C. Optical imaging in drug discovery and diagnostic applications. Adv. Drug
Deliv. Rev. 2005, 57, 1087–1108.
Molecules 2012, 17
8. Miyashiro, I.; Miyoshi, N.; Hiratsuka, M.; Kishi, K.; Yamada, T.; Ohue, M.; Ohigashi, H.;
Yano, M.; Ishikawa, O.; Imaoka, S. Detection of sentinel node in gastric cancer surgery by
indocyanine green fluorescence imaging: Comparison with infrared imaging. Ann. Surg. Oncol.
2008, 15, 1640–1643.
9. Ogasawara, Y.; Ikeda, H.; Takahashi, M.; Kawasaki, K.; Doihara, H. Evaluation of breast
lymphatic pathways with indocyanine green fluorescence imaging in patients with breast cancers.
Word J. Surg. 2008, 32, 1924–1929.
10. Sevick-Muraca, E.M.; Sharma, R.; Rasmussen, J.C.; Marshall, M.V.; Wendt, J.A.; Pham, H.Q.;
Bonefas, E.; Houston, J.P.; Sampath, L.; Adams, K.E.; Blanchard, D.K.; Fischer, R.E.;
Chiang, S.B.; Elledge, R.; Mawad, M.E. Imaging of lymph flow in breast cancer patients after
microdose administration of a near-infrared fluorophore. Radiology 2008, 246, 734–741.
11. Tagaya, N.; Yamazaki, R.; Nakagawa, A.; Abe, A.; Hamada, K.; Kubota, K.; Oyama, T.
Intraoperative identification of sentinel lymph nodes by near-infrared fluorescence imaging in
patients with breast cancer. Am. J. Surg. 2008, 195, 850–853.
12. Troyan, S.L.; Kianzad, V.; Gibbs-Strauss, S.L.; Gioux, S.; Matsui, A.; Oketokoun, R.; Ngo, L.;
Khamene, A.; Azar, F.; Frangioni, J.V. The FLARE intraoperative near-infrared fluorescence
imaging system: A first-in-human clinical trial in breast cancer sentinel lymph node mapping.
Ann. Surg. Oncol. 2009, 16, 2943–2952.
13. Ntziachristos, V.; Ripoll, J.; Wang, L.V.; Weissleder, R. Looking and listening to light: The
evolution of whole-body photonic imaging. Nat. Biotechnol. 2005, 23, 313–320.
14. Laidevant, A.; Hervé, L.; Debourdeau, M.; Boutet, J.; Grenier, N.; Dinten, J.-M. Fluorescence
time-resolved imaging system embedded in an ultrasound prostate probe. Biomed. Opt. Express
2011, 2, 194–206.
15. Liu, Y.; Bauer, A.Q.; Akers, W.J.; Sudlow, G.; Liang, K.; Shen, D.; Berezin, M.Y.; Culver, J.P.;
Achilefu, S. Hands-free, wireless goggles for near-infrared fluorescence and real-time
image-guided surgery. Surgery 2011, 149, 689–698.
16. Qin, C.; Zhu, S.; Tian, J. New optical molecular imaging systems. Curr. Pharm. Biotechnol.
2010, 11, 620–627.
17. Pierce, M.C.; Javier, D.J.; Richards-Kortum, R. Optical contrast agents and imaging systems for
detection and diagnosis of cancer. Int. J. Cancer 2008, 123, 1979–1990.
18. Gioux, S.; Choi, H.S.; Frangioni, J.V. Image-guided surgery using invisible near-infrared light:
Fundamentals of clinical translation. Mol. Imaging 2010, 9, 237–255.
19. te Velde, E.A.; Veerman, T.; Subramaniam, V.; Ruers, T. The use of fluorescent dyes and probes
in surgical oncology. Eur. J. Surg. Oncol. 2010, 36, 6–15.
20. Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P.L.; Urano, Y. New strategies for fluorescent
probe design in medical diagnostic imaging. Chem. Rev. 2010, 110, 2620–2640.
21. Bremer, C.; Ntziachristos, V.; Weissleder, R. Optical-based molecular imaging: Contrast agents
and potential medical applications. Eur. Radiol. 2003, 13, 231–243.
22. Desmettre, T.; Devoisselle, J.-M.; Mordon, S. Fluorescence properties and metabolic features of
Indocyanine Green (ICG) as related to angiography. Surv. Ophthalmol. 2000, 45, 15–27.
23. Nishioka, Y.; Yoshino, H. Lymphatic targeting with nanoparticulate system. Adv. Drug Deliv.
Rev. 2001, 47, 55–64.
Molecules 2012, 17
24. Gravier, J.; Navarro, F.; Delmas, T.; Mittler, F.; Couffin, A.C.; Vinet, F.; Texier, I. Lipidots: A
biocompatible alternative to quantum dots for in vivo fluorescence imaging. J. Biomed. Opt.
2011, 16, 096013.
25. Onishi, S.; Lomnes, S.J.; Laurence, R.G.; Gobashian, A.; Mariani, G.; Frangioni, J.V. Organic
alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node
mapping. Mol. Imaging 2005, 4, 172–181.
