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Nanoparticles in Photodynamic Therapy

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DOI: 10.1021/cr5004198

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Nanoparticles in Photodynamic Therapy
Sasidharan Swarnalatha Lucky,†,§ Khee Chee Soo,‡ and Yong Zhang*,†,§,∥
†
NUS Graduate School for Integrative Sciences & Engineering (NGS), National University of Singapore, Singapore, Singapore
117456
‡
Division of Medical Sciences, National Cancer Centre Singapore, Singapore, Singapore 169610
§
Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore, Singapore 117576
∥
College of Chemistry and Life Sciences, Zhejiang Normal University, Zhejiang, P. R. China 321004
CONTENTS
1. Introduction: Photodynamic Therapy for Cancer
2. Principle of Photodynamic Therapy
2.1. Photodynamic Reaction
2.2. Mechanism of Tumor Destruction
2.2.1. Direct Tumor Cell Kill
2.2.2. Vascular Damage
2.2.3. Inflammatory and Immune Response
3. Photosensitizers
3.1. 1st Generation Photosensitizers
3.2. 2nd Generation Photosensitizers
3.3. 3rd Generation Photosensitizers
4. Current Limitations of Photodynamic Therapy
4.1. Tumor Selectivity
4.2. Formulation of Photosensitizers
4.3. Tissue Penetration Depth
5. Nanoparticles in Photodynamic Therapy
5.1. Nanoparticles As Delivery Vehicles of Photo-
sensitizers
5.1.1. Biodegradable Nanoparticles
5.1.2. Nonbiodegradable Nanoparticles
5.2. Nanoparticles as Downconverting Photo-
sensitizers
5.2.1. Fullerenes
5.2.2. Titanium Dioxide Nanoparticle
5.2.3. Zinc Oxide Nanoparticle
5.3. Nanoparticles as Energy Transducers
5.3.1. X-ray Activatable Nanoparticle
5.3.2. Quantum Dots
5.3.3. Two-Photon Absorbing Nanoparticles
5.3.4. Upconversion Nanoparticles
6. Conclusion and Future Perspectives
Author Information
Corresponding Author
© XXXX American Chemical Society
Review
pubs.acs.org/CR
Notes AU
Biographies AU
Acknowledgments AV
References AV
1. INTRODUCTION: PHOTODYNAMIC THERAPY FOR
CANCER
Cancer accounted for 7.6 million deaths (around 13% of all
deaths) worldwide in 2008.1 Almost 13 million cancer cases are
newly diagnosed every year, and deaths are projected to rise,
with an estimated 13.1 million in 2030. Although regular
screening and surveillance programs as well as early
intervention are the best ways to improve the outcome and
A survival, efforts need to be divided at finding better cancer
B therapeutic options that are effective, efficient, affordable, and
B acceptable to patients. The conventional cancer treatment
B options are chemotherapy, radiotherapy, and surgery, and more
B recently small molecule-based therapies and immunotherapy
D along with a combination of these strategies are being practised.
E However, chemotherapy is often associated with systemic side-
F effects, high recurrence rate is associated with surgical resection
F of tumors, while radiation therapy is limited by the cumulative
F radiation dose. While refinement of the conventional cancer
G treatment modalities is important, research has also focused on
G developing alternate treatment modalities that are safe, potent,
H and cost-effective. Photodynamic therapy (PDT) is an
H alternative tumor-ablative and function-sparing oncologic
H intervention. Since its inception in early 1900s and its first
I modern demonstration by Dougherty et al. in 1975,2 PDT has
undergone extensive investigations and has emerged as a
I disease site specific treatment modality. Essentially, it involves
I the administration of a tumor-localizing photosensitizer (PS)
P followed by local illumination of the tumor with light of a
specific wavelength to activate the PS. The excited PS then
V transfers its energy to molecular oxygen, thus generating
V cytotoxic reactive oxygen species (ROS), such as singlet oxygen
W (1O2) that can oxidize key cellular macromolecules leading to
W tumor cell ablation.3 Hence, PDT employs 3 nontoxic
X components that by its own do not have any toxic effects on
X the biological systems, unlike chemotherapy drugs that induce
Y systemic toxicity and ionizing light of radiation therapy that
Y damages neighboring normal tissues. Clearly, PDT has its own
AA merits compared to the conventional treatment methods due to
AS
AU Received: August 3, 2014
AU
A DOI: 10.1021/cr5004198
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
Figure 1. Schematic illustration of a typical photodynamic reaction. Adapted from ref 10. 2002, Elsevier.
its minimal invasiveness, repeatability without cumulative
toxicity, excellent functional and cosmetic results, reduced
long-term morbidity, and improved quality of life of the
patients. Over the last four decades PDT has proven to be
effective in superficial bladder cancer,4 early and obstructive
lung cancer,5 Barrett’s esophagus,6 head and neck cancers,7 and
skin cancer.8 It is also being used as an adjunctive therapy
following surgical resection of tumor, to reduce residual tumor
burden.9
Despite the widespread and rapidly growing applications,
PDT has yet to gain clinical acceptance as a first-line
oncological intervention due to certain limitations including
lack of an ideal PS, challenges in formulating PS, choosing the
right light dosimetry for a complete and effective treatment,
difficulties in planning the treatment and monitoring the
treatment response; which will be discussed in detail in this
review. The application of nanoparticles in PDT has been a
major stride forward in resolving some of the challenges
associated with classic PS. Since a comprehensive review,
encompassing the theory behind the design of nanoconstruct to
its application in PDT, is beyond the scope of this review, we
have attempted to provide an overview of the present status
and prospects of such nanoparticles by highlighting its
development in each phase and giving special emphasis on
multifunctional theranostic agents, by taking specific illustra-
tions from recently published articles. The examples given in
this review do not mean that the pioneering contributions
made by a large number of researchers are neglected. Special
interest is devoted to the last section on “Upconversion
Nanoparticles” (UCNs), a multifaceted tool that due to its
recent accelerated progress shows great potential in augmenting
the scope of PDT in the treatment of solid tumors. For detailed
information on other nanoparticles, readers are requested to
refer to key publications and review articles provided in each
section.
2. PRINCIPLE OF PHOTODYNAMIC THERAPY
2.1. Photodynamic Reaction
A PS absorbs a photon of light and becomes activated from
ground state to a short-lived excited singlet state (1PS*; Figure
1). The excited PS may either decay back to the ground state by
emitting fluorescence, a property that can be clinically exploited
for imaging and photodetection; or it can undergo intersystem
crossing whereby the spin of its excited electron inverts to form
a relatively long-lived triplet state (3PS*).11 The triplet excited
B DOI: 10.1021/cr5004198
Review
PS can then directly interact with a substrate, such as the cell
membrane or a molecule, and transfer a proton or an electron
to form a radical anion or cation, respectively, which then reacts
with oxygen to produce oxygenated products such as
superoxide anion radicals, hydroxyl radicals, and hydrogen
peroxides (type I reaction). Alternatively, the energy of the
excited PS can be directly transferred to molecular oxygen
(itself a triplet in the ground state), to form 1O2 (type II
reaction). The energy required for the transition of oxygen
from triplet ground state to excited singlet state is 22 kcal
mol−1, which corresponds to a wavelength of 1274 nm.12 Thus,
a relatively small energy is only needed to produce 1O2.13 The
byproducts formed as a result of the type I and type II reactions
are responsible for the cell-killing and therapeutic effect in
PDT. It is to be noted that both type I and type II reactions can
occur simultaneously, and the ratio between these processes
depends on the type of PS, as well as the concentrations of
molecular oxygen and substrate present.11 However, most of
the studies indicate type II reactions, hence 1O2 play a
dominant role in PDT.14
2.2. Mechanism of Tumor Destruction
Following PDT, the extent of photoinduced tumor destruction
depends on various factors such as the type of PS, its
concentration and localization in the tumor microenvironment
or its subcellular location at the time of irradiation, the time
between PS administration and light irradiation (drug-light
interval, DLI), light fluence rate, the total fluence, the type of
tumor, and its level of oxygenation.3a,15 PDT’s downstream
targets include tumor cells, tumor as well as normal
microvasculature, and the inflammatory and immune host
system. The ROS generated by the photodynamic reaction is
the key effector causing irreversible damage to the tumor cells
and microvasculature, ensuing a plethora of inflammatory and
immune response; a combination of which often helps to
achieve a long-term tumor control.15b
2.2.1. Direct Tumor Cell Kill. Generally, the site of
photodamage is considered to coincide with the location of
accumulation of the PS in the tumor cells, as 1O2 has a very
short life (half-life: 0.03 to 0.18 ms),16 restricting its diffusion in
biological systems to a radius of <0.02 μm.17 Oxidation of the
lipids, amino-acids, and proteins by 1O2 induces irreversible
photodamage to vital subcellular targets such as plasma
membrane and organelles like mitochondria, lysosomes, Golgi
apparatus, and endoplasmic reticulum (ER). The location
where the PS ultimately resides within the cell generally
depends on its structural features such as the net ionic charge
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review

