Advanced Drug Delivery Reviews 62 (2010) 42–58

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Advanced Drug Delivery Reviews

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a d d r

Chitosan-based systems for molecular imaging☆

Prashant Agrawal ⁎, Gustav J. Strijkers, Klaas Nicolay
Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Molecular imaging enables the non-invasive assessment of biological and biochemical processes in living
Received 17 June 2009 subjects. Such technologies therefore have the potential to enhance our understanding of disease and drug
Accepted 29 September 2009 activity during preclinical and clinical drug development. Molecular imaging allows a repetitive and non-
Available online 25 October 2009
invasive study of the same living subject using identical or alternative biological imaging assays at different
time points, thus harnessing the statistical power of longitudinal studies, and reducing the number of
Keywords:
Chitosan
animals required and cost. Chitosan is a hydrophilic and non-antigenic biopolymer and has a low toxicity
Molecular imaging toward mammalian cells. Hence, it has great potential as a biomaterial because of its excellent
Imaging techniques biocompatibility. Conjugated to additional materials, chitosan composites result in a new class of
Imaging agents biomaterials that possess mechanical, physicochemical and functional properties, which have potential for
MRI use in advanced biomedical imaging applications. The present review will discuss the strengths, limitations
CT and challenges of molecular imaging as well as applications of chitosan nanoparticles in the field of
Ultrasound molecular imaging.
Optical imaging
© 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2. Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3. Molecular imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.1. Imaging techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.1.1. Computed tomography (CT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.1.2. Magnetic resonance (MR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.1.3. Positron emission tomography (PET) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.1.4. Single photon emission computed tomography (SPECT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.1.5. Ultrasound (US) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.1.6. Optical imaging (OI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.1.7. Imaging mass spectroscopy (IMS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2. Imaging agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2.1. Nanoparticle-based imaging agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.2.2. MRI contrast agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4. Current status of the use of chitosan composites in bioimaging applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

1. Introduction

Chitosan [poly(1,4-β-D-glucopyranosamine)], an abundant natural

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on
“Chitosan-Based Formulations of Drugs, Imaging Agents and Biotherapeutics”.
⁎ Corresponding author.
E-mail address: p.agrawal@tue.nl (P. Agrawal).
biopolymer, is produced by the deacetylation of chitin obtained from
the shells of crustaceans. It is a polycationic polymer that has one
amino group and two hydroxyl groups in the repeating hexosaminide
residue (Fig. 1). Chitosan has great potential as a biomaterial because