26. Kim, S.; Lim, Y.T.; Soltesz, E.G.; De Grand, A.M.; Lee, J.; Nakayama, A.; Parker, J.A.;
Mihaljevic, T.; Laurence, R.G.; Dor, D.M.; et al. Near-infrared fluorescent type II quantum dots
for sentinel lymph node mapping. Nat. Biotechnol. 2004, 22, 93–97.
27. Kobayashi, H.; Hama, Y.; Koyama, Y.; Barett, T.; Regino, C.; Urano, Y.; Choyke, P.L.
Simultaneous multicolor imaging of five different lymphatic basins using quantum dots.
Nano Lett. 2007, 7, 1711–1716.
28. Jain, R.; Dandekar, P.; Patravale, V. Diagnostic nanocarriers for sentinel lymph node imaging.
J. Control. Release 2009, 138, 90–102.
29. Hutteman, M.; Mieog, J.S.; van der Vorst, J.R.; Liefers, G.J.; Putter, H.; Löwik, C.W.;
Frangioni, J.V.; van de Velde, C.J.; Vahrmeijer, A.L. Randomized, double-blind comparison of
indocyanine green with or without albumin premixing for near-infrared fluorescence imaging of
sentinel lymph nodes in breast cancer patients. Breast Cancer Res. Treat. 2011, 127, 163–170.
30. Benson, R.C.; Kues, H.A. Fluorescence properties of indocyanine green as related to
angiography. Phys. Med. Biol. 1978, 23, 159–163.
31. Lee, B.T.; Matsui, A.; Hutteman, M.; Lin, S.J.; Winer, J.H.; Laurence, R.G.; Frangioni, J.V.
Intraoperative near-infrared fluorescence imaging in perforator flap reconstruction: Current
research and early clinical experience. J. Reconstr. Microsurg. 2010, 26, 59–65.
32. Unno, N.; Suzuki, M.; Yamamoto, N.; Inuzuka, K.; Sagara, D.; Nishiyama, N.; Tanaka, H.;
Konno, H. Indocyanine Green fluorescence angiography for intraoperative assessment of blood
flow: A feasibility study. Eur. J. Vas. Endovasc. Surg. 2008, 35, 205–207.
33. Matsui, A.; Tanaka, E.; Choi, H.S.; Winer, J.H.; Kianzad, V.; Gioux, S.; Laurence, R.G.;
Frangioni, J.V. Real-time intra-operative near-infrared fluorescence identification of the
extra-hepatic bile ducts using clinically available contrast agents. Surgery 2010, 148, 87–95.
34. Aoki, T.; Yasuda, D.; Shimizu, Y.; Odaira, M.; Niiya, T.; Kusano, T.; Mitamura, K.; Hayashi, K.;
Murai, N.; Koizumi, T.; et al. Image-guided liver mapping using fluorescence navigation system
with indocyanine green for anatomical hepatic resection. World J. Surg. 2008, 32, 1763–1767.
35. Matsui, A.; Tanaka, E.; Choi, H.K.; Kienzad, V.; Gioux, S.; Lomnes, S.J.; Frangioni, J.V.
Real-time, near-infrared, fluorescence-guided identification of the ureters using methylene blue.
Surgery 2010, 148, 78–86.
36. Rasmussen, J.C.; Tan, I.C.; Marshall, M.V.; Adams, K.E.; Kwon, S.; Fife, C.E.; Maus, E.A.;
Smith, L.A.; Covington, K.R.; Sevick-Muraca, E.M. Human lymphatic architecture and dynamic
transport imaged using near-infrared fluorescence. Transl. Oncol. 2010, 3, 362–372.
37. Alacam, B.; Yazici, B.; Intes, X.; Nioka, S.; Chance, B. Pharmacokinetic-rate images of
indocyanine green for breast tumors using near-infrared optical methods. Phys. Med. Biol. 2008,
Molecules 2012, 17
38. Poellinger, A.; Persigehl, T.; Mahler, M.; Bahner, M.; Ponder, S.L.; Diekmann, F.; Bremer, C.;
Moesta, T. Near-infrared imaging of the breast using omocianine as a fluorescent dye: Results of
a placebo-controlled, clinical, multicenter trial. Investig. Radiol. 2011, 46, 697–704.
39. van de Ven, S.; Wiethoff, A.; Nielsen, T.; Brendel, B.; van der Voort, M.; Nachabe, R.;
Van der Mark, M.; Van Beek, M.; Bakker, L.; Fels, L.; et al. A novel fluorescent imaging agent for
diffuse optical tomography of the breast: First clinical trial experience in patients. Mol. Imaging
Biol. 2010, 12, 343–348.
40. Gonçalves, M.S.T. Fluorescent labeling of biomolecules with organic probes. Chem. Rev. 2009,
41. Luo, S.; Zhang, E.; Su, Y.; Cheng, T.; Shi, C. A review of NIR dyes in cancer targeting and
imaging. Biomaterials 2011, 32, 7127–7138.
42. Pham, W.; Medarova, Z.; Moore, A. Synthesis and application of a water-soluble near-infrared
dye for cancer detection using optical imaging. Bioconjug. Chem. 2005, 16, 735–740.