Table 1. List of 1st and 2nd Photosensitizers That Are Approved for Clinical Use or Are under Clinical Trials

which can range from −4 to +4, the degree of hydrophobicity across the plasma membrane. PSs that are less hydrophobic
expressed as the logarithm of the octanol/water partition tend to be too polar to diffuse across the plasma membrane and
coefficient, as well as the degree of asymmetry present in the PS are therefore taken up by endocytosis.11 As most of the PSs do
molecule.11 Hydrophobic PSs with two or less negative charges not accumulate in cell nuclei, PDT typically has very low
tend to have the highest intracellular uptake and can diffuse chances of causing mutations.3a PDT induced cell death can
C DOI: 10.1021/cr5004198
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
Figure 2. Basic structures of porphyrin-based PS. Adapted from ref 27. 2013, MDPI.
occur by apoptosis, necrosis, or autophagy and does not
depend on the phase of the cell cycle.18 Necrotic cell death, also
known as accidental cell death, is often triggered when high PS
and/or light doses are used. It is typically characterized by
swelling of cytoplasm, devastation of organelles, and disruption
of the plasma membrane, leading to the release of intracellular
contents triggering an inflammatory response. Apoptotic cell
death (programmed cell death) remains the preferred mode of
cell death following PDT, which is characterized by cell
shrinkage, membrane blebbing, nuclear fragmentation, chro-
matin condensation, chromosomal DNA fragmentation, and
formation of apoptotic bodies that are scavenged by phagocytes
in vivo thus preventing an inflammatory response. The mode of
cell death also depends on the location of the PS within the
cell; for example if the PS is localized in the ER or
mitochondria, the cell death occurs via an apoptotic pathway,
while their localization in the plasma membrane or lysosomes
could delay or block apoptosis. This is when the cell takes
alternative routes of cell-death, such as the autophagic or
necrotic pathway.18b
2.2.2. Vascular Damage. The growth of solid tumors is
dependent on their capacity to induce the growth of blood
D DOI: 10.1021/cr5004198
Review
vessels (angiogenesis) to supply them with oxygen and
nutrients. Generally, due to poor and incomplete cellular
borders,19 these abnormal and leaky vessels may serve as an
additional means for PS accumulation in tumor region as it
leaks through. Moreover, PSs bound to carrier molecules such
as albumin, high-density lipoprotein (HDL), or low-density
lipoprotein (LDL), have high affinity to endothelial cells and
tumor microvascular endothelium because of the presence of
high number of specific receptors in their structure.20
Activation of PS either confined in the blood circulation,
localized in endothelial cells, or bound to the vessel walls results
in damage to the endothelial cells characterized by the loss of
tight junctions between cells and exposure of vascular basement
membranes.21 These primary changes within the vessel lumen
leads to the formation of thrombogenic sites and initiates a
cascade of reactions such as platelet aggregation, release of
vasoactive molecules, leukocyte adhesion, increase in vascular
permeability, and vessel constriction.22 All these effects
ultimately lead to partial or complete vascular shutdown,
blood flow stasis and tissue hemorrhages leading to persistent
post-PDT tumor hypoxia and long-term tumor control.23
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review

Figure 3. Chemical structures of some 2nd generation PSs. Adapted from ref 27. 2013, MDPI.

2.2.3. Inflammatory and Immune Response. While
direct tumor cell kill and vascular damage are mainly
responsible for initial tumor ablation, antitumor specific
immune process and enhancement of host immune system
might play important roles in secondary cytotoxicity which
could probably aid in the long term tumor control and/or
complete tumor response to PDT.24 The principal character-
istic of the inflammatory process is the release of inflammatory
mediators that occur from the treated region, which include
components of the complement system, proteinases, perox-
idases, cytokines, growth factors, and other immuno-regu-
lators.15c,25 This in turn stimulates various white blood cell
E DOI: 10.1021/cr5004198
components such as neutrophils and macrophages which
converge on the treated site. Macrophages remove the damaged
cancer cells by phagocytosis and present proteins from the
tumor cells to CD4 helper T lymphocytes, which then activate
CD8 cytotoxic T lymphocytes. The latter then not only induce
necrosis but may also induce apoptosis whenever tumor cells
are found, leading to long-term tumor control.25 Thus, PDT
has an added advantage of being an immune-stimulatory
modality, whereas surgery and chemotherapy are immunosup-
pressive.
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews Review