0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2009.09.007
 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58
Fig. 1. Chemical structure of chitosan. Individual atoms are numbered. Dashed lines
denote O3―O5 hydrogen bonds. Two dihedral angles (φ, ψ) defining the main chain
conformation and one dihedral angle χ defining the O6 orientation are indicated.
of its bio-compatible properties. It is hydrophilic, non-antigenic and
has a low toxicity toward mammalian cells [1–3]. In addition, chitosan
is known to facilitate drug delivery across cellular barriers and
transiently open the tight junctions between epithelial cells [4,5].
Chitin and chitosan are aminoglucopyrans composed of N-acetylglu-
cosamine (GlcNAc) and glucosamine (GlcN) residues. These poly-
saccharides are renewable resources which are currently also being
explored intensively for their applications in pharmaceutical, cos-
metics, biomedical, biotechnological, agricultural and food industries
[1,6].
These polymers have emerged as a new class of physiological mate-
rials of highly sophisticated functions and to exploit the properties of
these versatile polysaccharides, attempts are being made to derivatize
them [7]. Chemical modifications have done an excellent job for the
preparation of chitosan derivatives with higher solubility in water, such
as O-,N-carboxymethyl-chitosan, [8] N-carboxymethyl-chitosan, [9]
O-carboxymethyl-chitosan, [10,11] N-sulfate-chitosan, [12] O-sulfate
chitosan, [13] O-succinyl-chitosan, [14] N-methylene phosphonic
chitosan, [15] hydroxypropyl chitosan, [16] N-trimethyl chitosan, [17]
and others. The emergence of synthesis strategies for the fabrication of
nanosized particles leads to advancements in the nanotechnology,
which benefits molecular imaging for understanding of biological
processes at the molecular level. In addition, with the added multi-
functional features such particles may become an integral part of the
development of next generation therapeutic, diagnostic and imaging
technologies.
Molecular imaging holds the promise of the non-invasive assess-
ment of biological and biochemical processes in living subjects. Since
the inception of X-ray technology for medical imaging, many non-
invasive methodologies have been invented and successfully used for
applications ranging from clinical diagnosis to research in cellular
biology and drug discovery. Such technologies therefore have the
potential to enhance our understanding of disease and drug activity
during preclinical and clinical drug development. The advantage
of molecular imaging techniques over more conventional readouts
(e.g. immunohistochemistry) is that they can be performed in the
intact organism with sufficient spatial and temporal resolution for
studying biological processes in vivo. Furthermore, molecular imaging
allows a repetitive and non-invasive study of the same living subject
using identical or alternative biological imaging assays at different
time points, thus harnessing the statistical power of longitudinal
studies, and reducing the number of animals required and cost.
43
Molecular imaging usually exploits specific molecular probes as well
as intrinsic tissue characteristics as the source of image contrast, and
provides the potential for understanding of integrative biology, earlier
detection and characterization of disease, and evaluation of treatment
[18]. Molecular imaging could also aid decisions to select promising
drug candidates that seem most likely to be successful or to halt the
development of drugs that seem likely to ultimately fail [18].
Different imaging techniques are, in general, complementary
rather than competitive and the choice of imaging modality depends
primarily on the specific question that has to be addressed (Fig. 2).
Imaging of biological specimens both in vitro and in vivo has long
relied on light microscopy (fluorescence and luminescence imaging).
The presently leading non-invasive imaging techniques are computed
tomography (CT), magnetic resonance (MR), positron emission
tomography (PET), single photon emission CT (SPECT), ultrasound
(US) and optical imaging (OI), including their variations and sub-
categories [19–25]. Biomedical imaging research has prospered in
recent years because of the significant advances in electronics,
information technology and, more recently, nanotechnology.
The above imaging modalities can be broadly divided into two
groups, i.e., morphological/anatomical and molecular (i.e. functional)
imaging techniques. The morphological/anatomical imaging technol-
ogies, such as computed tomography (CT), MRI and ultrasound (US),
are characterized by high spatial resolution (Fig. 2). However, they
also share the limitation of not being able to detect diseases until
structural changes in the tissue (for example, growth of a tumor, or
extent of inflammation) are large enough to be morphologically
detected. On the other hand molecular imaging modalities, such as
optical imaging, PET and SPECT, offer the potential to detect molecu-
lar and cellular changes caused by disease (for example, before the
tumor is large enough to cause structural changes). However, these
molecular modalities suffer from a poor spatial resolution with cur-
rently available technology and do not provide anatomic information
(Fig. 2).
The present review will discuss the strengths, limitations and
challenges of molecular imaging as well as applications of chitosan
nanoparticles in the field of molecular imaging. We will first explain
the properties of chitosan, then will discuss different strategies of
molecular imaging, including their advantages and disadvantages. In
the last part of this review, agents for imaging will be reviewed as well
as the potential role and application of chitosan as a constituent of
molecular imaging contrast agents.
2. Chitosan
Chitosan is a natural polysaccharide derived from chitin and it has
been frequently employed as a polymer for self-assembling nano-
particles (Fig. 1). Most commonly, chitin represents the skeletal
material of invertebrates. R-Chitin occurs in the calyces of hydrozoa,
the egg shells of nematodes and rotifers, the radulae of mollusks, and
the cuticles of arthropods, while α-chitin is part of the shells of
brachiopods and mollusks, the cuttlefish bone, the squid pen, and the
pogonophora tubes [1]. Chitin is also found in exoskeletons,
peritrophic membranes, and cocoons of insects. All these organisms
synthesize chitin according to a common pathway that ends with the
polymerization of N-acetylglucosamine from the activated precursor
UDP-GlcNAc [1].
The industrial preparation of chitin uses the shells of crabs,
shrimps, prawns, and lobsters coming from the peeling machines in
canning factories. The isolation includes two steps: demineralization
with HCl and deproteination with aqueous NaOH [1]. After extraction
the chitin is deacetylated to chitosan, and the bio and cellular
compatibility of the materials is re-introduced by preparing chitosan
composites by conjugation of proteins and peptides of desirable
properties. Many of such composite scaffolds (chitosan–collagen
[26,27], chitosan–gelatin [28,29], chitosan–silk fibroin [30,31]) have
 44
P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58
Fig. 2. A collection of molecular imaging modalities. Each modality has particular characteristics, advantages and limitations, which are emphasized here. The different imaging modalities are generally considered complementary rather than
competitive.
Adapted with permission from Refs. [18,136].
 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58
been investigated for their ability to promote cell adhesion and
proliferation. Similarly, chitosan and other glycosaminoglycans
including dermatan sulfate, chondroitin sulfate, heparin and hyalur-
onan have been combined to form composites that provide specific
cellular adhesion and anti-thrombosis surfaces [32–35]. Kumar et al.
summarized the numerous works on the chemical modification of
chitosan and also indicated how this natural polymer is still being
modified to improve its utility, leading to various derivatives with
many applications [1].
Chitosan is a cationic copolymer of N-acetyl glucosamine and
D-glucosamine, varying in composition, sequence and molecular chain
length. This compound is nontoxic and biocompatible, thus offering
powerful potential for biomedical applications such as drug and gene
delivery, tissue engineering, wound healing, as well as for use in anti-
microbial, antiviral and immunoadjuvant strategies [36–40]. These
properties of chitosan attracted considerable attention also in the
areas of fisheries, textiles, food, ecology, academia and industry,
where chitin is upgraded to be exploited as a renewable resource and
at the same time contributing to the resolution of waste problems.
Chitosan's biocompatibility, its ability to prolong residence time in the
gastrointestinal tract through mucoadhesion, and its property to
enhance absorption by increasing cellular permeability have all been
major factors contributing to its widespread evaluation as an ingre-
dient of oral drug dosage forms [41]. With the rise of nanotechnology,
chitosan in conjunction with bioactive nanoparticles is fabricated into
various bio-nano-composites, providing alternatives to the new era of
regenerative medicine, as drug delivery vehicles and possibly as
contrast agent carriers for molecular imaging [35,39]. The increasing
interests in biomedical applications of chitosan create more oppor-
tunities for production of specialized products such as injectable
thermo-responsive chitosan hydrogel for cartilage tissue engineering
(Biosyntech Inc., Canada) and chitosan oligomers for gene delivery
(FMC Biopolymer AS, Norway) [35]. Moreover, environmentally
friendly chitosan-based antimicrobial textiles such as Crabyon from
Tec services (Italy) and Chitopoly from Fuji Spinning Co. Ltd (Japan)
are now available in the market [35]. Compared to these more tra-
ditional chitosan products already commercially introduced, chitosan
composites are relatively new and have not been fully commercialized
yet [35].
Despite these promising prospects, the application of chitosan and
chitosan-based materials is somewhat limited by their poor solubility,
reactivity, physical properties (e.g. rigidity and britility) and strong
intra- and intermolecular hydrogen bonds in aqueous media [42]. It is
expected that the biological potentialities of chitosan can be improved
dramatically, if a water-soluble chitosan or chitosan-based material
would be prepared [42]. With a pKa of approximately 6.5 on the
amine groups, chitosan is insoluble at neutral pH but is soluble and
positively charged at acidic pH [36,37]. It has been reported that after
acylation with fatty acid chlorides, the above inter- and intra-
molecular hydrogen bondings of chitosan are replaced by hydropho-
bic interactions, which are believed to increase its solubility [43].
Although water-soluble chitosan (WSC) is commercially available
now, it has seen limited biomedical use thus far.
3. Molecular imaging
The aim of molecular imaging is to visualize biological processes
non-invasively. Molecular imaging plays an important role in tackling
the challenges of the characterization of biological processes at the
cellular level in living objects. Most of the diagnostic techniques that
are applied for routine clinical use have a counterpart in the exper-
imental research setting. Hence, it is possible to design preclinical
experiments that not only help to define the clinical protocol, but can
also provide mechanistic understanding of the observed biological
response.
45
Although molecular imaging modalities, such as optical imaging,
PET and SPECT offer the potential to detect molecular and cellular
changes of diseases, they suffer from a poor spatial resolution. MRI, on
the other hand, has good spatial resolution, but in order to become
suitable as a molecular imaging modality the low sensitivity for the
detection of contrast agents is a major hurdle [19]. However, for
preclinical evaluation, the imaging technique must have both
adequate spatial resolution (usually in the range of tens of millimeters
to micrometers) and the sensitivity to detect biochemical events
(typically in the range of millimolar to nanomolar), as well as possess
the ability to detect small, clinically relevant changes over time. Hence,
the current trend is to design hybrid systems where the combined
strengths of morphological/anatomical and molecular imaging mo-
dalities are exploited. This allows for the detection of pathophysio-
logical changes in early disease phases at high sensitivity using, for
example, PET and with high spatial resolution, using CT or MRI.
3.1. Imaging techniques
Imaging techniques are an essential commodity in the day-to-day
activities of the biomedical scientist. Each imaging modality has
certain advantages as well as limitations, and the choice of an imaging
modality, or combination of techniques, is determined by the specific
biological questions being asked. Each modality has its own char-
acteristics in terms of sensitivity and resolution, complexity, time of
data acquisition and financial cost. Most of the techniques exhibit
useful overlap with each other. However, there is no single modality
that fits all applications (Fig. 2).
3.1.1. Computed tomography (CT)
Computed tomography (CT) is a powerful nondestructive evalu-
ation technique for producing 2-D and 3-D cross-sectional images of