43. Choi, H.S.; Nasr, K.; Alyabyev, S.; Feith, D.; Lee, J.H.; Kim, S.H.; Ashitate, Y.; Hyun, H.;
Patonay, G.; Strekowski, L.; et al. Synthesis and in vivo Fate of Zwitterionic Near-Infrared
Fluorophores. Angew. Chem. Int. Ed. 2011, 50, 6258–6263.
44. Ye, Y.; Bloch, S.; Kao, J.; Achilefu, S. Multivalent carbocyanine molecular probes: Synthesis
and applications. Bioconjug. Chem. 2005, 16, 51–61.
45. Zhang, Z.; Achilefu, S. Synthesis and evaluation of polyhydroxylated near-infrared carbocyanine
molecular probes. Org. Lett. 2004, 6, 2067–2070.
46. Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor vascular permeability and the EPR
effect in macromolecular therapeutics: A review. J. Control. Release 2000, 65, 271–284.
47. Jennings, L.E.; Long, N.J. ‘Two is better than one’-probes for dual-modality molecular imaging.
Chem. Commun. 2009, 3511–3524.
48. Liu, Y.; Yu, G.; Tian, M.; Zhang, H. Optical probes and the applications in multimodality
imaging. Contrast Media Mol. Imaging 2011, 6, 169–177.
49. Janib, S.M.; Moses, J.E.; MacKay, J.A. Imaging and drug delivery using theranostic
nanoparticles. Adv. Drug Deliv. Rev. 2010, 62, 1052–1063.
50. Kelkar, S.S.; Reineke, T.M. Theranostics: Combining Imaging and Therapy. Bioconjug. Chem.
2011, 22, 1879–1903.
51. Reiss, P.; Protiere, M.; Li, L. Core/Shell Semiconductor Nanocrystals. Small 2009, 5, 154–168.
52. Medintz, I.L.; Mattoussi, H.; Clapp, A.R. Potential clinical applications of quantum dots. Int. J.
Nanomed. 2008, 3, 151–167.
53. Smith, A.M.; Duan, H.; Mohs, A.M.; Nie, S. Bioconjugated quantum dots for in vivo molecular
and cellular imaging. Adv. Drug Deliv. Rev. 2008, 60, 1226–1240.
54. Zrazhevskiy, P.; Sena, M.; Gao, X. Designing multifunctional quantum dots for bioimaging,
detection, and drug delivery. Chem. Rev. 2010, 39, 4326–4354.
55. Xing, Y.; Rao, J. Quantum dot bioconjugates for in vitro diagnostics and in vivo imaging.
Cancer Biomark. 2008, 4, 207–319.
56. Wang, Y.M.; Chen, L. Quantum dots, lighting up the research and development of nanomedicine.
Nanomed.: Nanotechnol. Biol. Med. 2011, 7, 385–402.
Molecules 2012, 17
57. Li, L.; Daou, T.J.; Texier, I.; Chi, T.T.K.; Liem, N.Q.; Reiss, P. Highly luminescent CuInS2/ZnS
Core/Shell nanocrystals: Cadmium-free quantum dots for in vivo imaging. Chem. Mater. 2009,
58. Li, L.; Reiss, P. One-pot synthesis of highly luminescent InP/ZnS nanocrystals without precursor
injection. J. Am. Chem. Soc. 2008, 130, 11588–11589.
59. Choi, H.S.; Ipe, B.I.; Misra, P.; Lee, J.H.; Bawendi, M.G.; Frangioni, J.V. Tissue- and
organ-selective biodistribution of NIR fluorescent quantum dots. Nano Lett. 2009, 9, 2354–2359.
60. Gao, J.; Chen, K.; Xie, R.; Xie, J.; Lee, S.-W.; Cheng, Z.; Peng, X.; Chen, X. Ultrasmall
Near-infrared non-cadmium quantum dots for in vivo tumor imaging. Small 2010, 6, 256–261.
61. Pons, T.; Pic, E.; Lequeux, N.; Cassette, E.; Bezdetnaya, L.; Guillemin, F.; Marchal, F.;
Dubertret, B. Cadmium-free CuInS2/ZnS quantum dots for sentinel lymph node imaging with
reduced toxicity. ACS Nano 2010, 4, 2531–2538.
62. Schipper, M.L.; Iyer, G.; Koh, A.L.; Cheng, Z.; Ebenstein, Y.; Aharoni, A.; Keren, S.;
Bentolila, L.A.; Li, J.; Rao, J.; et al. Particle size, surface coating, and PEGylation influence the
biodistribution of quantum dots in living mice. Small 2009, 5, 126–134.
63. Yong, K.-T.; Roy, I.; Ding, H.; Bergey, E.J.; Prasad, P. Biocompatible near-infrared quantum
dots as ultrasensitive probes for long term in vivo imaging applications. Small 2009, 5, 1997–2004.
64. Zimmer, J.P.; Kim, S.W.; Ohnishi, S.; Tanaka, E.; Frangioni, J.V.; Bawendi, M.G. Size series of
small indium arsenide-zinc selenide core-shell nanocrystals and their applications to in vivo
imaging. J. Am. Chem. Soc. 2006, 128, 2526–2527.
65. Choi, H.S.; Liu, W.; Misra, W.; Tanaka, E.; Zimmer, J.P.; Ipe, B.I.; Bawendi, M.G.;
Frangioni, J.V. Renal clearance of quantum dots. Nat. Biotechnol. 2007, 25, 1165–1170.