3. PHOTOSENSITIZERS
Ideally, a PS is expected to have the ability to preferentially
accumulate in tumor tissue and rapidly clear from the normal
tissue. Amphilicity is another must have property for a clinically
successful PS, which when administered systemically will travel
to the target tissue (tumor) unhindered, for which it requires
some degree of hydrophilicity, and then bind to the target cells,
for which it requires some degree of lipophilicity. It must also
have negligible dark-toxicity, high quantum yield of triplet state
formation, and appropriate triplet lifetime to interact with
ground state oxygen or other substrates, to generate a sufficient
amount of ROS. Most importantly, the PS must be activated by
wavelengths of light above 700 nm to allow deeper penetration Figure 4. Chemical structures of some nonporphyrin 2nd generation
of light in tissues. This is because endogenous molecules, like PS. Adapted from ref 27. 2013, MDPI.
hemoglobin, have strong absorption of light below 700 nm,
thus limiting the effective penetration depth of light in target have absorption maxima at wavelengths longer than 630 nm, as
tissues. With so many disparate requirements, it is difficult to well as high extinction coefficients. This category also includes
find or develop any drug that can qualify as an ideal PS. metalated derivatives of PS such as aluminum phthalocyanine
However, many PSs that do not satisfy all these requirements tetrasulfonate (AlPcS4), Si(IV)-naphthalocyanine (SiNC), and
have been approved for clinical use, with some others being tin ethyl etiopurpurin (SnET2), although there is no consistent
tested in clinical trials (Table 1), and they largely belong to the correlation between metalation and increased photodynamic
first and second generation of PSs as discussed in the following activity.28 Generally, the second generation PS presented higher
sections. quantum yields of 1O2, higher tumor-to-normal tissue
3.1. 1st Generation Photosensitizers concentration compared to HpD and, consequently, exhibit a
better antitumor effect. Furthermore, owing to the shorter
Generally, first generation PSs include porphyrin-based PSs
tissue accumulation time of the second generation PS,
that were developed in the 1970s and early 1980s.
treatment time could be reduced as PDT can be carried out
Hematoporphyrin derivative (HpD) (containing a proprietary
on the same day as the administration of the drug. This gives an
mix of porphyrin monomers, dimers, and oligomers) or
opportunity to conduct PDT as an out-patient procedure,
porfimer sodium (the active material in HpD) are examples
making it convenient and accepatable for patients. In addition
of first generation PSs. They were the earliest and most useful
to the rapid treatment time, the second generation PSs
PSs in clinical trials.15c There are several advantages to porfimer
displayed a shorter window of cutaneous photosensitivity (<2
sodium in that it destroys tumors effectively, exhibits negligible
weeks). The properties of the PS largely depends on chemical
dark toxicity, and can be easily formulated in a water-soluble
and physical parameters such as lipophilicity, type and number
preparation for intravenous delivery. However, these PSs have
of charged groups, charge-to-mass ratio, and type and number
relatively weak absorption of light in the red portion of the
of ring and core substituents.29
spectrum where tissue penetration of light is optimal, thus
Although, some second generation PSs such as amino-
significantly reducing the penetration abilities and thus the
levulinic acid (ALA), mono-L-aspartyl chlorin e6 (MACE),
effectiveness of the treatment. Due to their low extinction
Lutexaphyrin, AIPcS4, tetraphenylporphine (TPPS3, TPPS4),
coefficients, administration of a large amount of the drug is
etc. are relatively hydrophilic, most of them especially those
often required to obtain a satisfactory phototherapeutic
composed of porphyrin ring structures such as Chlorin e6
response. Furthermore, DLI is typically 48−72 h, during
(Ce6), meta-tetrahydroxyphenylchlorin (mTHPP), bacterio-
which the patient must be protected from light. Moreover, they
chloropyll-a, 2-[1-hexyloxyethyl]-2-devinylpyropheophorbide-a
readily accumulate and are retained in the normal tissue and
(HPPH), Tookad, SnET2, and unsubstituted phthalocyanines
skin for prolonged period, leading to severe photosensitivity for
are highly hydrophobic. It is well-known that the degree of PS
approximately 4−6 weeks after treatment. But, these side effects
hydrophobicity affects its route of administration as well as the
could be prevented by avoiding sunlight and high-energy light,
biodistribution/pharmacokinetic profile,13,30 and it was re-
or by wearing protective clothing and sunglasses for
ported that hydrophobic PSs exhibited a higher tumor to
approximately 6 weeks after treatment. However, lack of
normal ratio of 7:1 to 8:1, whereas equivalent hydrophilic PS
tumor selectivity, poor bioavailability, and unfavorable bio-
exhibited a mere 2:1 ratio.31 Although, hydrophobic character-
distribution, as well as prolonged photosensitivity was a major
istic could allow the PS to penetrate the cell membrane and
limitation during the initial clinical trials with first generation
locate in the photosensitive subcellular compartment, highly
PSs.26
hydrophobic PS could form aggregates in aqueous solution
3.2. 2nd Generation Photosensitizers particularly under physiological milieu, which could in turn
Porphyrinoid compounds comprising of porphyrin or porphyr- affect its photophysical (1O2 yield) and photokilling properties
in-based macrocyclic structures such as chlorins, bacteriochlor- due to its inadequate pharmacokinetics. Furthermore, hydro-
ins, phthalocyanines, pheophorbides bacteriopheophorbies, phobicity could hamper its solubility in physiological solvents
texaphyrins (Figures 2 and 3), as well as nonporphyrinoid and body fluids, limiting their clinical applications.32 Thus,
compouds like anthraquinones, phenothiazines, xanthenes, clinically successful PS must have balance between the degree
cyanines, and curcuminoids (Figure 4) with known chemical of hydrophilicity and lipophilicity. In order to improve the
structures are collectively known as second generation PSs.27 solubility of these PSs, various hydrophilic polar substituents
They were mostly developed since the late 1980s, to overcome were introduced in the lead PS structure to synthesize
the shortcomings of the first generation PSs. These PSs usually derivatives that were amphiphilic. The porphyrin ring system
F DOI: 10.1021/cr5004198
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
inherently possess 12 positions, which could be substituted
with various groups such as sulfonic acid, carboxylic acid,
hydroxyls, quarternary ammonium salts, carbonyl, pyridinium
substituents, and so forth, giving countless number of possible
derivatives.33 Apart from the addition of various side groups,
the porphyrin ring system can also be oxidized, extended, and/
or modified to carry a central ion to alter the photophysical and
pharmacological properties of the molecule. Similar modifica-
tions also apply for other second generation PS compounds like
perylenequinones, dyes, phenothiazines, anthraquinone deriva-
tives, etc. In general, a PS containing no charged peripheral
groups tends to be more hydrophobic; PSs with three or more
charged substituents are likely to be hydrophilic; and that with
around two charged peripheral groups could probably be
amphiphilic. In addition to this, PS with anionic substituents,
such as sulfonate or carboxyl groups, have been reported to
localize preferentially in the cytoplasm,34 whereas PSs function-
alized with cationic groups traverse the mitochondrial
membrane and accumulate in the mitochondrion.35 However,
the exact structural characteristics and mechanism behind these
specific distributions and localizations are very much unclear,
and the question still remains as to how to maximize tumor
tissue selectivity over normal tissue accumulation.
3.3. 3rd Generation Photosensitizers
Much of the research in this field now focuses on development
of third generation PSs that can be activated with light of a
longer wavelength, provoke shorter generalized photosensitivity
and more importantly, have better tumor specificity. This can
be achieved either by (a) modifying existing PS with biologic
conjugates such as peptides-/antibody-/antisense-conjugates to
achieve tumor specific targeting of PSs,36 or (b) chemical
conjugation/encapsulation of PS in delivery vehicles or carriers
that can transport the drug efficiently in blood from the
administration site to the target tissue. In short, the third
generation PS will be an improvement of the current second
generation PSs in terms of their delivery or targeting abilities.
Although, many of the third generation PSs have been widely
studied and considered to enable selective targeting in vitro,
very few have been evaluated for clinical applications as their in
vivo selectivity was not found to be sufficiently high.37
4. CURRENT LIMITATIONS OF PHOTODYNAMIC
THERAPY
Although, PDT has the potential to be a stand-alone modality
for the treatment and management of cancers at different
stages, currently it is only being used in the treatment of
superficial and flat lesions, lesions that are accessible through
endoscopes or as a surgical adjuvant and prophylactic treatment
of dysplasias and primary lesions. The fundamental problem
lies in the inability of PDT to treat solid, bulky tumors or deep-
seated tumors. It also cannot be used for the treatment of
advanced disseminated disease because whole body irradiation
is not possible at least with the currently available technologies.
This is partly due to the short-comings in the properties of the
PSs that are currently available, and partly due to the
incomplete knowledge about the light dose to be delivered to
the tumor without affecting the surrounding normal tissue;
making it difficult for the clinicians to plan and customize the
treatment. As PDT is a localized treatment, it essentially
requires an appropriate dose of light to reach every point in the
tumor, for it to be effective in eradicating the tumor. Despite
advanced Monte Carlo calculations, often clinical PDT fails to
G DOI: 10.1021/cr5004198
Review
control residual tumor regrowth and prevent tumor recurrence
due to under-illumination of the tumors.38 In some other cases,
overillumination could induce excessive toxicity to adjacent
normal tissue, again leading to unfavorable response. Unlike
radiation therapy, in which the biological response is well
correlated with the energy absorbed per unit mass of tissue,
there is a clear lack of a widely accepted definition of PDT light
dose. This is a fairly complicated problem mainly due to the
inherent complexity of the PDT mechanism along with lack of
suitable techniques to measure it clinically. Yet, a frequently
practiced, but a rather crude method involves measuring the
light fluence, amount of PS and oxygen, which are then used to
calculate the 1O2 yield (explicit dosimetry).39 The distribution
of light is determined by the light source characteristics and the
tissue optical properties. However, due to the complexity of
tissue architecture and the relatively sparse knowledge on tissue
optical properties, measurement and interpretation of light
fluence seems rather challenging. Although the distribution of
oxygen and PS can be measured by optical spectroscopy, the
actual distribution could vary due to oxygen consumption
during the photodynamic reaction and photobleaching,
respectively. Hence, a more dynamic modeling of the
photodynamic process is required. An alternative approach,
involves measuring a “surrogate” directly associated with
biological response (implicit dosimetry).40 For example,
measuring the fluorescence photobleaching of the PS, indirectly
predicts the production of 1O2.40b A third method - direct
dosimetry, relies on the direct detection of 1O2 itself, either
through its own phosphorescence emission or via 1O2-sensitive
chromophores.41 Hence, there is a clear need to develop
integrated dosimetry systems with light delivery devices as well
as multimodality imaging (anatomic and diffuse optical
tomography) systems with real-time PDT response monitoring
capabilities, which will enable clinicians to customize and plan
PDT treatment regimens on a case by case basis.
It is well-known that PDT is an oxygen consuming modality.
In other words, the antitumor effects of PDT will be severely
affected in the absence of tissue oxygen. Tumor hypoxia can
occur either due to the presence of pre-existing hypoxic cells
that formed as a results of exhaustion of blood supply through
the rapid tumor growth; or could be induced during PDT
through rapid shut down of tumor vasculature and fast
depletion of local oxygen supply.21 Solid tumors with high
fraction of hypoxic cells are reported to have a poor prognosis
to PDT, as hypoxic tumor cells in solid tumors are PDT-
resistant.42 Among the various methods adopted to deal with
depletion of tissue oxygenation, fractionation of light irradiation
into controlled light and dark periods,43 as well as reducing the
fluence rate to 20 mW cm−2,44 has been extensively studied.
These techniques are considered to promote reperfusion of
tissue oxygen, to compensate for oxygen depletion. However,
the hypoxia caused by pre-existing hypoxic cells is not affected
by these techniques.45 Besides, both reduction in fluence rate
and light fractionation would lead to significant increase in time
of irradiation required for delivery of specific light dose. A third
method involves hyperoxygenation therapy, which is found to
compensate tumor hypoxia in both pre-existing hypoxic cells
and those that are acutely induced by PDT. When C3H mouse
transplanted with mammary carcinoma cells MCa were
subjected to hyperoxygenation, up to 80% of the tumors
showed no regrowth after 60 days; whereas in animals that
inhaled room air, only 20% did not regrow tumors.45 Following