an object. Characteristics of the internal structure of an object such as
dimensions, shape, internal defects, and density are readily available
from CT images. CT creates the image by using an array of individual
small X-Ray sensors and a computer. By spinning the X-ray source and
the detectors around the patient, data is collected from multiple
angles. A computer then processes this information to reconstruct an
image. These images are called “sections” or “cuts” because they
appear to resemble cross-sections of the body. This technique elim-
inates the problem of conventional X-rays, which produce a pro-
jection image [18].
Although multimodality imaging approaches are increasing for the
diagnostic assessment, multislice computed tomography (MS-CT) is
the most frequently used single modality for, e.g., imaging of cancer.
Primary staging usually implies CT of the neck, thorax, and abdomen/
pelvis in axial orientation, completed by multiplanar reconstructions
and secondary data reconstruction using a bone kernel, thus covering
the main routes of lymphatic and hematogenous metastatic spread.
Yet, a limitation of CT is the low soft tissue contrast outside the lung,
which may cause difficulties when assessing distant metastases or
tumor extension into surrounding structures.
3.1.2. Magnetic resonance (MR)
Another technology that has seen tremendous growth as a tool in
medical diagnostics, especially for soft tissues, is magnetic resonance
imaging (MRI). MRI is the most versatile imaging technique since the
introduction of X-rays. Following its debut in the clinic in the 1980s, it
became an irreplaceable tool in diagnostic medicine and more
recently in basic research. In medicine, MRI is primarily used to
produce anatomical images of organs and it can also provide
information on the physicochemical state of tissues, their vascular-
ization, as well as function [44]. The success of MRI is based on the
unsurpassed soft tissue differentiation, the versatility of the slice
orientation, and the enormous variety of pulse sequences leading to
46 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58
information on morphology, motion, flow, diffusion, function, integ-
rity of vessel walls and more [44].
One of the most informative parameters encoded in the magnetic
resonance signal is the so-called chemical shift. While this informa-
tion is only partially used in MRI, it is the very basis of magnetic
resonance spectroscopy (MRS). In vivo MRS is a combination of
diagnostic MRI and high-resolution NMR. An MR spectrum represents
signals of different metabolites ordered in a plot along a chemical shift
axis, and represents a unique tool to obtain biochemical information
non-invasively, from living tissue [44].
MRI relies on the counterbalance between the exceedingly small
magnetic moment of the proton and the large number of protons
present in a biological tissue. This produces a measurable effect in the
presence of a large magnetic field. The MRI signal can be sensitized to
the longitudinal (T1) and transverse (T2) proton relaxation times of
mainly water and differences in proton relaxation times within and
among tissues are the source of contrast in MR images [45]. The
intrinsic relaxation times of tissue water are dependent on the
physiological environment, which can be used to monitor disease
progression and regression. The MRI contrast of tissues can be altered
with contrast agents that shorten the longitudinal and/or transverse
relaxation time. This ability of a contrast agent to alter T1 and T2 is
frequently used to enhance the sensitivity and specificity of disease
detection by MRI [45]. Apart from T1 and T2, MRI offers many other
contrast parameters that can be exploited to gain a better under-
standing of tissue structure, function and metabolism, both in health
and in disease.
3.1.3. Positron emission tomography (PET)
For PET a short-lived positron-emitting radioisotope of commonly
used atoms of Carbon (C), Oxygen (O), Nitrogen (N), or Fluorine (F),
contained in a compound of interest, is injected into the body. The
radioactive decay of such a radiopharmaceutical involving positron
emission and subsequent annihilation with an electron into two
511 keV γ-photons irradiated in opposite directions is quantitatively
measured by means of photomultiplier–scintillator detectors that
enclose the object of study. The line drawn through space between
two detected γ-quanta gives the line of points at which the
annihilation event occurred; thus, effective collimation is achieved.
The creation of positron emitters that are incorporated in biologically
active molecules allows PET to probe the functional biochemistry of
the living organism. The data from the detectors are analyzed,
integrated, and reconstructed to produce images of the spatial
distribution of the PET tracer in the object being scanned. The
advantage of PET over SPECT is the improved resolution due to the
natural “collimation” provided by the physics of positron annihilation.
The best way to monitor the pharmacokinetics and pharmacody-
namics of a drug is to follow the drug itself and with PET this is
possible for all drugs by incorporating suitable radionuclides (e.g., 18F,
11
C and 15O) [46]. Tai et al. have recently described a new high-
resolution PET camera to image small animals, using detectors based
on lutetium oxyorthosilicate crystals [47]. The imaging of gene
expression is of particular interest with the recent developments of
localized genetic therapies for the treatment of a variety of disorders.
Using microPET, the level of expression can be quantified in serial
studies in the same animals. This has obvious implications for the
development and tuning of expression profiles in genetic therapy
[48]. A particular advantage of PET over anatomical imaging
techniques is that it can assess disease response to molecularly
targeted drugs (e.g. Herceptin and Erbitux). Several studies have used
PET imaging for evaluation of the optimal doses and by using the
direct radio-labeling of different drugs, PET has also been used for an
analysis of the biodistribution of drugs, when using different routes of
administration. PET has been used extensively for imaging cancer,
neurological function, and cardiovascular disease [49].
PET will always require well-registered anatomical images,
acquired with another modality, to pinpoint the location of the
activity measured with the PET detector. Also precise attenuation
correction in the PET reconstruction algorithm can be obtained from
CT images, which has led to the development of co-linear PET/CT
scanners that are now considered the standard option [50]. Systems
like this as well as their PET/MRI counterparts that are currently under
development will play an important role in advancing local therapy
and drug delivery. For example, with PET/MRI co-registered data it
would be possible to measure the local functional improvements, as
measured with MRI, at locations of stem cell homing, as detected with
PET [48].
3.1.4. Single photon emission computed tomography (SPECT)
A nuclear imaging alternative to PET that is frequently used in the
clinical setting is SPECT [48]. While the sensitivity of SPECT systems is
intrinsically about two orders of magnitude less than that of PET
systems (because of the need to collimate the emitted photons), the
necessary radiopharmaceuticals and imaging systems are more readily
available. A radionuclide tracer injected into the blood, accumulates at
different locations in the body depending on the relative perfusion and
affinity for the compound containing it. The SPECT camera then
estimates the relative amount of activity at each position in the body
by detecting the emission of gamma rays from a series of angular
views. The resolution of the resulting image is dependent on the
aperture through which the radiation is detected [48].
Weber et al. have been developing pinhole SPECT systems for over
a decade [51]. Also, a cylindrical fixed camera system with multiple
pin-holes has been developed [52]. For this system, there is no need to
correct for the center of rotation of the SPECT camera head, and there
is obviously better efficiency at capturing the gamma rays emitted
from the subject [48]. Liu and colleagues showed that their high-
resolution fixed detector SPECT system was able to delineate infarct
sizes in the rat myocardium with a very high correlation to the TTC
staining of those regions [53]. These rats had undergone left coronary
ligation for 90 min, followed by 30 min of reperfusion before an
injection of 5 to 10 mCi 99mTc-sestambibi into the femoral vein was
given. 99mTc-sestambibi is a technetium imaging agent used to reveal
blood-starved cardiac tissue during a heart attack. This creates the
possibility of serial imaging of rats that have undergone revascular-
ization therapy, or the evaluation of myocardial protection with drugs
[48]. The spatial resolution of the SPECT images is clearly sufficient to
image the ischemic zone with high precision. Recently, micro-SPECT
was used with 99mTc-glucarate for sizing myocardial infarcts and
measuring differences in washout kinetics depending on the severity
of the infarct [54].
The future applications of micro-SPECT could include serial
imaging of infarcts to measure the evolution of the injury zone during
different treatment regimes, and gated blood pool studies to acquire
simultaneous estimates of ventricular function. Further development
in gated studies will also decrease the effect of cardiac motion on
infarct assessment [48].
3.1.5. Ultrasound (US)
The basic principle of ultrasound (US) imaging is the detection of
features within the body as reflecting surfaces for the sound waves
transmitted from an oscillating transducer pressed up against the
body surface. US imaging is a trade-off between depth of penetration
and spatial resolution, which are both related to the frequency used to
evaluate tissue. When high-frequency sound waves are used, the
wavelengths are shorter yielding a more precise estimate of the
distances between reflecting surfaces. US, like CT, is primarily a tool
for anatomical and physiological imaging or for real-time interven-
tion. Its role in molecular imaging includes evaluation of disease
progress and image-guided drug delivery, both of which may be
performed in the early developmental phases.
 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58
High-frequency ultrasound imaging also holds promise for asses-
sing microvascular blood flow in vivo. With frequencies in the range
of 20 to 60 MHz, the imaging resolution is improved sufficiently to
reconstruct dynamic signals from cylindrical volumes in the order of
43 µm diameter and 66 µm depth, over a field of view of 10 mm
(width) by 5 mm (depth) [48]. This increased resolution is obtained
by the design of high-frequency transducers and these new systems
have the ability to follow branching patterns of closely spaced micro-
vessels in the heart and tumors from 30 µm to 100 µm in diameter
[48,55].
Targeted local drug delivery has attracted much attention, since it
represents a strategy to increase the drug concentration at the desired
location and to decrease the unwanted systemic toxicity effects.
Focused US presents a powerful non-invasive strategy to trigger local
drug delivery, acting via cavitation, radiation forces, and/or heat.
Ultrasound can be focused deep inside the body into a small region
with dimensions on the order of 1 mm. This approach can be used to
induce the local drug release from a carrier vehicle and the local
increase of cell membrane permeability either by a mechanical action
or by a temperature increase. Combined US and MRI methods play an
important role here. MRI allows precise targeting of (focused)
ultrasound through real-time temperature monitoring, and gives
access to a variety of imaging biomarkers that may be used to assess
drug action [46]. Future applications of US could include dynamic
imaging of vessel wall morphology, longitudinal studies of athero-
sclerotic plaque formation, and the real-time guidance of microsur-
gical interventions in small animals [48].
3.1.6. Optical imaging (OI)
The desire for a diagnostic optical imaging modality has motivated
the development of 3D image reconstruction procedures for optical
imaging [56]. This requires solving an inverse problem based on the
assumption that, given a set of measurements of transmitted light
between pairs of points on the surface of an object, there exists a
unique three-dimensional distribution of internal scatterers and
absorbers which would yield that set. Thus imaging becomes a task
of solving an inverse problem using an appropriate model of photon
transport [57]. There are a number of distinct approaches for probing
tissue by using optical wavelengths. Phased-array [58,59] and diffuse
optical tomographic techniques [60] have opened parts of the human
body to imaging by means of near-infrared light, allowing imaging to
be done centimeters from the surface. Other optical imaging
approaches rely on fluorescence, absorption, reflectance, or biolumi-
nescence as the source of contrast, while imaging systems can be
based on surface-weighted imaging (reflectance diffuse tomography)
[61,62], confocal imaging [63–65], multiphoton imaging [66,67], or
microscopic imaging with intravital microscopy [68,69].
At the microscopic level, 10 µm2 resolution is achievable with
optical coherence tomography [70,71], a technique analogous to US.