66. Yang, R.S.H.; Chang, L.W.; Wu, J.-P.; Tsai, M.-H.; Wang, H.-J.; Kuo, Y.-C.; Yeh, T.-K.;
Yang, C.S.; Lin, P. Persistent tissues kinetics and redistribution of nanoparticles, quantum dot
705, in mice: ICP-MS quantitative assessment. Environ. Health Perspect. 2007, 115, 1339–1343.
67. Schipper, M.L.; Cheng, Z.; Lee, S.-W.; Bentolila, L.A.; Iyer, G.; Rao, J.; Chen, X.; Wu, A.M.;
Weiss, S.; Gambhir, S.S. micro-PET based biodistribution of quantum dots in living mice.
J. Nucl. Med. 2007, 48, 1511–1518.
68. Gao, J.; Chen, K.; Luong, R.; Bouley, D.M.; Mao, H.; Qiao, T.; Gambhir, S.S.; Cheng, Z.
A novel clinically translatable fluorescent nanoparticle for targeted molecular imaging of tumors
in living subjects. Nano Lett. 2011, 12, 281–286.
69. Hauck, T.S.; Anderson, R.E.; Fischer, H.C.; Newbigging, S.; Chan, W.C.W. In vivo Quantum-dot
toxicity assessment. Small 2010, 6, 138–144.
70. Jokerst, J.V.; Lobovkina, T.; Zare, R.; Gambhir, S.S. Nanoparticle PEGylation for imaging and
therapy. Nanomedicine 2011, 6, 715–728.
71. Daou, T.J.; Li, L.; Reiss, P.; Josserand, V.; Texier, I. Effect of poly(ethylene glycol) length on
the in vivo behavior of coated quantum dots. Langmuir 2009, 25, 3040–3044.
72. Tavares, A.J.; Chong, L.; Petryayeva, E.; Algar, W.R.; Krull, U.J. Quantum dots as contrast agents
for in vivo tumor imaging: Progress and issues. Anal. Bioanal. Chem. 2011, 399, 2331–2342.
73. Gao, X.; Cui, Y.; Levenson, R.M.; Chung, L.W.; Nie, S. In vivo cancer targeting and imaging
with semiconductor quantum dots. Nat. Biotechnol. 2004, 22, 969–976.
Molecules 2012, 17
74. Ballou, B.; Ernst, L.A.; Andreko, S.; Harper, T.; Fitzpatrick, J.A.; Waggoner, A.S.;
Bruchez, M.P. Sentinel lymph node imaging using quantum dots in mouse tumor models.
Bioconjug. Chem. 2007, 18, 389–396.
75. Kobayashi, H.; Kosaka, N.; Ogawa, M.; Morgan, N.Y.; Smith, P.D.; Murray, C.B.; Ye, X.C.;
Collins, J.; Kumar, G.A.; Bell, H.; Choyke, P.L. In vivo multiple color lymphatic imaging using
upconverting nanocrystals. J. Mater. Chem. 2009, 19, 6481–6484.
76. Choi, J.; Burns, A.; Williams, R.M.; Zhou, Z.; Zipfel, W.R.; Wiesner, U.; Nikitin, A.Y.
Core-shell silica nanoparticles as fluorescent labels for nanomedicine. J. Biomed. Opt. 2007, 12,
77. Friedman, R. Nano dot technology enters clinical trials. J. Natl. Cancer Inst. 2011, 103, 1428–1429.
78. Kumar, S.; Roy, I.; Ohulchanskyy, T.Y.; Goswani, L.N.; Bonoiu, A.C.; Bergey, E.J.;
Tramposch, K.M.; Maitra, A.; Prasad, P. Covalently dye-linked, surface-controlled, and
bioconjugated organically modified silica nanoparticles as targeted probes for optical imaging.
ACS Nano 2008, 2, 449–456.
79. Lee, C.H.; Cheng, S.H.; Wang, Y.J.; Chen, Y.-C.; Chen, N.-T.; Souris, J.; Chen, C.-T.; Mou, C.-Y.;
Yang, C.-S.; Lo, L.-W. Near-Infrared Mesoporous Silica Nanoparticles for Optical Imaging:
Characterization and in vivo Biodistribution. Adv. Func.Mater. 2009, 19, 215–222.
80. Kumar, R.; Roy, I.; Ohulchansky, T.; Vathy, L.A.; Bergey, E.; Sajjad, M.; Prasad, P. In vivo
biodistribution and clearance studies using multimodal organically modified silica nanoparticles.
ACS Nano 2010, 4, 699–708.
81. He, X.; Nie, H.; Wang, K.; Tan, W.; Wu, X.; Zhang, P. In vivo study of biodistribution and
urinary excretion of surface-modified silica nanoparticles. Anal. Chem. 2008, 80, 9597–9603.
82. Liong, M.; Lu, J.; Kovochich, M.; Xia, T.; Ruehm, S.G.; Nel, A.E.; Tamanoi, F.; Zink, J.I.
Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery. ACS Nano
2008, 2, 889–896.
83. Sharma, P.; Brown, S.; Walter, G.; Santra, S.; Moudgil, B. Nanoparticles for bioimaging.
Adv. Colloid. Interf. Sci. 2006, 471–485.