hyperoxygenation, oxygen was found to reach critical, chronic
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
hypoxic cells and also resulted in better tumor response to PDT
due to enhanced direct and secondary tumor cell kill.46 In the
molecular level, the effect of hypoxia is mediated by hypoxia-
inducible factor-1 (HIF-1),47 a heterodimeric protein, compris-
ing of HIF-1α and HIF-1β subunits; both of which are
transcription factors. While HIF-1α is cytoplasmic protein
responsive to oxygen tension, HIF-1β is a nuclear protein that
is constitutively expressed and independent of oxygen level.48
In normoxic cells, HIF-1α is continuously degraded, whereas
under hypoxic conditions it translocates to the nucleus and
heterodimerizes with HIF-1β to form active HIF-1 protein. The
HIF-1 protein, in turn binds and activates various hypoxia
responsive genes such as vascular endothelial growth factor
receptor (VEGF), other angiogenic growth factors, glycolytic
enzymes and cytokines, as an adaptive response.47,49 This will
in turn diminish the therapeutic responses by triggering
angiogenesis and tumor relapse. Several reports have
documented the upregulation of HIF-1α and VEGF following
PDT as a result of vascular damage and photochemical
consumption of oxygen.50 Integrating PDT with antiangiogenic
agents, such as Avastin (bevacizumab), Celecoxib, Prinomastat
(AG-3340), PI3−K inhihtors, etc. has clearly demonstrated
improved therapeutic efficacy in preclinical studies.50a However,
blocking one or two molecular pathways at a time might not be
sufficient, as multiple pathways might be involved in the
proliferation of tumor cells. Therefore, combination therapy
that targets multiple molecular pathways at the same time could
be an attractive therapeutic strategy to achieve complete and
effective tumor response following PDT. Apart from these
difficulties, lack of an ideal PS continues to be a major
roadblock restricting the clinical utility of PDT. In fact, the
search for an ideal PS, with majority of the characteristics as
summarized in Table 2,26,38,51 still continues even after nearly 4
Table 2. List of Properties Required in a Clinically
Successful PS
chemical purity for regulatory approval
ease of synthesis for commercial and large-scale production
no degradation upon light activation to form toxic byproducts
synthesis method with minimal batch variations
chemical stability for transport, storage and reconstitution
solubility in injectable solvents and body’s fluids
amphilicity for tissue penetration
good selectivity toward tumor tissue
clinically useful half-life in tissues
rapid elimination from nontarget tissue, minimizing skin photosensitivity
negligible dark toxicity or systemic toxicity
high quantum yield of singlet oxygen generation
high molar extinction coefficient
good photostability
excitable by near-infrared wavelengths (700−1300 nm)
multiple administration routes (oral, intravenous, intratumoral, or inhalational)
decades of PDT. Although many first and second generation
PSs have been effective in establishing the viability and
feasibility of this technique, it has not been optimum in
terms of effectiveness. This is because PSs developed for PDT
seems to have been driven chemically, rather than clinically.
Focus is needed on solving the problems of the PS or
modifiying the existing PS to overcome the 3 major problems
that might be the potential impediment to the clinical
H DOI: 10.1021/cr5004198
Review
application of PDT. The areas that require improvement in
terms of clinical PDT are as follows.
4.1. Tumor Selectivity
Due to the generalized photosensitivity associated with most of
the earlier generation PSs, patients find it difficult to make
lifestyle changes and stay indoors for a prolonged period, which
generally prevents them from consenting to PDT. Although
some photosensitizing drugs are found to be preferentially
retained by the tumor cells compared to the surrounding
healthy tissue, the exact mechanisms that drive this process are
not fully understood. However, the abnormal physiology of
tumors such as leaky vasculature, poor lymphatic drainage,
abnormal stromal composition, decreased pH, and increased
number of low density lipoprotein receptors are often
considered to contribute to the selectivity of photosensiti-
zers.15c Tumor selectivity could be improved by targeted
delivery agents that can transport the PS from the site of
administration to the tumor tissue, leading to the selective
accumulation of the PS in the tumor. In this way, unfavorable
biodistribution can be avoided, improving bioavailability of the
PS. Consequently, undesirable side effects such as prolonged
skin photosensitivity and damage to surrounding healthy tissues
can be prevented.
4.2. Formulation of Photosensitizers
To a large extent, the efficacy of PDT treatment depends on
the photochemical, photobiological, and pharmacokinetic
properties of the PS. In general most of the potent PSs have
extended delocalized aromatic π electron system, a character-
istic property that allows them to absorb light efficiently. At the
same time due to π−π stacking and hydrophobic interactions,
they easily form aggregates in aqueous media. On one hand,
hydrophobicity could be a desirable characteristic, as
solubilization of the PS in the lipid bilayer of the cell membrane
has been pointed out to be one of the main factors of PS
efficacy. On the other hand, this feature leads to lack of
solubility of PS and contributes to its aggregation in aqueous
solution.33,52 This affects the bioavailability of the dye and
hampers its ROS generation ability, resulting in suboptimal
PDT treatment outcome. Hydrophobicity also complicates
preparation of pharmaceutical formulations for parenteral
administration.10,53 Again this could be solved by formulating
the PS in suitable carrier systems that can transport PS in its
stable monomeric form without altering its spectroscopic and
functional properties. To be therapeutically useful, a PS
formulation should deliver the PS in a form that can be readily
and selectively internalized by hyperproliferative cancer cells, in
such a way that it facilitates accurate and convenient dosing. At
the same time, the carrier should be biodegradable, nontoxic or
must not release toxic degradation products, or must have no
immunogenic properties.
4.3. Tissue Penetration Depth
Clinical application of PDT for the treatment of solid or deep-
seated tumors has been hampered by the tissue penetration
depth limit of the visible light required by most of the
conventional PSs for its activation. The typical penetration
depth of red light in living tissue used for PDT is only 1−3 mm.
Hence, even if the surface of the target area is illuminated
evenly, the energy of the incident light will fall dramatically at
increasing depths below the surface (or at increasing distances
within tissue from an interstitial fiber). This is because most
tissue chromophores (hemoglobin, melanin, fat etc.) absorb
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Chemical Reviews
light strongly in the visible spectrum.54 Thus, effectiveness of
PDT greatly decrease with tissue thickness due to strong
attenuation of visible light with increasing tissue depth, leading
to incomplete treatment and tumor relapse.55 Apparently, for
deep penetration of light into tissues, wavelength of the
irradiated beam should be in the near-infrared (NIR) region
between 700−1300 nm, which is known as the “optical
window” of biological tissue.56 Although some efforts have been
made to utilize PSs that absorb in the NIR range to improve the
tissue penetrability,57 the low energy associated with the NIR
light at the single-photon level does not fulfill the energy
requirement to efficiently excite PSs for sufficient ROS
generation. This underscores the need of an efficient energy
or light transducers that can absorb light in the NIR region and
emit in the visible region, which could then activate the PS
near-by.
Different strategies have evolved to overcome the current
shortcomings with regard to the issues associated with the PS,
often by means of a delivery vehicle that enables a stable
dispersion of PS in aqueous systems. Despite enhanced drug
loading and improved tumor uptake over free drugs, the
traditional delivery systems such as oil-based drug formulations
(micellar systems) using low molecular weight surfactants such
as the polyoxyethylated castor oils (e.g., polysorbate 80
(Tween-80), Cremophor- EL etc.) have been reported to elicit
acute hypersensitivity (anaphylactic) reactions in vivo.58 Hence,
more stable, biocompatible and tumor selective carrier systems
are required for efficient delivery of PS.
5. NANOPARTICLES IN PHOTODYNAMIC THERAPY
Nanoparticles represent an emerging technology in the field of
PDT that can overcome most of the limitations of classic PS.59
Typically, they are defined as submicroscopic particles between
1 and 100 nm in size. Nanoparticles are designed out of a
variety of naturally occurring or synthetic materials and can be
engineered to carry multiple theranostic agents, in a targeted
manner. Depending on the type of nanocarrier and the mode of
attachment or loading of PS in it, the use of nanoparticles in
conjunction with PDT could have at least some of the following
advantages:
a. They have large surface to volume ratios,60 thus could
effectively increase the amount of PS that can be
delivered to the target cells.61
b. Nanoparticles may prevent the premature release of PS
and potential inactivation of the drug by plasma
components,62 thereby preventing its nonspecific accu-
mulation in normal tissues, and reducing overall
photosensitivity.
c. They bestow amphilicity to PS, a property that some
potentially outstanding PS agents lack. This allows
nanoparticles to travel unhindered through the blood-
stream carrying the PS payload and localize in the tumor
tissue.63
d. They generally take advantage of the enhanced
permeability and retention (EPR) effect, a phenomenon
caused by the abnormal and leaky tumor neovasculature
and poor lymphatic drainage of the tumor tissue,
facilitating both diffusion of PS carriers into and their
retention within tumor tissue.64
e. Their surface can be further modified with functional
groups or targeting agents for altering its biological or
physical properties thereby improving the biodistribu-
I DOI: 10.1021/cr5004198
Review
tion, pharmacokinetics, cell uptake, and targeting
abilities.32a,65
f. They can be designed as multifunctional nanoplatforms
that carry multiple components, for example, imaging
agents, chemo-drugs, targeting ligands, and “cloaking”
agents (agents that avoid interference with the immune
system).66
In recent years, there have been several reviews that
highlighted the various approaches for utilizing nanoparticles
for PDT, which generally categorized nanoparticles into passive
and active nanoparticles either depending on the absence or
presence of any targeting moieties on their surface, or based on
their involvement in the excitation of PS.32a,66,67 Here, we have
broadly classified the nanoparticles utilized in PDT based on
the functions or tasks they perform into three: carriers of PSs,
PSs by itself and energy transducers of PS. We have further
attempted to provide comprehensive information on the
design, advantages, perspectives and challenges of each of the
nanoformulations along with a critical discussion in order to aid
the future research in the field of photodynamic nanomedicine.
5.1. Nanoparticles As Delivery Vehicles of Photosensitizers
The first report on nanoparticles as delivery vehicles in 1976
focused on the development of nanoparticle-based vaccines for
controlled and slow release of the vaccine, leading to a better
immune response.68 This marked a significant technological
and medical breakthrough, as until then it was considered
impossible to administer pharmaceutical suspensions (solid
particles dispersed in a liquid) intravenously, due to the obvious
risks of embolism. Today, the development of nanoparticle
based delivery vehicles has made it possible to deliver many
chemotherapeutic drugs of low therapeutic index, such as
doxorubicin (DOX) and paclitaxel, in patients by selectively
directing them toward the tumors cells. Currently, there are
numerous nanoformulations that have been either approved or
under clinical trials for various oncological indications.
In 1991, Labib et al. was the first to report the use of cyano-
acrylic nanospheres (150 to 250 nm) or nanocapsules (10−380
nm) synthesized by interfacial polymerization in an oil-in-water
emulsion, encapsulating phthalocyanine or naphthalocyanine
derivatives.69 The immediate and subsequent studies focused
mainly on the development of a sensitizer-delivery system
based on such biodegradable polymeric nanoparticles. Unlike
chemotherapeutic drugs, that need to be released from the
carrier to exert its effect on the target cells, the PS need not be
released from the carrier as long as molecular oxygen and 1O2,
is able to diffuse in and out of the carrier. Due to this fact, two
different strategies are often adopted to develop nanoparticle
carriers for PDT: a) biodegradable nanoparticles (natural or
synthetic polymer-based nanoparticles) carrying the PS pay-
load, which could undergo enzymatic or hydrolytic degradation
and thereby excreted out, minimizing long-term accumulation
of carriers in biological system; or b) nonbiodegradable
nanoparticles (ceramic- or metal-based nanoparticles), which
does not readily degrade in the biological system, but quite
often offer in-built multiple functionalities that make them
valuable as theranostic agents.”
5.1.1. Biodegradable Nanoparticles. 5.1.1.1. Polyester
and Polyacrylamide Based Nanoparticles. Biopolyesters are
biocompatible polymers that can be naturally occurring such as
poly(hydroxyalkanoates) (PHAs) or synthetic polymers like
poly(orthoesters), poly(β-amino esters) (PbAE), as well as
poly(α-hydroxy esters); the latter which includes poly(D,L-
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Chemical Reviews Review