Subsurface imaging of fluorescence [62] can be brought to the sub-
cellular level in mouse models with intravital microscopy [68,69].
Bioluminescence imaging can be used to follow transgene expres-
sion in the whole animal, albeit at a considerably lower resolution
[55,72,73]. Overall, the size of the mouse is advantageous for OI,
especially for the detection of near-infrared photons for which the
mean scattering and attenuation lengths are on the order of 1 mm and
1 cm, respectively. The more invasive approaches of optical coherence
tomography and intravital microscopy, with their improved resolu-
tions can also be exploited to help evaluate disease in mouse model
systems [55]. Because of the availability of many optical tracers and
high detection sensitivity, optical imaging techniques are very
powerful and popular.
3.1.7. Imaging mass spectroscopy (IMS)
Imaging mass spectroscopy (IMS) has emerged as a powerful tool
for biomarker discovery on excised tissue specimen [74]. A main
47
strength of this technique is its ability to probe the proteome directly
from a tissue section with preservation of the spatial relationships of
the sample and minimal sample preparation. This allows for direct
correlation of protein expression with histology and makes IMS a
seemingly ideal tool for biomedical diagnostics and molecular his-
tology [75]. IMS allows for high throughput analysis of tissue samples
and is fully compatible with biostatistical analysis without prior
knowledge of protein expression. Several studies have demonstrated
that direct tissue mass spectral analysis provides insight into clinical
questions that is not readily available by other means. Examples
include the determination of lymph node status from investigation of
primary breast tumors, prediction of response of breast tumors to
chemotherapy, classification and prediction of progression of lung
lesions, and exploration of ‘molecular’ margins in invasive disease
[74,75].
3.2. Imaging agents
Molecular imaging techniques in general need a contrast agent to
report on the biological process of interest. These imaging agents are
commonly classified in two main categories, i.e. endogenous and ex-
ogenous agents. Endogenous agents are either naturally present in
the tissue of interest, or they are produced in situ following genetic
manipulation. Exogenous contrast agents are injected, in most cases
intravenously, and are not intrinsic to biological tissues.
Endogenous agents. The most commonly employed reporter sys-
tems are the fluorescent proteins, such as green fluorescent protein
(GFP) (and its mutants) [19,76–80] and the luciferin/luciferase
bioluminescent system [81–84], both of which are introduced into
cells of interest via molecular biological techniques. These systems
can either be constitutively expressed or their expression can be made
to depend on the presence or absence of other factors. GFP proteins
require no further substances or substrates to work. Although
fluorescent proteins offer excellent and very diverse opportunities
for extracting molecular and cellular level information in small
animals, they presently have no role in clinical applications [85,86].
On the other hand in bioluminescence imaging, a substrate (typically
luciferin) is administered to an animal that has been designed to
produce the luciferase enzyme such that when the substrate and
enzyme meet, the luciferin is oxidized. This leads to emission of light,
thus enabling detection of sites of luciferase expression [19]. Bio-
luminescence can be used to overcome some of the issues encoun-
tered with endogenous fluorophores and has promising applications
in in vivo tracking of injected cells. However, current drawbacks
include positional uncertainty of light emitting cells due to non-
homogenous scattering, light penetration issues and an apparent need
for a stable expression of optical reporter [19]. MRI is well-known for
its superb intrinsic soft tissue contrast. However, it also makes use of
a number of endogenous contrast agents that are subject to phys-
iological or pathophysiological modulations. The most used is the
contrast change brought about by changes in the oxygenation level of
hemoglobin in red blood cells, which forms the basis of functional
MRI.
Exogenous imaging agents. Exogenous agents are the more familiar
contrast agents ranging from simple dyes used for colorimetric
contrast to sensitive fluorescent probes and beyond. The common
exogenous agents used in research today are the nuclear agents, (PET/
SPECT tracers), organic fluorophores used in fluorescence imaging,
microbubbles in US imaging as well as gadolinium chelates and
superparamagnetic iron oxide used in MRI. There are a number of
limitations associated with the use of conventional contrast agents
such as organic dyes and gadolinium chelates. However, innovations
through nanotechnology have helped overcome these issues and have
opened doors to new applications.
The conventional optical imaging agents such as organic dyes have
many disadvantages. For example: (a) they are prone to rapid
48 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58
photobleaching and are unsuitable for extended periods of bioima-
ging observations [87]; (b) organic fluorophores are not well suited
for simultaneous multicolor imaging applications; (c) emission/
excitation wavelengths are often altered by changes in local chemical
environment (e.g., pH, interacting ions, etc.); and (d) emission from
dyes can overlap with auto-fluorescence from tissues [19]. The devel-
opment of nanoparticle targeted imaging agents made it possible to
overcome these disadvantages. For example, targeted fluorescent
probes have made it possible to selectively view specific biological
events in vivo with improved detection limits, imaging modalities
and engineered biomarker functionality. Hence, the development of
multifunctional nanoparticles is a step further in the field of imaging
agents.
Multimodality, i.e. the ability to be detected with several different
imaging techniques as well as their biocompatibility, stability and
non-toxicity make nanoparticles ideal tools for molecular imaging.
Thus, these imaging agents have become more and more important
for modern biomedical research.
3.2.1. Nanoparticle-based imaging agents
Like colloid particles used for drug delivery, imageable nanoparticles
can be conjugated with ligands to target them to a molecular marker
of interest, allowing indirect marker detection by different imaging
techniques. Furthermore, pH- and temperature-sensitive nanoparticles
can be used to visualize regional differences in these parameters with
modern imaging techniques [45]. Self-assembling nanoparticles are
generally composed of copolymers with both hydrophobic and hydro-
philic segments. The core of the self-assembled nanoparticles provides
an effective loading compartment for many hydrophobic and hydro-
philic drugs, fluorescent probes, and contrast agents, while the shell
improves the suspension stability of nanoparticles in aqueous solution
[88–90].
Self-assembling nanoparticles and colloidal particles provide a
promising platform for effective drug delivery, gene therapy, and
diagnostic imaging [91,92]. They can be used as blood pool agents
with long circulation times for magnetic resonance angiography
(MRA), as well as for the detection of pathological tissues with
enhanced vascular permeability, which occurs in inflammation,
myocardial infarction, atherosclerosis, breakdown of the blood–
brain barrier and tumors [45]. These nanoparticles are generally
composed of copolymers with both hydrophobic and hydrophilic
segments. The important features of a nanoparticle-based optical
imaging agent are (a) in vitro and in vivo stability, (b) resistance to
metabolic disintegration and non-toxicity, (c) high quantum yield and
high absorbance and (d) sufficient dispersibility in the biological
environment. GdIII is known to be toxic in its free, uncomplexed form;
it binds to serum proteins and, consequently, most of it concentrates
in the bone where it becomes tightly and irreversibly associated.
Hence, for Gd-based MRI contrast agents, it is important that the
imaging agent is permanently chelated to the largest extent possible.
Recently Hak et al. have synthesized the Gd(III)DOTA-DSPE lipid and
incorporated it in a liposomal formulation [93]. The resulting
liposomes were extensively characterized in vitro in terms of contrast
agent stability, efficiency and structural properties. The liposomes
were shown to have a high longitudinal relaxivity, which is crucial for
the detection of low concentration molecular markers in molecular
imaging studies [93]. Moreover, to limit the toxicity due to non-
specific uptake of the contrast agent, it is desirable that the imaging
agent be sequestered preferentially at the targeted site [19].
Nanoparticle-based optical contrast agents such as quantum dots
(QDs), gold nanoparticles, organically modified dye-doped silica, up-
converting phosphors and lanthanide-based contrast agents are
recent additions to the list of exogenous optical contrast agents
which combine a number of the desired features listed above [19].
The stabilized imaging agents in aqueous solutions are promising
candidates for biomedical applications. One possible way to make
them soluble in aqueous media is by encapsulating them with
polymeric materials that can form nanoparticles. Ideally, these
polymeric materials should be biocompatible and possess reactive
functional groups for the further attachment of other biomolecules.
These particles are composed of lipids and/or other amphiphilic
molecules, which have both hydrophobic and hydrophilic parts that
spontaneously assemble into aggregates in an aqueous environment.
A variety of polymers are used to form nanoparticles carrying imaging
agents. Out of these, the most commonly used in chitosan-based
systems, i.e. microbubbles, micelles and liposomal nanoparticles, are
discussed in more detail below.
3.2.1.1. Microbubbles. Perfluorocarbon (PFC) nanoparticles can serve
as a platform technology for molecular imaging and targeted drug
delivery applications [94,95]. Most microbubbles consist of air- or
perfluorocarbon-filled microspheres stabilized by an albumin or lipid
shell with a size in the range of 1–10 µm [96]. By combining targeted
molecular imaging and localized drug delivery, PFC nanoparticles
provide diagnosis and therapy with a single agent [94,95]. Morawski
et al. have applied particles that are 280 nm in size and can both be
used for 1H-MRI (via the Gd-lipid) and 19F-MRI (via the F-19 agent in
the emulsion) [95]. For extravascular targeting, particles with much
smaller diameters are needed. Hence, due to their size, microbubbles
are mainly suited for intravascular targeting. Microbubbles can
drastically enhance US bioeffects, notably in the case of cavitation.
Microbubbles, either non-specific or targeted, may also be used as a
drug delivery system [46].
There are several different approaches for the use of microbubbles
as drug carriers. Drugs may be attached to the membrane surrounding
the microbubble by a charge-dependent, noncovalent binding [97] or
by ligands embedded in the membrane [98]. Drugs may also be
incorporated within the membrane itself [99], or loaded into the
interior of the microbubble, either in a hydrophobic or hydrophilic
phase [100]. Thus, the use of microbubbles as drug carriers or imaging
agents has two components. First, the transported substances may be
released from the microbubbles by destruction in a temporally and
spatially controlled manner, increasing the local concentration.
Second, the destruction of microbubbles by US may cause cavitation
effects in the surrounding tissue, with increasing extravasation of the
released substances [46].
3.2.1.2. Micelles. Micelles are an interesting system for carrying poorly
soluble pharmaceutical agents [45,101–103]. Micelles are built of
amphiphilic molecules that contain both a hydrophobic and hydro-
philic part. Because of this dual character and the energetically
unfavorable contact between water and the hydrophobic part, the
center of the micelles can be loaded with hydrophobic molecules. The
diameters of micelles range between 5 and 30 nm, large enough to
escape renal excretion and small enough to extravasate at sites of
disease, including cancer tissue [46]. Pegylation of the micelles lets
them circulate in the blood for a relatively long time without being
recognized by certain plasma proteins and/or phagocytic cells [45]. In
this way, they can be used as long-circulating blood pool agents,
which nonspecifically target to areas with a leaky vasculature. Ligands
can also be coupled to the surface of the micelles for targeting to
specific sites and these properties as well as flexibility make micelles
excellent candidates to function as imaging agent carriers [45].
3.2.1.3. Liposomes. Like micelles, liposomes are also formed of
amphiphilic lipids, but in this case the hydrophobic part of the lipids
occupies more space than that of the micelle-forming lipids, leading to
the creation of a lipid bilayer. Liposomes can be defined as spherical,
self-closed structures formed by one or several concentric lipid bi-
layers with an aqueous phase enclosed by the lipid bilayers.
Liposomes have been used extensively as a model to study the
properties of biological membranes [45]. In contrast to micelles,
 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58 49