84. Larson, D.R.; Ow, H.; Vishwasrao, H.D.; Heikal, A.A.; Wiesner, U.; Webb, W.W. Silica
nanoparticle architecture determines radiative properties of encapsulated fluorophores.
Chem. Mater. 2008, 20, 2677–2684.
85. Rampazzo, E.; Bonacchi, S.; Montalti, M.; Prodi, L.; Zaccheroni, N. Self-organizing core-shell
nanostructures: Spontaneous accumulation of dye in the core of doped silica nanoparticles. J.
Am. Chem. Soc. 2007, 129, 14251–14256.
86. Bringley, J.F.; Penner, T.L.; Wang, R.; Harder, J.F.; Harrison, W.J.; Buonemani, L. Silica
nanoparticles encapsulating near-infrared emissive cyanine dyes. J. Colloid. Interf. Sci. 2008,
87. Burns, A.; Vider, J.; Ow, H.; Herz, E.; Penate-Medina, O.; Baumgart, M.; Larson, S.M.;
Wiesner, U.; Bradbury, M. Fluorescent silica nanoparticles with efficient urinary excretion for
nanomedicine. Nano Lett. 2009, 9, 442–448.
88. Altinoglu, E.I.; Russin, T.J.; Kaiser, J.M.; Barth, B.M.; Eklund, P.C.; Kester, M.; Adair, J.H.
Near-infrared emitting fluorophore-doped calcium phosphate nanoparticles for in vivo imaging of
human breast cancer. ACS Nano 2008, 2, 2075–2084.
Molecules 2012, 17
89. Barth, B.M.; Altinoglu, E.I.; Shanmugavelandy, S.S.; Kaiser, J.M.; Crespo-Gonzalez, D.;
DiVittore, N.A.; McGovern, C.; Goff, T.M.; Keasey, N.R.; Adair, J.H.; Loughran, T.P.;
Claxton, D.F.; Kester, M. Targeted indocyanine-green-loaded calcium phosphosilicate
nanoparticles for in vivo photodynamic therapy of leukemia. ACS Nano 2011, 5, 5325–5337.
90. Epple, M.; Ganesan, K.; Heumann, R.; Klesing, J.; Kovtun, A.; Neumann, S.; Sokolova, V.
Application of calcium phosphate nanoparticles in biomedicine. J. Mater. Chem. 2010, 20, 18–23.
91. Schwiertz, J.; Wiehe, A.; Gräfe, S.; Gitter, B.; Epple, M. Calcium phosphate nanoparticles as
efficient carriers for photodynamic therapy against cells and bacteria. Biomaterials 2009, 30,
92. Lee, S.Y.; Cha, E.J.; Park, K.; Lee, S.Y.; Hong, J.K.; Sun, I.C.; Kim, S.Y.; Choi, K.; Kwon, I.C.;
Kim, K.; Ahn, C.H. A near-infrared fluorescence quenched gold nanoparticle imaging probe for
in vivo drug screening and protease activity determination. Angew. Chem. Int. Ed. 2008, 47,
93. Zhou, C.; Long, M.; Qin, Y.; Sun, X.; Zheng, J. Luminescent gold nanoparticles with efficient
renal clearance. Angew. Chem. Int. Ed. 2011, 50, 3168–3172.
94. Faure, A.-C.; Dufort, S.; Josserand, V.; Perriat, P.; Coll, J.L.; Roux, S.; Tillement, O. Control of
the in vivo biodistribution of hybrid nanoparticles with different poly(ethyleneglycol) coatings.
Small 2009, 5, 2565–2575.
95. le Masne de Chermont, Q.; Chanéac, C.; Seguin, J.; Pellé, F.; Maîtrejean, S.; Jolivet, J.P.;
Gourier, D.; Bessodes, M.; Scherman, D. Nanoprobes with near infrared persistent luminescence
for in vivo imaging. Proc. Natl. Acad. Sci. USA 2007, 104, 9266–9271.
96. Texier, I.; Heinrich, E.; Berger, M.; Tillement, O.; Louis, C.; Peltié, P. Luminescent
up-converting nano-crystals for in vivo imaging. Proc. SPIE 2007, 6449, 64490D:1–64490D:11.
97. Hilderbrand, S.A.; Shao, F.; Salthouse, C.; Mahmood, U.; Weissleder, R. Upconverting
luminescent nanomaterials: Application to in vivo bioimaging. Chem. Commun. 2009, 4188–4190.
98. Xiong, L.; Chen, Z.; Tian, Q.; Cao, T.; Xu, C.; Li, F. High contrast upconversion luminescence
targeted imaging in vivo using peptide-labeled nanophosphors. Anal. Chem. 2009, 81, 8687–8694.
99. Zhou, J.; Sun, Y.; Du, X.; Xiong, L.; Hu, H.; Li, F. Dual-modality in vivo imaging using
rare-earth nanocrystals with near-infrared to near-infrared (NIR-to-NIR) upconversion
luminescence and magnetic resonace properties. Biomaterials 2010, 31, 3287–3295.
100. Sahoo, S.K.; Parveen, S.; Panda, J.J. The present and future of nanotechnology in human health
care. Nanomed.: Nanotechnol. Biol. Med. 2007, 3, 20–31.