Figure 5. General PS loading approaches in biodegradable polymeric nanoparticles.

Figure 6. Conceptual illustration of the mechanism of bioreducible nanoparticles for the switchable photoactivity of PpA. The nanoparticles do not
exhibit photoactivity during blood circulation. However, when internalized by cancer cells, the intracellular reduction-triggered cleavage of the
disulfide bonds accelerates the dissociation of the polymeric nanoparticle, causing the rapid release of PpA and restoration of its photoactivity.
Reproduced with permission from ref 79. 2014 American Chemical Society.

lactide) (PLA), poly(glycolide) (PGA), and poly(ε-caprolac-
tone) (PCL) are the most widely employed polymers for
micelle and nanoparticle preparation. Earlier reports using such
polyester-based (PLA) colloidal delivery systems of PS
exhibited greater photodynamic efficiency and reduced photo-
sensitivity compared to surfactant based carrier systems.10
Encapsulation of PSs within these systems (Figure 5) can
substantially increase its solubility/dispersibility in aqueous
media, thus improving their pharmacokinetic properties.
Further advantages of using biodegradable polymer based
nanoparticles is the ability to deliver a reasonably good payload
of the multiple agents (therapeutic PS and diagnostic agents) to
the tumor, shielding it from premature leakage in the blood
thereby maintaining the photostability of photoactive agents
and fluorophores. In addition, it offers the possibility of further
J DOI: 10.1021/cr5004198
modification of its surface with a variety of biomolecules for
targeted PS delivery.
The initial success of using polymeric micelles and
nanoparticles for the delivery of PS along with good PDT
outcome saw a surge in the number of articles involving
research in this field, demonstrating how their targeting ability
and pharmacokinetic functions in vivo are controlled by its
composition, size, surface charge, morphology and hydro-
phobicity.70 Most of the second generation PSs have been
tested out in combination with such polymeric nanoparticles
especially poly(D,L-lactic-co-glycolic acid) (PLGA),71 a biode-
gradable copolymer of PLA and PGA, that has been approved
by FDA for use in humans.72 Such studies on PLGA
nanoparticles encapsulating different PSs have shown varying
degree of success in terms of improved photodynamic activity
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Chemical Reviews Review

Figure 7. (A) Schematic of fabrication of SPION encapsulated nanoparticle following a LbL deposition of PAH and DP and removal of the
polystyrene (PS) template. (B) TEM images of SPIONs⊂NC3. (C) T2-weighted MR images with various concentration of contained Fe. (D)
Fluorescence images of ROS generation in HeLa cells treated with SPIONs⊂NC3 and light irradiation. (E) Quantification of induced ROS.
Reproduced with permission from ref 83. 2014 American Chemical Society.

and dramatic tumor-inhibiting effect, enhanced 1O2 production
and increased plasma circulation time offering the possibility of
reducing the amount of PS administered as well as shortening
the DLI. A detailed review by Bechet et al.66 and Chatterjee et
al.67b in 2008 and book chapter by Lee and Kopelman73 in
2011, outlined each stage in the development of these
nanoconstructs and further discussed its stability, photo-
cytotoxicity, biodistribution and PDT efficiency. Degradation
of the polymeric nanoparticle is generally believed to occur via
a hydrolytic process, although the possibility of an enzyme-
catalyzed degradation has also been considered.74 Once
degraded to biocompatible nontoxic products, they are
removed from the body by physiological pathways and
clearance mechanisms. The rate of degradation can be
modulated by changing the composition, molecular weight
and ratio of the copolymeric system. Systemic use of such
synthetic polymeric nanoparticles has been limited due to their
rapid opsonisation and removal from the systemic circulation
by the macrophage cells of the mononuclear phagocytic system
(MPS). In order to evade the recognition and removal by the
MPS, the nanoparticles are often further surface modified by
polyethylene glycol (PEG) or poly(ethylene oxide) to confer
“stealth properties”.70e PEGylated PLGA nanoparticles have
K DOI: 10.1021/cr5004198
been shown to have enhanced circulation time and remain in
the blood compared to bare PLGA nanoparticles.75 For
example, PLGA (poly(lactide-co-glycolide)) nanoparticles with
sizes above 100 nm, there occurs a very rapid elimination from
the blood with the half-life ranging from 13 to 35s, whereas
PEGylated PLGA had much longer half-life of close to 7 h.76
Generally, nanoformulations accumulate within the tumor
stroma via EPR effect. However, they can re-enter the
bloodstream via diffusion, resulting in decreased drug
concentration at the target tissue over time. Intracellular uptake
of these nanoconstructs from the extracellular space within the
tumor requires time-resolved cell membrane-mediated pro-
cesses. Active targeting of nanoparticles to cancer cell-specific
receptor could promote rapid intracellular uptake of the EPR-
accumulated nanoformulations. Along these lines, Master et al.
synthesized epidermal growth factor receptor (EGFR) targeting
GE11 peptide-decorated PEG−PCL micelles encapsulating the
PS Pc4, for rapid intracellular uptake and enhanced PDT of
EGFR-overexpressing A431 human epidermoid carcinoma
cells.77 Premature biodegradation of the polymers while still
in circulation could lead to PS leakage from the nano-
formulation, again resulting in decreased bioavailability in the
target tissue. To tackle this problem, bioresponsive polymeric
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Chemical Reviews
systems that make use of the difference between the normal
and tumor physiology could be utilized. Peng et al. utilized the
pH differences between the normal (pH remains constant at
7.4) and tumor (pH below 7.2) tissue, to develop 2-
(diisopropylamino)ethyl methacrylate (DPA) based poly-
(ethylene glycol) methacrylate-co-DPA (PEGMA-co-DPA)
copolymers that self-assembled to form pH sensitive nano-
particles in an aqueous environment, encapsulating hydro-
phobic m-THPC.78 The nanoparticles were found to be
unstable only below a pH of 6.89, thus releasing the PS in a
site-specific manner. Recently, Kim et al. developed a technique
for bioreducible activation of encapsulated PS Pheophorbide-A
(PpA) upon intracellular uptake of the polymeric nano-
particle.79 Here, 2 PpA molecules were chemically conjugated
to one mPEG molecule with biarmed linkages (Figure 6). The
amphiphilic construct self-assembled to form core−shell-
structured nanoparticles under aqueous conditions, where the
hydrophobic PpA remains in a photoinactive state due to
fluorescence resonance energy transfer (FRET) mediated
intramolecular and intermolecular quenching. This property
would minimize deleterious effect due to any 1O2 generation
during its systemic circulation. The PS payload carrying
polymeric construct accumulates in the tumor stroma via the
EPR effect. As the nanoconstruct enters the cytosol of the
tumor cell that has a higher concentration of glutathione
(GSH) than those in extracellular fluids, the thiol responsive
disulfide linkers are rapidly cleaved releasing the photoactive PS
molecule; resulting in enhanced cell-kill.
Of late, multifunctional polymeric nanoparticle based
delivery systems are gaining interest and several studies
reported application of polymeric nanoparticle in carrying
both diagnostic and therapeutic payload. Gupta et al. reported a
postloading approach of a NIR excitable cyanine dye (CD)
fluorophore and red-light absorbing HPPH, at a ratio of 1:2, in
a biodegradable polyacrylamide (PAA) nanoplatform for
simultaneous tumor-imaging (NIR fluorescence imaging) and
long-term tumor cure by PDT.80 A different approach was
adopted by Lee et al. where multifunctional PLGA nano-
particles (PLGA coupled with methoxy-PEG (mPEG) or Ce6),
synthesized by the Steglich esterification method, were utilized
to carry an inorganic cargo−iron oxide (Fe3O4).81 The
synthesized multifunctional PLGA nanoparticles exhibited
potential in simultaneous high contrast magnetic resonance
imaging (MRI) and therapy, demonstrating significant
regression of tumor volume in human nasopharyngeal
epidermal carcinoma-KB tumor-bearing nude mice, following
PDT. Similarly, Wang et al. developed a tumor targeting
multifunctional nanocarrier based on amine-functionalized
biodegradable PAA nanoparticles, for simultaneous fluores-
cence imaging and PDT in a “see and treat” approach.82 The
HPPH conjugated PAA nanoparticles were synthesized by a
modified water-in-oil microemulsion method. The presence of
a tumor cell and tumor blood vessel (angiogenic vasculature)
homing F3 peptide, targeted the nanoparticle specifically to the
tumor site. Furthermore, chemical conjugation of CD
molecules to both surface and inside the matrix of the
nanoparticles, enabled fluorescence imaging. The synthesized
tumor-specific nanocarriers containing both fluorophores and
PS, emitted bright fluorescence and also generated 1O2 upon
light irradiation, resulting in an irreversible but selective
destruction of the cancer cells. A novel layer-by-layer (LbL)
deposition of positively charged poly(allylamine hydrochloride)
(PAH) and negatively charged PS−dendrimer porphyrin (DP)
L DOI: 10.1021/cr5004198
Review
onto a sacrificial template (polystyrene nanoparticles)
embedded with superparamagnetic iron oxide nanoparticles
(SPIONs), followed by dissolution of the template (Figure 7),