Fig. 3. Collection of nanoparticles for molecular imaging using multiple modalities: (i) cross-linked iron oxide (CLIO) (MRI), (ii) micellular iron oxide (MCIO) (MRI), (iii)
superparamagnetic iron oxide (SPIO) (MRI), (iv) multimodal micelle (MRI, PET, SPECT, OI), (v) multimodal liposome (MRI, PET, SPECT, OI), (vi) multimodal lipid coated emulsion
(MRI, PET, SPECT), (vii) quantum dot micelle (OI), and (viii) microbubble (US). Nanoparticles with iron oxide are traditionally employed as negative MR contrast agents; while those
with Gd have positive contrast properties. Nanoparticles ii, iv, v, vi and vii additionally possess fluorescent properties by incorporation of a fluorescent dye or quantum dot. Oil-filled
microspheres can be designed such that their interior is loaded with drugs or gas. A stabilizing layer, in this case a monolayer of lipid, surrounds the perfluorocarbon bubble. The
bottom of the figure shows the legend with the constituents of the nanoparticles. Particles and components are not drawn to scale.

liposomes are recognized and cleared by the cells of the reticuloen-
dothelial system due to their larger size (50–400 nm). However, it is
possible to increase the circulation half-lives by incorporating
polyethylene glycol (PEG)-lipids in the bilayer, which provides
them with ‘stealth’-properties [104–106]. Because of their striking
biological properties and possibility to manipulate them according to
the need, liposomes were suggested for use as carriers for drugs and
imaging agents shortly after their introduction in the 1970s [107].
Liposomes are versatile vehicles, as they can carry water-soluble
pharmaceutical agents in the aqueous interior and amphiphilic or
hydrophobic agents in the lipid bilayer. Focused US can release the
content of liposomes by two ways, first by mechanical force and
second by thermal energy [46]. Surface modifications, like coupling of
targeting ligands, make delivery of liposomes to diseased tissue and
into cells more specific. These properties also make liposomes
excellent candidates to carry or deliver contrast agents for molecular
imaging techniques like MRI or SPECT [45].
3.2.2. MRI contrast agents
Within the emerging field of cellular and molecular MRI, contrast
agents are essential elements and, at present, most of the chitosan-
based imaging agents are used as MRI contrast agents. Hence, here we
focus on a more detailed description of the properties and char-
acteristics of MRI contrast agents. Materials with paramagnetic prop-
erties act indirectly to provide contrast enhancement by accelerating
T1 and T2 relaxation processes via alteration of the local magnetic
environment. Many of the metals belonging to the lanthanide and
transition metal series have paramagnetic properties, which make
them suitable as MRI contrast agent (Fig. 3).
Roughly, MR contrast agents can be divided into four groups [45].
The first group consists of non-specific contrast agents and includes
both the low molecular weight contrast agents, e.g. Gd-DTPA, and the
high molecular weight blood pool agents [108], such as high gen-
eration Gd-loaded dendrimers [109]. These agents can be used for MR
angiography and to measure the perfusion and permeability proper-
ties of tissues. The second group of molecules is targeted contrast
agents, which are actively directed to a specific molecular target with
an appropriate ligand. The third group consists of the so-called smart
contrast agents, also referred to as activated or responsive agents,
which most often require a specific enzyme to become more or less
active, or exhibit a change in relaxivity when subjected to altered
physicochemical conditions (e.g. pH). The fourth group is the cell