101. Wagner, V.; Dullaart, A.; Bock, A.-K.; Zweck, A. The emerging nanomedicine landscape.
Nat. Biotechnol. 2006, 24, 1211–1217.
102. Peer, D.; Karp, J.M.; Hong, S.; Farokhzad, O.C.; Margalit, R.; Langer, R. Nanocarriers as an
emerging platform for cancer therapy. Nat. Nanotechnol. 2007, 2, 751–760.
103. Bricarello, D.A.; Smolowitz, J.T.; Zivkovic, A.M.; German, J.B.; Parikh, A.N. Reconstituted
lipoprotein: A versatile class of biologically-inspired nanostructures. ACS Nano 2011, 5, 42–57.
104. Larson, N.; Ghandehari, H. Polymeric conjugates for drug delivery. Chem. Mater. 2012, 24,
105. Blasi, P.; Giovagnoli, S.; Schoubben, A.; Ricci, M.; Rossi, C. Solid lipid nanoparticles for
targeted brain drug delivery. Adv. Drug Deliv. Rev. 2007, 59, 454–477.
Molecules 2012, 17
106. Constantinides, P.P.; Chaubal, M.V.; Shorr, R. Advances in lipid nanodispersions for parenteral
drug delivery and targeting. Adv. Drug Deliv. Rev. 2008, 60, 757–767.
107. Huynh, N.T.; Passirani, C.; Saulnier, P.; Benoit, J.P. Lipid nanocapsules: A new platform for
nanomedicine. Int. J. Pharm. 2009, 379, 201–209.
108. Joshi, M.D.; Müller, R.H. Lipid nanoparticles for parenteral delivery of actives. Eur. J. Pharm.
Biopharm. 2009, 71, 161–172.
109. Sawant, K.K.; Dodiya, S.S. Recent advances and patents on solid lipid nanoparticles. Recent pat.
Drug Deliv. Formul. 2008, 2, 120-135.
110. Couvreur, P.; Vauthier, C. Nanotechnology: Intelligent design to treat complex disease.
Pharm. Res. 2006, 23, 1417–1450.
111. Torchilin, V.P. Multifunctional nanocarriers. Adv. Drug Deliv. Rev. 2006, 58, 1532–1555.
112. Moghimi, S.M.; Hunter, A.C.; Murray, J.C. Long-circulating and target-specific nanoparticles:
Theory to practice. Pharmacol. Rev. 2001, 53, 283–318.
113. Tung, C.H. Fluorescent peptide probes for in vivo diagnostic imaging. Biopolymers 2004, 76,
114. Tung, C.-H.; Bredow, S.; Mahmood, U.; Weissleder, R. Preparation of a cathepsin D sensitive
near-infrared fluorescence probe for imaging. Bioconjug. Chem. 1999, 10, 892–896.
115. Tung, C.-H.; Gerszten, R.E.; Jaffer, F.A.; Weissleder, R. A novel near infrared fluorescence
sensor for detection of thrombin activation in blood. ChemBioChem 2002, 3, 207–211.
116. Tung, C.-H.; Mahmood, U.; Bredow, S.; Weissleder, R. In vivo imaging of proteolytic enzyme
activity using a novel molecular reporter. Cancer Res. 2000, 60, 4953–4958.
117. Miki, K.; Oride, K.; Inoue, S.; Kuramochi, Y.; Nayak, R.R.; Matsuoka, H.; Harada, H.;
Hiraoka, M.; Ohe, K. Ring-opening metathesis polymerization-based synthesis of polymeric
nanoparticles for enhanced tumor imaging in vivo: Synergistic effect of folate-receptor targeting
and PEGylation. Biomaterials 2010, 31, 934–942.
118. Saxena, V.; Sadoqi, M.; Shao, J. Enhanced photo-stability, thermal-stability and aqueous-stability
of indocyanine green in polymeric nanoparticulate systems. J. Photochem. Photobiol. B: Biol.
2004, 74, 29–38.
119. Saxena, V.; Sadoqi, M.; Shao, J. Polymeric nanoparticulate delivery system for indocyanine
green: Biodistribution in healthy mice. Int. J. Pharm. 2006, 308, 200–204.
120. Larush, L.; Magdassi, S. Formation of near-infrared fluorescent nanoparticles for medical
imaging. Nanomedicine 2011, 6, 233–240.
121. Schadlich, A.; Rose, C.; Kuntsche, J.; Caysa, H.; Mueller, T.; Gopferich, A.; Mader, K. How
stealthy are PEG-PLA nanoparticles? An NIR in vivo study combined with detailed size
measurements. Pharm. Res. 2011, 28, 1995–2007.
122. Tong, R.; Coyle, V.J.; Tang, L.; Barger, A.M.; Fan, T.M.; Cheng, J.J. Polylactide nanoparticles
containing stably incorporated cyanine dyes for in vitro and in vivo imaging applications.
Microsc. Res. Tech. 2010, 73, 901–909.
123. Longmire, M.R.; Ogawa, M.; Choyke, P.L.; Kobayashi, H. Biologically optimized nanosized
molecules and particles: More than just size. Bioconjug. Chem. 2011, 22, 993–1000.