was reported by Yoon et al.83 The multifunctional hollow
nanoconstruct showed potential in MR imaging and PDT and
could further be utilized to carry additional therapeutic tools
with in their inner void space.
Xiang et al. developed polymeric nanovesicle poly(ethylene
glycol)-block-poly(D,L-lactic acid) [PEG−PDLLA] loaded with
a chemotherapeutic agent, DOX and a lipophilic PS
hematoporphyrin monomethyl ether (HMME) [PEG−
PDLLA-DOX-HMME], for combined PDT and chemotherapy,
and confirmed that a synergistic effect of both agents led to an
increased in vivo cell kill in human hepatocellular carcinoma
(HepG2) cells.84 Yet another approach for triple modal
fluorescence, MR, and photoacoustic imaging (PAT)-guided
combined photothermal (PTT) and PDT, was very recently
demonstrated by Gong et al. utilizing a nanomicellar system.85
Ce6 grafted to an amphiphilic polymer composed of poly-
(maleic anhydride-alt-1-octadecene) (C18PMH) and PEG
chains (abbreviated as C18PMHPEG-Ce6), was utilized to
encapsulate IR825 (an indocyanine green (ICG) analog); for
combined PDT and PTT. In addition to being a PDT and
fluorescence imaging agent, Ce6 also serves as a chelating agent
to capture gadolinium-III, enabling T1 -contrast in MRI. Apart
from fluorescence and MR imaging the synthesized nano-
construct could also offer contrast in PAT imaging due to its
strong NIR absorbance attributed from IR825. Combined PDT
(660 nm light at a power density of 2 mW cm−2 for 1 h; 7.2 J
cm−2) and PTT (808 nm laser at a power density of 0.3 W
cm−2; 108 J cm−2) in vivo, 12 h post intravenous injection of
IR825@C18PMH−PEG−Ce6-Gd nanomicelles, demonstrated
remarkable synergist effect in inhibiting tumor growth. In
conclusion, these nanoparticles present as one of the most
versatile drug/PS carrying nanoplatforms that could be further
manipulated to incorporate a variety of functionalities.
5.1.1.2. Liposomal Nanoparticles. Liposomes are one of the
first nanoparticle-based delivery platforms to be applied in
medicine,86 and there are over 11 liposomal formulations
approved for clinical use today, with many more in clinical and
preclinical development. They are concentric phospholipid
vesicles consisting of single or multiple bilayered membrane
structure composed of natural or synthetic lipids (Figure 8A).87
Their unique ability to contain hydrophilic drugs in their
aqueous core and hydrophobic agents within their lamellae
makes them excellent therapeutic carriers.
Various studies have compared the tumor uptake and PDT
outcome of liposomal versus nonliposomal PSs under identical
conditions since the late 1980s. Although liposomes have
proved to be biocompatible carriers of PS, they did not emerge
as an ultimate PS delivery system. This is mainly due to three
reasons. First of all most of the conventional or unmodified
liposomes often included cholesterol in its structure to improve
the rigidity of the bilayer membrane. But, the presence of
cholesterol reduced the permeability of encapsulated PS.88
Second, there exists an exchange of lipids between the
liposomes and HDLs, leading to an irreversible disintegration
of the liposome.89 This causes premature release of the PS in
the bloodstream before reaching the target tissue. Conventional
liposomes are also prone to opsonisation by plasma proteins
after which they are quickly removed from the circulation by
cells of the MPS. Thus, they generally exhibit short plasma half-
life, reducing the time for tumor cell uptake, hampering their
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Chemical Reviews
Figure 8. PS loading strategies in (A) liposome and (B) and (C)
dendrimer.
generalized use as tumoritropic carriers of PSs.90 However, in
the year 2000, a liposomal formulation of PS, labeled as
Visudyne, hit the clinics for the treatment of patients with age-
related macular degeneration or subfoveal choroidal neo-
vascularisation secondary to pathological myopia. The lip-
osomal formulation was composed of zwitterionic dimyristoyl-
phosphatidylcholine (DMPC), negatively charged egg phos-
phatidylglycerol (EPG), and a hydrophobic PS−Verteporfin or
benzoporphyrin derivative monoacid (BPD-MA), a second
generation PS derived from protoporphyrin IX (PpIX)
dimethyl ester.91 The incorporation of BPD-MA in the
liposome enabled its partition into plasma lipoproteins more
rapidly, ensuring higher levels of the PS in the tumor tissues
and the neovasculature. As liposomal membrane provides
sufficient sites to conjugate multiple functional ligands,
liposomes with a specifically modified design, like PEG grafted
long-circulating liposomes92 or antibody,93 peptide,94 folic acid
(FA),95 or glycoprotein (transferrin)96 conjugated tumor
targeting liposomes can be designed for enhanced pharmaco-
kinetic properties as well as improved therapeutics. This has
been reviewed by Derycke and de Witte89 and more recently by
Jin and Zheng.97 In a yet another review, Skupin-Mrugalska et
al. summarized the modifications of the PS properties in
liposomal formulations and further discussed the development,
preclinical studies, and future perspective of such liposomal
formulations.98 Active targeting of liposome was expected to
improve the drug delivery and overall PDT efficacy by
enhancing tumor selectivity of the PS, but the results of these
studies are not always very straightforward. In addition, the PS
concentration in liposomes is generally less than 10%. This
underscores the need of liposomal vesicles with better stability
and PS loading efficiency.
Liposomal carriers with triggered-release mechanism, that
selectively release PS upon internal (pH or enzymes) or
external stimuli (light or temperature), are also gaining more
attention due to its potential in being truly tumoritropic
carriers.99 Such mechanisms seem to enhance cellular internal-
M DOI: 10.1021/cr5004198
Review
ization of the PS, which consequently contributes to increased
tumor accumulation and minimizes the nonspecific photo-
sensitivity of normal tissues. Although, progress has been made
in the comprehension of critical parameters for design and
development of a variety of liposomal formulations of PSs, the
critical parameter to consider when optimizing liposomal PDT
for clinical application such as its pharmacokinetics, biodis-
tribution, drug release, stability of the nanocarrier, and
intratumoral PS localization is still unclear. Reshetov et al.
compared two liposomal formulations of temoporfin, a
conventional liposome (Foslip) and polyethylene glycosylated
liposome (Fospeg), in tumor-grafted mice and found
PEGylated liposomal formulation to be far superior in terms
of blood circulation time, resulting in a rapid and efficient
accumulation of PS in the tumor via EPR effect and thus
significantly shortening the DLI, a characteristic that would
enhance patients interest in PDT.100 On the other hand, Foslip
was soon eliminated from the circulation due to premature
drug release and liposome destruction.
Another area which is gaining popularity is multifunctional
liposomal formulation for theranostic application such as
nanocells101 and the porphysomes.102 While, nanocells are
comprised of PS incorporated polymeric nanoparticles
encapsulated in a PEGylated liposome, porphysomes are
formed by self-assembly of PS (pyropheophorbide)-phospho-
lipid conjugate into liposome-like structures. In addition to
their drug delivery capabilities, these novel classes of liposomes
have shown potential in multimodal imaging and therapeutics.
Recently, Liang et al. reported yet another multifunctional
liposomal carrier named cerasome, developed by chemical
conjugation of porphyrin to an organoalkoxysilylated lipid, with
a high drug loading efficiency of 33.46%.103 This is significantly
higher than that obtained by physically entrapping PS within
the cerasomes (generally less than 10%). Second, covalent
linkage could prevent premature release of PSs during systemic
circulation. The silica-like surface endows the cerasome
remarkably high stability, whereas the inherent porosity allows
oxygen molecules to diffuse in and out of the vesicles freely.
Unlike porphysomes that had almost no photosensitizing effect
as the energy that is normally released for 1O2 generation is
dissipated thermally, cerasomes exhibited good fluorescence
even at an extremely high number of porphyrins, thus proving
to be a powerful tool for simultaneous photodynamic
diagnostics and therapy. However, no studies were performed
to prove its efficacy in vivo. A different approach by Hasan et al.
combined the targeting as well as anticancer properties of
monoclonal antibody Cetuximab (C225), to develop a
liposomal formulation of BPD-MA for simultaneous photo-
immune/PDT.104 Here, BPD-MA was encapsulated in a
Cetuximab antibody attached preformed plain liposome
(PPL) by physical adsorption. Cetuximab antibody not only
facilitated selective binding of the nanoconstruct to EGFR
overexpressing ovarian cancer cells, but also blocked the EGFR
signal transduction pathway, which in combination with PDT
resulted in synergistically enhanced cancer cell destruction.
5.1.1.3. Dendrimer Based Nanoparticles. Dendrimers are
three-dimensional tree-like branched macromolecules that can
be tailored in size with functional peripheral groups and inner
cavity for incorporation of a variety of molecules. Unlike
conventional polymers, dendrimers are prepared by a stepwise
synthetic procedure, leading to a highly ordered branching
pattern with consistent and well-defined architecture. Owing to
its three-dimensional structure and relatively large size, ranging
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Chemical Reviews Review