Fig. 4. MR images of phantoms containing control hMSCs (5000 cells/mL) without nanoparticles (A) and with CMCS-SPION-labeled hMSCs at a cell density of 500 (B), 1000 (C), 2500
(D), and 5000 cells/mL (E). The cells were suspended in 1% low-melting agarose.
Reproduced with permission from Ref. [136].
50 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58

Fig. 5. MR images of the central region of mouse liver before (A) and after (B–C) injection of SPION-loaded WSC–LA nanoparticles. Images were obtained at (B) 30 min and (C) 1 h
after injection of the nanoparticles. L = left.
Reproduced with permission from Ref. [39].

labeling contrast agents, such as TAT–peptide conjugated iron oxide
particles that are used to label cells ex vivo followed by implantation
and MRI monitoring in vivo [110].
Out of the large variety of metals and compounds tested, due to its
large magnetic moment the rare-earth metal ion GdIII forms the basis
of some of the most powerful MRI contrast agents. Gd-based agents
are so-called T1 contrast agents, which permit signal enhancement on
T1-weighted images. Small Gd-based complexes such as Gd-DTPA
(diethyl triaminepenta acetic acid, DTPA is a chelating group which
binds tightly with the GdIII ion) and Gd-DOTA (tetra azacyclodode-
canetetra acetic acid, DOTA being a chelating group again binding the
GdIII ion) are widely used as contrast agents for clinical MRI. However,
Gd-DTPA has some safety issues, mainly because Gd itself is toxic in
ionic form with a half-life of several weeks [93]. Although Gd-DTPA
is administered in a chelated form, complex stability is influenced
by temperature, pH, concentration of surrounding ions and ligands.
Moreover, ions of endogenous metals, such as ZnII and CuII, can
displace the Gd ion from the complex [93,111]. In patients with
normal kidney function, these problems are prevented by rapid renal
clearance of the Gd-chelates. However, in patients with renal failure,
in whom the Gd complexes are only slowly cleared, this may lead to a
severe disorder called nephrogenic systemic fibrosis [112].
Among the transition metals commonly used, particle-based
systems comprise superparamagnetic iron oxide (SPIO, 50–500 nm)
or ultra-small paramagnetic iron oxide (USPIO, < 50 nm) (Fig. 3).
They are so-called T2 or T*2 contrast agents, which permit negative
contrast enhancement on T2-weighted scans and, thus, yield less
signal on images when present. Magnetic iron oxide nanoparticles
have been extensively utilized for many applications, especially
superparamagnetic iron oxide nanocrystals in biomedical fields such
as MRI, magnetic drug delivery, cancer diagnosis and treatment, and
biomolecular separation [39,113–115].
Fig. 3 shows several additional imaging agents such as fluorescent
dye-doped silica nanoparticles, quantum dots and gold nanoparticles.
These particles have overcome many of the limitations of conventional
contrast agents such as poor photostability in case of optical imaging,
low quantum yield, insufficient in vitro and in vivo stability, etc.
Current developments in optical imaging focus on new agents that
contain molecular structures for absorbance and emission in the near-
infrared region. This is expected to allow for real-time as well as deep

Fig. 6. Scanning electron micrographs (SEMs) of CMS-Gd-DTPA prepared at various Gd-DTPA:chitosan ratios: (A) placebo chitosan microspheres (PCMSs), (B) 1.5:1 (gadolinium
content, 9.1%), (C) 1.7:1 (gadolinium content, 11.6%), and (D) 15:1 (gadolinium content, 13.3%). Bar size, 10 µm.
Reproduced with permission from Ref. [139].
 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58 51

Fig. 7. Chitosan nanobeads encapsulating QDs and Gd-DTPA. (A) Transmission electron microscopy (TEM) image and (B) higher magnification. Inset (B) shows multiple QDs
encapsulated within a single chitosan nanobead. (C) Excitation and emission spectra of CdSe/ZnS-3MPA QDs and chitosan nanobeads encapsulating CdSe/ZnS-3MPA and Gd-DTPA.
Reproduced with permission from Ref. [141].

tissue imaging via optical routes. Other efforts to facilitate deep tissue
imaging with pre-existing technologies have led to the development of
multimodal nanoparticles which are both optical and MRI active [19].
4. Current status of the use of chitosan composites in
bioimaging applications
Chitosan is an exemplary polymer in biological applications owing
to its biocompatible properties. It is a natural polycationic polymer
and composed of D-glucosamine and N-acetyl-D-glucosamine linked
by b-(1.4)-glycosidic bonds, and thus has one free amino group and
two free hydroxyl groups in the repeating hexosaminide residue.
These groups can be modified with hydrophobic segments to improve
the self-assembling capabilities by increasing intermolecular hydro-
phobic interactions between segments [116–118]. Conjugated to
additional materials, chitosan composites result in a new class of
biomaterials that possess mechanical, physicochemical and functional
properties, which cannot be achieved either by native chitosan or the
incorporated material alone. The incorporation of hard and brittle
calcium phosphate or hydroxyapatite into chitosan yields a bior-
esorbable composite with favorable mechanical properties for bone
and cartilage tissue engineering [119–121]. Moreover, incorporation
of imaging agents such as Fe3O4 for MRI into the self-assembled
nanoparticles could enhance hepatocyte-targeted imaging [39] and
the particle could serve as MR molecular imaging agent.
Various inorganic materials including metals can be incorporated
in the chitosan composite preparations and their combined char-
acteristics have proven beneficial for several biomedical applications.
Chitosan polyion complex composites can be prepared by interactions
of chitosan with natural and synthetic polyanion molecules [35].
Preparing fluorescent chitosan quantum dot composites enables the
combination of targeted drug and gene delivery with optical imaging
[122,123]. Polyacrylic acid (Carbopol), an anionic synthetic polymer
having mucoadhesive properties, is extensively used with chitosan to
form polymer composites, which have longer circulation times in vivo,
resulting in higher bioavailability of incorporated therapeutic agents
[35,124–126]. The latter composites can also be made to contain
contrast agents for imaging purposes. Guiot et al. investigated the
ability of oxygen-loaded chitosan bubbles to promote the exchange of
oxygen in the presence of US. Oxygen delivery is enhanced by
sonication and both frequency and time duration of US affected the
exchange kinetics [127].

Fig. 8. A schematic drawing of the RGD-HGC nanoparticles (left) which are directly administered in solid tumor (right). The RGD peptides released from the HGC nanogel bind to
tumor endothelial cells, and prevent the formation of microvessels in tumors, thereby suppressing tumor growth.
Reproduced with permission from Ref. [145].
52 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58

As described in Section 3.2.2, the SPIO MRI contrast agents allow
researchers and clinicians to enhance the tissue contrast of an area of
interest by increasing the relaxation rates of water protons. For these
applications, the SPIO based contrast agents should be suspended in
aqueous media and have a good stability as well as a narrow size
distribution [128]. To achieve these properties, several polymers such
as dextran, polyethylene glycol (PEG), alginate, and pullulan have
been used for surface modification [129–132]. Modified nanoparticles
with increased blood half-life are beneficial for in vivo applications.
During the last decade the potential of chitosan as a delivery vehicle
for biocompatible MRI contrast agent, in particular SPION nanopar-
ticles, has been explored [39,133–135].
Shi et al. prepared biocompatible (carboxymethyl) chitosan
(CMCS)-coated SPIO that show promise for the use in visualizing
hMSCs with MRI [136]. The carboxymethylation of chitosan increases
its solubility in water, and hence the CMCS-coated SPIO will disperse
better in aqueous media. Fig. 4 shows MR images of various
concentrations of CMCS-SPION-labeled hMSCs suspended in a low-
melting agarose. Compared with control preparations of the agarose
medium with unlabeled cells (Fig. 4A), the images of the labeled cells
displayed tiny distinct punctuate signal extinctions that clearly
contrasted against the high signal intensity of the aqueous agarose
medium. The increase in the density of the black dots observed in the
MR images of the CMCS-SPION-labeled cells was clearly dependent on