Molecules 2012, 17
124. Almutairi, A.; Akers, W.J.; Berezin, M.Y.; Achilefu, S.; Fréchet, J.M.J. Monitoring the
biodegradation of dendritic near-infrared nanoprobes by in vivo fluorescence imaging.
Mol. Pharm. 2008, 5, 1103–1110.
125. Quadir, M.A.; Radowski, M.R.; Kratz, F.; Licha, K.; Hauff, P.; Haag, R. Dendritic multishell
architectures for drug and dye transport. J. Control. Release 2008, 132, 289–294.
126. Rodriguez, V.B.; Henry, S.M.; Hoffman, A.S.; Stayton, P.S.; Li, X.; Pun, S.H. Encapsulation and
stabilization of indocyanine green within poly(styrene-alt-maleic anhydride) block-poly(styrene)
micelles for near-infrared imaging. J. Biomed. Opt. 2008, 13, 014025.
127. Zheng, X.; Xing, D.; Zhou, F.; Wu, B.; Chen, W.R. Indocyanine Green-containing
nanostructures as near infrared dual-functional targeting probes for optical imaging and
photothermal therapy. Mol. Pharm. 2011, 8, 447–456.
128. Tanisaka, H.; Kizaka-Kondoh, S.; Makino, A.; Tanaka, S.; Hiraoka, M.; Kimura, S.
Near-infrared fluorescent labeled peptosome for application to cancer imaging. Bioconjug. Chem.
2008, 19, 109–117.
129. Wang, D.; Qian, J.; He, S.; Park, J.S.; Lee, K.-S.; Han, S.; Mu, Y. Aggregation-enhanced
fluorescence in PEGylated phospholipid nanomicelles for in vivo imaging. Biomaterials 2011,
130. Hilderbrand, S.A.; Kelly, K.; Niedre, M.; Weissleder, R. Near infrared fluorescence-based
bacteriophage particles for ratiometric pH imaging. Bioconjug. Chem. 2008, 19, 1635–1639.
131. Ghoroghchian, P.; Frail, P.; Susumu, K.; Blessington, D.; Brannan, A.; Bates, F.; Chance, B.;
Hammer, D.; Therien, M. Near IR emissive polymersome: Self-assembled soft matter for in vivo
optical imaging. Proc. Natl. Acad. Sci. USA 2005, 102, 2922–2927.
132. Duncan, T.V.; Ghoroghchian, P.; Rubtsov, I.V.; Hammer, D.; Therien, M. Ultrafast excited-state
dynamics of nanoscale near-infrared emissive polymersomes. J. Am. Chem. Soc. 2008, 130,
133. Ghoroghchian, P.P.; Frail, P.R.; Li, G.; Zupancich, J.A.; Bates, F.S.; Hammer, D.A.;
Therien, M.J. Controlling bulk optical properties of emissive polymersomes through
intramembranous polymer-fluorophore interactions. Chem. Mater. 2007, 19, 1309–1318.
134. McNeil, S.E. Nanoparticle therapeutics: A personal perspective. Wiley Interdiscip. Rev.:
Nanomed. Nanobiotechnol. 2009, 1, 264–271.
135. Weyenberg, W.; Filev, P.; van den Plas, D.; Vandervoort, J.; De Smet, K.; Sollie, P.; Ludwiga, A.
Cytotoxicity of submicron emulsions and solid lipid nanoparticles for dermal application. Int. J.
Pharm. 2007, 337, 291–298.
136. Devoisselle, J.-M.; Soulié-Bégu, S.; Mordon, S.; Desmettre, T.; Maillols, H. A preliminary study
of the in vivo behaviour of an emulsion formulation of Indocyanine Green. Lasers Med. Sci.
1998, 13, 279–282.
137. Mulder, W.; Strijkers, G.; van Tilborg, G.; Cormode, D.P.; Fayad, Z.A.; Nicolay, K.
Nanoparticulate assemblies of amphiphiles and diagnostically active materials for multimodality
imaging. Acc. Chem. Res. 2009, 42, 904–914.
Molecules 2012, 17
138. Akers, W.J.; Kim, C.; Berezin, M.Y.; Guo, K.; Fuhrhop, R.; Lanza, G.M.; Fischer, G.M.;
Daltrozzo, E.; Zumbusch, A.; Cai, X.; Wang, L.V.; Achilefu, S. Noninvasive photoacoustic and
fluorescence sentinel lymph node identification using dye-loaded perfluorocarbon nanoparticles.
ACS Nano 2011, 5, 173–182.
139. Delmas, T.; Couffin, A.C.; Bayle, P.A.; de Crécy, F.; Neumann, E.; Vinet, F.; Bardet, M.;
Bibette, J.; Texier, I. Preparation and characterisation of highly stable lipid nanoparticles with
amorphous core of tuneable viscosity. J. Colloid Interf. Sci. 2011, 360, 471–481.
140. Delmas, T.; Piraux, H.; Couffin, A.C.; Texier, I.; Vinet, F.; Poulin, P.; Cates, M.E.; Bibette, J.
How to prepare and stabilize very small nanoemulsions. Langmuir 2011, 27, 1683–1692.
141. Navarro, F.; Mittler, F.; Berger, M.; Josserand, V.; Gravier, J.; Vinet, F.; Texier, I. Cell
tolerability and biodistribution in mice of Indocyanine Green-loaded lipid nanoparticles.