Figure 9. (A) Synthesis of mono-(PcSi(OH)(mob) 2) and disubstituted (PcSi(mob)2 3) derivatives of the silicon phthalocyanine (PcSi(OH)2 1)
and schematic representation of tumor targeted theranostic platform based on phthalocyanine-loaded dendrimer (Pc−LHRH). (B)
Phototherapeutic effects of Pc−LHRH on A2780/AD cancer cell incubated with different drug concentrations and irradiated for 5 min at 670
nm (36 J cm−2). (C) The influence of laser irradiation time on A2780/AD cells treated with 0.6 μg mL−1 Pc−LHRH and irradiated at 670 nm. (D)
In vivo images of tumor-bearing mouse and ex vivo images of major organs acquired before and 10 h after intravenous injection of Pc−LHRH.
Organs: 1, liver; 2, spleen; 3, kidneys; 4, heart; 5, lungs; 6, tumor. Reproduced with permission from ref 112. 2014 American Chemical Society.

from first up to larger seventh-generation, dendrimers are
expected to be internalized in membrane-limited organelles,
thereby achieving controlled localization in the intracellular
compartment.105 Dendrimers have received much attention as a
PS carrier due to their unique structure, which consists of
several surface groups that can be used to tag different targeting
molecules or functional groups. Furthermore, the size and
lipophilicity of the dendrimer-conjugate can be controlled and
modified for optimizing cellular uptake and tissue biodistribu-
tion.106 Thus, the characteristics of the conjugated PS can be
modulated for optimizing the drug delivery, and thereby
improve the therapeutic efficiency. In their recent review,
Klajnert et al. discussed in detail how the application of
dendrimers in PDT can aid in overcoming the obstacles in PS
delivery to improve PDT outcome.107 The PS can be either
tagged to the functional groups on the surface of the dendrimer
by covalent linkages or encapsulated in the core of the
N DOI: 10.1021/cr5004198
dendrimer (Figure 8B and C). PS molecules loaded on the
exterior are generally released, either upon photoexcitation and
induction of 1O2 that dissolves the covalent linkage between the
PS and dendrimer108 or by cellular esterases that hydrolyses
these bonds.109 Conjugating the PS to the surface of the
dendrimer serves several advantages: first, it reduces the toxicity
of otherwise toxic dendrimer like polypropyleneimine (PPI);
the PS can be loaded in its monomeric form; prevents
premature release of PS and increases the intracellular
accumulation of PS compared to free PS.110 However, the
photosensitizing activity of the PS could be affected by such
chemical complexing. Several studies also reported chemical
synthesis of a built in central PS with in the dendrimer core as
well as PS centered ionic dendrimers capable of forming
polyion micelles through electrostatic interactions with
oppositely charged block copolymers.111 However, such
synthesis method often involves long and tedious multistep
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Chemical Reviews
processes required for preparation and purification of the PS-
core dendrimer. Moreover, this strategy only allows embedding
of a single PS molecule per dendrimer, resulting in low drug
loading efficiency.
Recently, Taratula et al. reported a simple and efficient
approach to encapsulate hydrophobic silicon-based phthalo-
cyanine (PcSi) into PPI dendrimer (Figure 9).112 The synthesis
step involved modification of the PcSi molecule with a
hydrophobic linker (4-methoxy-4-oxobutanoic acid /(mob)),
which significantly enhanced physical encapsulation of the PS
into the hydrophobic pockets of a fourth generation PPI
dendrimer with drug encapsulation efficiency of 20% w/w.
Further modification of the complex with PEG and luteinizing
hormone-releasing hormone (LHRH) peptide, improved its
biocompatibility and tumor-targeted delivery. The prepared
formulation required no release of the drug from the carrier,
preventing leakage of SiPc into the blood circulation, and at the
same time exhibited significant ROS production upon laser
activation. Furthermore, the nanocarrier-PS conjugate exhibited
a distinct NIR absorption (700 nm) and fluorescence emission
(710 and 815 nm) spectra, required for efficient PDT and
fluorescence imaging in the NIR range.
5.1.1.4. Natural Macromolecule Based Nanoparticles.
Various nanoparticles made of cross-linked natural polymers
such as proteins and polysaccharides have also been tested as
carriers of PSs.
Albumin. Human serum albumin (HSA) based nanoparticles
have attracted interest as theranostic drug carrier particularly
for applications involving lipophilic drugs. This technology
allows for transportation of hydrophobic drugs without the
need for potential toxic solvents. Moreover, it exploits
endogenous albumin pathways for selective delivery of large
amounts of chemotherapeutic drugs to the tumor, thus skipping
the issue of possible accumulation of these drug-nanoparticles
conjugates in other parts of the body.113 This forms the basis of
the first U.S. FDA approved nanoparticle-albumin bound
paclitaxel (nab-paclitaxel), sold under the trade name of
Abraxane, for the treatment of metastatic breast cancer after
the failure of first line standard chemotherapy. Wacker et al.
employed albumin based nanoparticles as carriers of two
lipophilic PSs namely, mTHPP and m-tetrahydroxyphenyl-
chlorin (mTHPC).114 The PSs were loaded into a preformed
albumin nanoparticle by an absorptive drug loading technique.
Although both formulations generated relatively low amount of
1
O2 in aqueous solution due to low local oxygen concentration,
efficient 1O2 generation and consequently effective cell death
was reported when the PS loaded nanoparticles were incubated
with cells. Despite being an efficient carrier for PSs, there are
still a number of problems associated with the development of
albumin nanoparticles as delivery agents, including complicated
fabrication methods, wide size distribution, instability under
physiological conditions, and the unintended release of PSs
before it reaches the target site.115 Thereafter, Jeong et al.
devised an alternative method of conjugating Ce6 to lysine
residues within HSA so as to form self-assembling nano-
particles.116 The developed Ce6 bound albumin nanoparticles
exerted higher tumor specificity mainly due to the prolonged
circulation in the blood in addition to the EPR effect and
exhibited better therapeutic efficacy compared with free Ce6
following irradiation. Recently, Portilho et al.117 fabricated an
albumin-based nanosphere containing zinc-phthalocyanine
tetrasulfonate (ZnPcS4-AN) and compared PDT using this
PS loaded nanoconstruct with standard chemotherapeutic
O DOI: 10.1021/cr5004198
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agent DOX following an intratumoral administration of both
formulations, for a head to head comparison. Although, the PS
loaded albumin nanoparticles did not induce a significant tumor
volume reduction, it resulted in a remarkable increase in the
necrotic area both in the central region as well as the peripheral
region of the tumor, unlike the other experimental groups in
which the necrosis was localized only to the central part.
Furthermore, it exhibited very low toxic side effects compared
to treatment with DOX, and thus could be developed in to a
promising PS delivery agent.
Chitosan. Chitosan is a polysaccharide derived from the
partial deacetylation of chitin, primarily from crustacean and
insect shells. It consists of repeating units of glucosamine and
N-acetyl-glucosamine. Although chitosan is insoluble at neutral
pH, it is soluble and positively charged at acidic pH.118 Several
studies have reported the use of amphiphilic chitosan-based
nanoparticles as carriers of hydrophobic anticancer drugs in
their inner cores.119 For PDT, the PS was either entrapped in
the inner core of self-assembled chitosan nanoparticles120 or
chemically linked to chitosan which then self-assembles to form
a nanoparticle.121 The latter design has given rise to the
possibility of developing “switchable quenching/dequenching”
or “on/off” carrier systems. Such a system remains in a
quenched or “off” state with no fluorescence signal and
phototoxicity with light exposure due to FRET based
quenching effect of PS within the nanoparticle core. However,
upon cellular uptake the fluorescence signal and 1O2 generation
was restored due to the cleavage of bonds and release of the
PSs from the chitosan nanoparticles. Although, for self-
assembly of chitosan derivatives into nanoparticles, often
hydrophobic moieties such as bile acids or fatty acids have to
be conjugated to it. Lee et al. conjugated ursodeoxycholic acid
(UDCA), a secondary bile acid, to chitosan (ChitoUDCA) via
EDC-NHS chemistry and incorporated highly anionic and
hydrophobic Ce6 into the cationic UDCA-chitosan nano-
particles through ion-complex formation and hydrophobic
interaction.122 PDT at 664 nm (2 J cm−2) following 2 h
incubation of Ce6-incorporated ChitoUDCA nanoparticles in
HuCC-T1 human cholangiocarcinoma cells, enhanced Ce6
uptake in the cells and correspondingly high ROS generation
and phototoxicity compared to free Ce6. Recently, Chen et al.
coloaded a NIR absorbing PS−ICG and a gold nanorod in
chitosan nanospheres (CS-AuNR-ICG NSs) prepared by a
nonsolvent counterion complexation method and electrostatic
interaction, for combined PTT/PDT.123 The 180 nm CS-
AuNR-ICG NSs were designed to be triggered by single NIR
laser at 808 nm, allowing efficient heat generation and ROS
production at the same time. NIR radiation (2 W cm−2 for 10
min) of murine hepatic H22 xenograft tumors in mouse model,
8 h post intravenous injection of CS-AuNR-ICG NSs (7 mg
kg−1 Au equivalent plus 6 mg kg−1 ICG equivalent), led to
significant reduction in tumor growth and prolonged survival
due to a synergistic PTT/PDT effect.
Gelatin. Gelatin, a water-soluble macromolecule obtained by
partial hydrolysis of porcine or bovine collagen, is a
proteinaceous polymer widely utilized as a food additive.
They make good drug carrier systems because of their
biocompatibility, relatively low antigenicity, and the presence
of free amino groups on their surface which can be exploited for
surface modification with target molecules.124 Babu et al.
explored the potential of PEG modified-gelatin nanoparticles
loaded with hypocrellin B (HB) for PDT.125 Despite being an
efficient PDT agent, problems like poor drug loading and rapid
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Chemical Reviews
drug release from the gelatin nanoparticles called for further
improvements to this carrier system. These issues were
addressed in a subsequent paper by the same group, in which
they developed a carrier system based on PEG modified-gelatin
nanoparticles associated with hydrophobic PLA biopolymers
and loaded with hypocrellin B (HB).126 Here, incorporation of
PLA was considered to support enhanced loading of hydro-
phobic drugs, and thereby the therapeutic efficacy of the
associated hydrophobic PS. The average size of the nanocarriers
were comparatively smaller, with relatively better drug loading
capabilities exhibiting slow release of drug from the nano-
particles. Furthermore, the HB loaded nanocariers evoked a
superior phototoxicity compared to free-HB in vitro.
5.1.2. Nonbiodegradable Nanoparticles. Due to their
inability to degrade and release drugs in a controlled manner,
nonbiodegradable nanoparticles are not traditionally used as a
drug carrier. However, nonbiodegradable nanoparticles have
gained much attention in the field of PDT as multifunctional
theranostic carriers due to their unique characteristics such as
optical properties and tunability in its shape, size, porosity, etc.
They usually include inorganic, metallic, or composite nano-
particles that may not degrade readily in the biological system
when compared to polymeric biodegradable nanoparticles.
While, the latter undergo degradation into biologically benign
components, nonbiodegradable nanoparticles do not degrade
readily and do not release the accompanying PS at the time of
their activation but allow the generated 1O2 to diffuse out of the
carrier. Thus, these nanoplatforms can be considered as a
modular system that contains thousands of PS molecules,
bringing them as a single, nondepleting synergistic unit to the
tumor cells.
5.1.2.1. Silica Nanoparticles. For application in PDT, silica
based nanoparticles such as Stö ber silica nanoparticles,
organically modified silica (ORMOSIL), and mesoporous silica
nanoparticles (MSN) are frequently used. These nanoparticles
are quite promising as vectors for PDT applications due to its
chemical inertness, transparency of the matrix to light
absorption and porosity that is not susceptible to swelling or
alterations with a varying pH. They can be prepared using a
variety of precursors and many routes of synthesis are available
offering great flexibility. The nanoparticles are tunable in terms
of their size, shape, porosity, and dispersibility. Furthermore,
they can be surface modified with a variety of functional
moieties, polymers such as PEG, or targeting biomolecules for
tumor-cell targeting.
Spherical and monodisperse silica nanoparticles are generally
prepared following the sol−gel procedure proposed by Stöber
et al. in 1968.127 By this method, silica nanoparticles of different
sizes ranging from 50 nm to 1 μm can be prepared from
aqueous alcohol solutions of silicon alkoxides in the presence of
ammonia as a catalyst, where the size of particles depends on
the type of silicon alkoxide and alcohol. This method has
several advantages such as the synthesis may be carried out at
low temperature, as well as desired pH to yield high purity
products and the reaction kinetics of the process may be
controlled by varying the composition of the reaction mixture.
ORMOSILs, on the other hand, are prepared from the
precursor alkoxy organosilane in an oil-in-water microemulsion,
through a complex purification process. The presence of both
hydrophobic and hydrophilic groups on the precursor helps
them to self-assemble as both normal micelles and reverse
micelles under appropriate conditions, giving rise to nano-
particles of about 160 nm in size. ORMOSILs can possibly be
P DOI: 10.1021/cr5004198
Review
degraded through biochemical decomposition of the silicon−
carbon bond128 and presence of the organic group confers
some degree of flexibility to the otherwise rigid silica matrix,
which enhances the stability of such particles in aqueous
systems. MSNs are typically synthesized by sol−gel processes in
the presence of a surfactant such as C12-trimethylammonium
bromide or C16-trimethylammonium bromide, to control pore
sizes.
In the 2010 review by Couleaud et al., the different method
of synthesis of silica nanoparticles and the two main PS loading
strategies (noncovalent and covalent) are discussed in detail.129
Physical entrapment of PS inside the silica network may lead to
premature release of the PS from the carrier resulting in
reduced treatment efficiency and side-effects. Covalent coupling
of the PS inside or at the surface of the nanoparticles is
expected to overcome these drawbacks. One of the first reports
in this field by Prasad et al. discussed the synthesis of 30 nm
ORMOSIL nanoparticles encapsulating water insoluble
HPPH.130 The entrapped PS was found to generate 1O2
efficiently upon irradiation, due to the inherent porosity of
the nanoparticles. The fine pores (0.5−1 nm in diameter) in
the ORMOSIL nanoparticles are small enough to block drug
release but permeable to molecular oxygen and the generated
1
O2. In a subsequent study by the same group, the PS
iodobenzylpyropheophorbide was covalently incorporated to
the ORMOSIL nanoparticles (20 nm), to avoid premature
release of PS often associated with noncovalent encapsulation
of PS.131 Again, the incorporated PS molecules retained their
spectroscopic and functional properties and could generate 1O2
robustly upon photoexcitation. This was followed by a series of
studies using ORMOSIL nanocarriers embedding different PS
such as phthalocyanine 4 (Pc4),132 PpIX,133 and mTHPC.134
Tang et al. compared PS loading and PDT efficiency of
hydrophilic methylene blue (MB) in 20−30 nm hydrophilic
polyacrylamide nanoparticles, 190 nm sol−gel silica nano-
particles and 160 nm ORMOSIL nanoparticles.135 As expected,
MB loading in the smaller polyacrylamide nanoparticle was
found to be the lowest. The more spacious and negatively
charged sol−gel nanoparticles demonstrated higher loading of
the positively charged MB compared to ORMOSIL nano-
particles that has a unique internal hydrophilic and external
hydrophobic layer. Although, the Stöber nanoparticles gen-
erated a higher amount of 1O2 delivered per milligram of
nanoparticle due to their high PS loading efficiency, the
reaction of the generated 1O2 with ADPA after irradiation at
650 nm showed a higher kinetic rate with ORMOSIL
nanoparticles than with Stöber method.
MSNs possess large surface area and high accessible pore
volume, making them very suitable as drug delivery agents.
Qian et al. reported the synthesis of ultrasmall (25 nm) and
highly monodispersed MSNs encapsulating PpIX.136 These
nanoparticles were efficiently taken up by HeLa cells and
caused cell-death via necrotic pathway upon photoexcitation of
PpIX at 532 nm light within 8 min post irradiation. Recently,
targeted delivery of such PS loaded multifunctional MSNs are
gaining interest. Brevet et al. developed mannose-functionalized
MSNs encapsulating a water-soluble anionic porphyrin, for
cancer cell specific targeting.137 These nanocarriers presented
much higher photokilling efficiency in MDA-MB-231 cancer
cells in vitro, through mannose dependent endocytosis of the
functionalized nanocarriers. However, when physically ad-
sorbed into the mesoporous structures of MSNs, there is a
high probability of PS molecules being prematurely released
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Chemical Reviews Review