Fig. 9. In vivo non-invasive NIR fluorescence imaging and quantification of local concentrations of RGD peptide. (A) In vivo fluorescence imaging of athymic nude mice bearing s.c.
tumors after i.v. or i.t. injection of RGD peptide or i.t. injection of RGD-HGC nanoparticles. (B) Histological evaluations of RGD-HGC nanoparticle-treated tumors stained with DAPI
and CD31 antibody, and NIR fluorescence images of RGD peptide in tumors. (C) The fluorescence intensity for the region-of-interest (tumor) was recorded as total photon counts per
tumor. (D) Tumor contrast (tumor-to-normal tissue ratio) measured as a function of time post-injection. (●) RGD-i.v., (▲) RGD-i.t., and (■) RGD-HGC nanoparticles-i.t.
Reproduced with permission from Ref. [145].
 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58 53

the amount of cells used, while the image intensity of control their toxicity [19]. The inherent cytotoxicity of the individual ions
preparations had a uniform appearance not influenced by the number (including Cd2+, Se2+ and Te2+) which the QDs are composed of
of cells present. According to the authors in parts c–e of Fig. 4, some prevented their widespread application in bioimaging [143,144].
spots are observed to be darker and larger than others, owing to both Nanoparticle technology has been utilized to overcome the hydro-
partial volume effects inherent in the imaging parameters and phobic property of these dyes, but problems still remain in their
different amounts of the iron label incorporated into each cell. fabrication.
Other possibilities are that there is some overlap between labeled cells Tan et al. successfully prepared biocompatible chitosan nanopar-
and some labeled cells that did not provide sufficient contrast for ticles encapsulating both CdSe–ZnS QDs and Gd-DTPA [141]. These
detection. The authors concluded that the results presented suggest multifunctional nanobeads can be used as fluorescent biomarkers and
that CMCS-SPIONs as contrast agent have a very high labeling also as contrast agents for MRI. The detailed characterization done by
efficiency, allowing the detection of very low numbers of labeled the authors demonstrated that their chitosan-based nanobeads are
cells [136]. Furthermore, CMCS has been shown to enhance interac- spherical, about 50 nm in size and possess a much improved quantum
tions with the cell membrane [137], which may facilitate the uptake of yield as well as favorable relaxivity of the Gd-DTPA (Fig. 7) [141].
the CMCS-coated SPIOs by stem cells [136]. Fig. 7C shows the optical properties of the QDs and chitosan
Lee et al. have developed novel self-assembling nanoparticles nanobeads. The 3MPA-modified water-soluble CdSe/ZnS-3MPA QDs
composed of amphipathic water-soluble chitosan–linoleic acid exhibit an intense, narrow emission spectrum with a peak at 565 nm.
(WSC–LA) conjugates for encapsulation of SPIOs as a contrast agent Similarly, nanobeads encapsulating both QDs and Gd-DTPA possess an
to target hepatocytes [39]. The WSC–LA conjugates self-assembled equally intense and narrow emission spectrum, with its peak position
into core–shell structures in aqueous solution. The SPIO clusters at 567 nm [141]. These multifunctional chitosan nanobeads are
formed inside the hydrophobic core had a very high MRI relaxivity. promising for future biomedical applications.
Fig. 5 shows T2-weighted MR images of the central region of the liver Kim et al. prepared self-assembled glycol chitosan nanoparticles
before and after injection of SPIO-loaded WSC–LA nanoparticles and explored whether these constructs might function as a prolonged
(SCLNs) via the tail vein [39]. The signal intensity of T2-weighted
images clearly dropped after injection of SCLNs. The T2-weighted MR
images of the liver showed a signal drop of 69.2, 78.2, and 62.9% at
30 min, 1 h, and 2 h after injection of the nanoparticles, respectively
[39]. These SPIO-loaded WSC–LA nanoparticles can be used as a
contrast agent to aid in the diagnosis of hepatic diseases [39].
As described before, Gd-DTPA is a widely used MRI contrast agent.
Since its incorporation in nanoparticles, its potential for in vivo
molecular imaging applications has increased tremendously. In a
similar fashion, chitosan-based Gd-nanoparticles have been prepared
by incorporating Gd-DTPA using the emulsion-droplet coalescence
technique [1]. Their release properties and their ability for long-term
retention of Gd-DTPA in the tumor indicated that these Gd-
nanoparticles might be useful as an i.t. injectable device for gadolinium
neutron-capture therapy (Gd-NCT) [138]. The extent of Gd loading
was different for different types of chitosans used in the preparation.
The highest Gd load was achieved with 100% deacetylated chitosan in
15% Gd-DTPA aqueous solution, and the particle size was 452 nm,
whereas chitosan with lower deacetylation level produced much
larger particles with decreased Gd-DTPA content. Fig. 6 shows
scanning electron micrographs (SEMs) of placebo chitosan micro-
spheres (PCMSs) and chitosan microspheres (CMS)-Gd-DPTA pre-
pared at various applied weight ratios of Gd-DPTA/chitosan. Similarly,
CMS-Gd-DTPA prepared at Gd-DTPA:chitosan ratios of 1.5:1 and 1.7:1
had fairly regular, spherical shapes (Fig. 6B and C, respectively);
however, all CMS-Gd-DTPA prepared at a ratio of 15:1 Gd-DTPA:
chitosan were nonspherical, rough-faced, collapsed and/or aggregated
(Fig. 6D) [139]. Process optimization facilitated production of
gadolinium-loaded chitosan nanoparticles with an extremely high
Gd-DTPA content (45.3%) and a suitable size for i.v. injection (452 nm)
[1]. Gadolinium-loaded chitosan nanoparticles displayed prolonged
retention in tumor tissue after in vivo intratumoral injection [139,140].
Kumar et al. also described the chemistry and preparations of
Holmium-166 and Samarium-153 chitosan complexes, which are
mainly suited for radiopharmaceutical applications [1].
Fluorescence techniques are widely used tools in biology. The
drive to measure several biological processes simultaneously imposes
new demands on the fluorescent probes used in these experiments. Fig. 10. In vivo imaging of atherosclerotic lesion targeting by AP-tagged HGC-Cy5.5 nano-
Quantum dots (QDs) have emerged as promising fluorescent probes particles. (A) Representative NIR fluorescence images of the exposed aorta an Ldlr−/−
for bioimaging, and particularly for multiplexing (Fig. 3). Compared to mouse (1) and a normal mouse (2). AP-tagged HGC-Cy5.5 nanoparticles (5 mg/kg) were
conventional organic fluorophores, QDs have strong, narrow and injected via the lateral tail vein. After 6 h, the exposed aortas were imaged. (B) Oil Red O
lipid staining of aortas of an Ldlr−/− mouse (1) and of a normal mouse (2). (C) NIR
symmetric fluorescence emission as well as high photochemical fluorescence microscopy of aortas of an Ldlr−/− mouse (1) and of a normal mouse (2)
stability [141]. However, negative photophysical aspects of these injected with AP-tagged HGC-Cy5.5 nanoparticles.
particles have been fluorescence intermittency (blinking) [142] and Reproduced with permission from Ref. [146].
54 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58