J. Biomed. Nanotechnol. 2012, in press.
142. Navarro, F.; Berger, M.; Guillermet, S.; Josserand, V.; Guyon, L.; Goutayer, M.; Neumann, E.;
Rizo, P.; Vinet, F.; Texier, I. Lipid nanoparticle vectorization of IndoCyanin Green improves non
invasive fluorescence imaging. J. Biomed. Nanotechnol. 2012, in press.
143. Goutayer, M.; Dufort, S.; Josserand, V.; Royère, A.; Heinrich, E.; Vinet, F.; Bibette, J.;
Coll, J.L.; Texier, I. Tumor targeting of functionalized lipid nanoparticles: Assessment by in vivo
fluorescence imaging. Eur. J. Pharm. Biopharm. 2010, 75, 137–147.
144. Texier, I.; Goutayer, M.; Da Silva, A.; Guyon, L.; Djaker, N.; Josserand, V.; Neumann, E.;
Bibette, J.; Vinet, F. Cyanine loaded lipid nanoparticles for improved in vivo fluorescence
imaging. J. Biomed. Opt. 2009, 14, 054005.
145. Cormode, D.P.; Jarzyna, P.A.; Mulder, W.; Fayad, Z.A. Modified natural nanoparticles as
contrast agents for medical imaging. Adv. Drug Deliv. Rev. 2010, 62, 329–338.
146. Kenneth, K.N.G.; Lovell, J.F.; Zheng, G. Lipoprotein-inspired nanoparticles for cancer
theranostics. Acc. Chem. Res. 2011, 44, 1105–1113.
147. Cao, W.; Kenneth K.; Corbin, I.; Zhang, Z.; Ding, L.; Chen, J.; Zheng, G. Synthesis and
Evaluation of a Stable Bacteriochlorophyll-Analog and Its Incorporation into High-Density
Lipoprotein Nanoparticles for Tumor Imaging. Bioconjug. Chem. 2009, 20, 2023–2031.
148. Corbin, I.R; Chen, J.; Cao, W.; Li, H.; Lund-Katz, S.; Zheng, G. Enhanced cancer-targeted
delivery using engineered high-density lipoprotein-based nanocarriers. J. Biomed. Nanotechnol.
2007, 3, 367–376.
149. Zhang, Z.; Chen, J.; Ding, L.; Jin, H.; Lovell, J.F.; Corbin, I.; Cao, W.; Lo, P-C.; Yang, M.;
Tsao, M-S.; Luo, Q.; Zheng, G. HDL-mimicking peptide-lipid nanoparticles with improved
tumor targeting. Small 2010, 6, 430–437.
150. Zheng, G.; Chen, J.; Li, H.; Glickson, J.D. Rerouting lipoprotein nanoparticles to selected
alternate receptors for the targeted delivery of cancer diagnostic and therapeutic agents.
Proc. Natl. Acad. Sci. USA 2005, 102, 17757–17762.
151. Chen, J.; Corbin, I.; Li, H.; Cao, W.; Glickson, J.D.; Zheng, G. Ligand conjugated low-density
lipoprotein nanoparticles for enhanced optical cancer imaging in vivo. J. Am. Chem. Soc. 2007,
152. Kirchherr, A.-K.; Briel, A.; Mäder, K. Stabilization of indocyanine green by encapsulation within
micellar systems. Mol. Pharm. 2009, 6, 480–491.
Molecules 2012, 17 Download full-text
153. Deissler, V.; Rüger, R.; Frank, W.; Fahr, A.; Kaiser, W.A.; Hilger, I. Fluorescent liposomes as
contrast agents for in vivo optical imaging of edemas in mice. Small 2008, 4, 1240–1246.
154. Sandanaraj, B.S.; Gremlich, H.-U.; Kneuer, R.; Dawson, J.; Wacha, S. Fluorescent nanoprobes as
a biomarker for increased vascular permeability: Implications in diagnosis and treatment of
cancer and inflammation. Bioconjug. Chem. 2010, 21, 93–101.
155. Portnoy, E.; Lecht, S.; Lazarovici, P.; Danino, D.; Magdassi, S. Cetuximab-labeled liposomes
containing near-infrared probe for in vivo imaging. Nanomed.: Nanotechnol. Biol. Med. 2011, 7,
156. Proulx, S.T.; Luciani, P.; Derzsi, S.; Rinderknecht, M.; Mumprecht, V.; Leroux, J.-C.;
Detmar, M. Quantitative imaging of lymphatic function with liposomal indocyanine green.
Cancer Res. 2010, 70, 7053–7062.
157. Lovell, J.; Jin, C.S.; Huynh, E.; Jin, H.; Kim, C.; Rubinstein, J.L.; Chan, W.C.; Cao, W.;
Whang, LV.; Zheng, G. Porphysome nanovesicles generated by porphyrin bilayers for use as
multimodal biphotonic contrast agents. Nat. Mater. 2011, 10, 324–332.
158. Frias, J.C.; Lipinski, M.J.; Lipinski, S.E.; Albeda, M.T. Modified lipoproteins as contrats agents
for imaging atherosclerosis. Contrast Media Mol. Imaging 2007, 2, 16–23.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license