and sustained drug delivery system for RGD peptide, used as an
antiangiogenic model drug in cancer therapy [145]. Glycol chitosan
hydrophobically modified with 5β-cholanic acid (HGC) formed
nanoparticles with a diameter of 230 nm. RGD peptide was easily
encapsulated into the HGC nanoparticles (yielding RGD-HGC nano-
particles) with a high loading efficiency (≥85%). Fig. 8 shows a
schematic diagram of the molecular structure of the nanosized HGC
drug carrier, which was generated by the chemical conjugation of an
average of 120 hydrophobic cholanic acid residues to a hydrophilic
glycol chitosan polymer chain [145]. The resulting amphiphilic HGC
conjugates formed nanoparticles in aqueous solution and the average
diameter of the nanoparticles was ca. 200 nm with a narrow size
distribution. Fig. 9 shows time-dependent in vivo biodistribution
profiles of RGD peptide in tumor-bearing athymic nude mice which
were obtained using whole-body NIR fluorescence intensity moni-
toring as a function of time after agent administration [145]. The
authors showed with an in vivo study that the antiangiogenic peptide
drug formulation of RGD-HGC markedly inhibited bFGF-induced
angiogenesis and decreased hemoglobin content in Matrigel plugs.
Additionally, the intratumoral administration of RGD-HGC signifi-
cantly decreased tumor growth and microvessel density compared to
native RGD peptide injected either intravenously or intratumorally,
because the RGD-HGC formulation strongly enhanced the antiangio-
genic and antitumoral efficacy of RGD peptide by affording prolonged
and sustained RGD peptide delivery locally in the solid tumors [145].
A new imaging probe for atherosclerotic lesion imaging by
chemically conjugating an atherosclerotic plaque-homing peptide
(termed the AP peptide) to hydrophobically modified glycol chitosan
(HGC) nanoparticles was developed by Park et al. [146]. HGC
nanoparticles were labeled with the near-infrared (NIR) fluorophore
Cy5.5, yielding nanoparticles 314 nm in diameter. In vivo NIR
fluorescence imaging demonstrated that AP-tagged HGC-Cy5.5
nanoparticles bound better to atherosclerotic lesions in a low-density
lipoprotein receptor-deficient (Ldlr−/−) atherosclerotic mouse than

Fig. 11. In vivo NIR fluorescence images of tumor accumulation and tissue distribution for DTX-HGC nanoparticles in A549 human lung tumor-bearing athymic nude mice. (A) In vivo
non-invasive NIR fluorescence images of real-time tumor targeting characteristics of CPT-HGC nanoparticles. NIR images were obtained at different times after i.v. injection of DTX-
HGC nanoparticles. The tumor location is specified with an arrow. (B) TBR [tissue to background (muscle) ratio] value versus time. The TBR value was determined as follows: TBR =
(tumor signal − background signal)/(background signal). (C) Representative fluorescence images of tumor tissue and normal muscle tissue. (D) Representative ex vivo NIR
fluorescence images of dissected organs and tumor of mice bearing A549 human lung tumor sacrificed at 72 h after i.v. injection of DTX-HGC nanoparticles.
Reproduced with permission from Ref. [147].
 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58
to such lesions in a normal mouse (Fig. 10). These results suggest that
the newly designed AP-tagged HGC-Cy5.5 nanoparticles may be
useful for atherosclerotic lesion imaging, and may also be employed to
elucidate pathophysiological changes, at the molecular level, on
atherosclerotic endothelium [146].
Hwang et al. loaded HGC nanoparticles with the anticancer drug
docetaxel (DTX) using a dialysis method, and the resulting docetaxel-
loaded HGC (DTX-HGC) nanoparticles had a mean diameter of
350 nm in aqueous solution [147]. The authors showed that these
nanoparticles were well dispersed and stable for 2 weeks in vitro
under physiological conditions (pH 7.4 and 37 °C) with a sustained
drug release profile. The DTX-HGC nanoparticles were reasonably
stable in the presence of excess bovine serum albumin, which
suggested that these nanoparticles might also be stable in the blood
stream. The authors also evaluated the time-dependent excretion
profile, in vivo biodistribution, the circulation time, and tumor
targeting ability of DTX-HGC nanoparticles by using a non-invasive
live animal imaging technology (Fig. 11). Under optimal conditions
for cancer therapy, the DTX-HGC nanoparticles showed higher
antitumor efficacy such as reduced tumor volume and increased
survival rate in A549 lung cancer cells-bearing mice and strongly
reduced anticancer drug toxicity compared to that of free DTX [147].
Ex vivo labeling of cells with nanoparticles for subsequent in vivo
detection is of enormous interest to various biomedical applications.
To visualize these cells, Ge et al. prepared novel fluorescent/magnetic
nanoparticles coated with modified chitosan possessing a magnetic
iron oxide core and a covalently attached fluorescent dye [4]. In order
to localize the magnetic nanoparticles in the cells, the authors
performed fluorescence microscopy and electron microscopy. Fig. 12
(left panel) shows the magnetic particles located inside the cells as
well as on the cell surface. Fig. 12 (right panel) shows that the
nanoparticles were internalized within the cell inside late endosomes
or lysosomes [4]. The feasibility and efficiency in labeling cancer cells
(SMMC-7721) was also evaluated, showing a high level of internal-
ization, as demonstrated by flow cytometry and magnetic resonance
imaging [4]. These new magneto-fluorescent nanoagents thus have
potential for advanced bioimaging studies.
Fig. 12. (Left) Fluorescent images of SMMC-7721 cells incubated with (A) control (blank); B and C FITS-CS@MNPs for 8 h. C is zoomed in as compared to B. (Right) TEM images of
SMMC-7721 cells when incubated with the (D) control; (E) FITS-CS@MNPs for 8 h. Black arrows point to MNPs.
Reproduced with permission from Ref. [4].
55
5. Conclusions
In this review, the basic principles and major use of different imaging
techniques, nanoparticulate imaging agents and chitosan as possible
nanocarrier system for imaging agents were discussed. Molecular
imaging is now increasingly being applied in preclinical studies.
However, to successfully exploit the opportunities for molecular
imaging in drug development, several challenges need to be addressed
[18]. The molecular imaging techniques are fulfilling an important
criterion for a bench-to-bedside translational approach and are now
becoming an integral part of the drug development process as well as in
diagnostics. Established imaging modalities are benefiting greatly from
developments in other areas of science, including nanotechnology and
biochemistry. Advances in drug development, drug delivery and
imaging agent research are interdependent, and therefore a close
partnership between the pharmaceutical and imaging communities is
crucial for the development of new molecular imaging tracers for
diagnostic and therapeutic purposes. Many components of drug and
imaging probe development are alike, and therefore joint development
strategies are justified and necessary. For example, libraries of failed
drugs could become available for mining for imaging probe develop-
ment [18]. New developments in the imaging agents will also require
new carrier systems, which are biocompatible. Chitosan and its
derivatives are likely to play an important role in this area.
Different groups have focused on different molecular weight
chitosans, with different degrees of deacetylation, resulting in
nanoparticles of varying sizes and charge ratios [148]. While much
work has been done in the last few years to achieve successful
chitosan nanoparticle formations for imaging agents, the field has yet
to progress beyond animal models and demonstrate relevant efficacy
in humans. However, the many advantages of chitosan, including the
ease of availability, low cost, the flexibility and, most importantly, the
biocompatibility of chitosan nanoparticles make them very promising
for biomedical imaging applications. These nanoparticles could also
provide an excellent template for new imaging nanoparticle formula-
tions. Ongoing developments in science, especially nanotechnology,
may lead to modifications of these nanoparticles for improved
56 P. Agrawal et al. / Advanced Drug Delivery Reviews 62 (2010) 42–58
imaging, better stability and more defined size. The studies in this
review indicate the feasibility of using chitosan nanoparticles for in
vivo biomedical imaging applications. However, more insights in the
parameters that influence the macroscopic transport of these
nanoparticles, their biodistribution in different tissues, types of cells
transfected, transgene expression kinetics, and extra- and intracellu-
lar release of the imaging agents from the nanoparticles are required.
In summary, chitosan and its derivatives provide an attractive
toolkit for advanced molecular imaging applications. Although there
is clearly much work to be done before the full potential of chitosan
can be realized, the benefits are significant enough to make the effort
worthwhile.
Acknowledgements
The authors' work in the field of MRI contrast agents and MRI-based
molecular imaging is funded in part by the European Commission FP6-
projects DiMI (project number LSHB-CT-2005-512146) and MediTrans
(project number NMP4-CT-2006-026668), as well as by the BSIK
program entitled Molecular Imaging of Ischemic Heart Disease (project
number BSIK03033). Parts of these studies are performed in the
framework of the European Cooperation in the field of Scientific and
Technical Research (COST) D38 Action Metal-Based Systems for
Molecular Imaging Applications.
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