Figure 10. (A) Synthetic process of Ce6-doped mesoporous silica nanorods (CMSNRs). (B) TEM images of CMSNRs (the upper row). Those
samples with different aspect ratios were prepared by adding increasing amount of Ce6. (C) Schematic illustration for of the loading of DOX into
CMSNRs. (D) Relative viabilities of 4T1 cells after incubation with different concentrations of Ce6 and CMSNR4 under 660 nm light irradiation (5
mW cm−2, 1 h). (E) Relative viabilities of 4T1 cells after incubation with various concentrations of free DOX and CMSNRs4/DOX in the dark. (F)
Relative viabilities of 4T1 cells after treated with CMSNRs4 plus light, CMSNRs4/DOX in the dark, and CMSNRs4/DOX plus light (660 nm, 5
mW cm−2, and 1h). P values were calculated by Tukey’s post-test (***p < 0.001, **p < 0.01, or *p < 0.05). (G) Tumor growth curves of different
groups of mice after various treatments indicated (5 mice per group). Error bars were based on standard errors of the mean. Reproduced with
permission from ref 141. 2014 Springer-Verlag Berlin Heidelberg.

from the carrier, leading to a reduced efficiency of PDT. Zhang
et al. developed multifunctional core−shell structured nano-
carrier composed of a nonporous FITC dye-doped silica core
and a mesoporous silica shell containing PS, hematoporphyrin
(HP).138 Such a design allowed simultaneous fluorescence
imaging and PDT. The HP molecules were covalently linked to
the mesoporous silica shell, exhibited excellent photo-oxidation
efficiency and cell imaging abilities. However, cell killing
efficiency following PDT was not tested in vitro. Teng et al.
Q DOI: 10.1021/cr5004198
synthesized a folate conjugated phospholipid-capped PpIX-
loaded and FITC-sensitized mesoporous silica nanocarriers, for
targeted intracellular delivery of the nanocarriers in folic-acid
receptor overexpressing HeLa cells.139 The developed nano-
construct was found to be superior to free PpIX in terms of its
intracellular delivery efficiency and decreased dark toxicity in
vitro. In vivo PDT at 630 nm following intratumoral injection
of folate tagged nanoconstruct in nude mice bearing B16F10
melanoma tumors, caused around 65% destruction of tumors.
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Chemical Reviews Review

Figure 11. (A) Design of PEG-SiPc4-AuNP. (B) In vivo fluorescence imaging of PEG-SiPc4-AuNP conjugate injected mouse at various time points
within 24 h. Arrows indicate tumor location. (C) Assessment of PEG-AuNP-SiPc4 biodistribution on Au content evaluated by GFAAS. (D)
Histology studies of the organ tissues from mice injected with PEG-AuNP-SiPc4 after 4 h, 24 h, and 7 days postinjection. The black and brownish
spots indicate AuNPs in the tissues. Images were captured at 40× magnification. Scale bar represents 100 μm. (E) fluorescence images of organs
from the injected mice showing biodistribution and elimination of PEG-SiPc4-AuNP based on Pc 4 fluorescence. Reproduced with permission from
ref 146. 2011 American Chemical Society.

Another dual functional nanoconstruct based on mesoporous
silica composite nanoparticles was fabricated by Zhao et al. for
the codelivery of tetra-substituted carboxyl aluminum phthalo-
cyanine (AlC4Pc) for PDT and small Pd nanosheets for
PTT.140 While the PS was covalently conjugated to a
mesoporous silica network, the Pd nanosheets were coated
on the surface of mesoporous silica via coordination and
electrostatic interaction. As both AlC4Pc and Pd nanosheets
displayed maximum absorptions in the 600−800 nm region, the
fabricated 170 nm nanocomposites could be excited at the same
time using a single 660 nm laser irradiation. Viability of HeLa
cell incubated with the dual functional nanocomposites (200 μg
mL−1 for 12 h) dropped to below 35% following 10 min
irradiation at power density of 0.5 W cm−2, which was
significantly higher than the means of either PTT or PDT, thus
displaying a synergistic effect.
Very recently, Yang et al. reported an interesting approach
for precisely modulating the morphology of MSNs by varying
the amount of organic PS molecules covalently doped in the
silica matrix (Figure 10).141 In their study, the increase in the
R DOI: 10.1021/cr5004198
amount of Ce6 during the synthesis step was found to change
the shape of the MSN from spheres to rods with aspect ratios
ranging from about 1.2 to 4.6. A chemotherapeutic drug DOX
was then noncovalently adsorbed on the mesoporous structure
of MSNs. The Ce6-doped mesoporous silica nanorods
(CMSNR) with the highest aspect ratio was found to have
highest Ce6 loading efficiency of 7.51% (w/w) as well as faster
uptake in to the cancer cells. In vitro PDT using CMSNR
under 660 nm (5 mW cm−2 for 1 h), showed comparable or
slightly better cell killing effects compared to free Ce6.
Moreover, the synergistic effect of CMSNR/DOX was found
to be much stronger than the monotherapies both in vitro and
in vivo. The tumor growth inhibition in 4T1 tumor models
following intratumoral injection of CMSNR/DOX and 660 nm
light irradiation (5 mW cm−2 for 1 h) was 60%, compared to
42%, 12%, and 27%, for Ce6+DOX (light), CMSNR (light),
and CMSNR/DOX (light) groups, respectively.
5.1.2.2. Gold Nanoparticles. Gold nanoparticles (AuNPs)
possess a combination of unique properties such as the surface
plasmon resonance (SPR) effect and the ability to convert light
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Chemical Reviews
energy to heat for PTT applications, due to which they have
gained much popularity in the development of combinatorial
hyperthermia-phototherapy agents. Moreover, due to localized
surface plasmon resonances, field enhancement of the incident
light around AuNPs could be used to increase the excitation
efficiency of the accompanying PS. Russell et al. was the first to
report the synthesis of a three-component (PS/gold/phase
transfer reagent) system, where a phase transfer reagent
(tetraoctylammonium bromide; TOAB) was utilized to stabilize
phthalocyanines (Pc) on AuNPs.142 The developed 2−4 nm
construct generated 1O2 with enhanced quantum yields
compared to the free Pc, due to the presence of TOAB that
helps to increase the triplet energy transfer to molecular
oxygen. In a follow-on work, they reported in vitro uptake and
photodynamic efficiency of these AuNPs in HeLa cells.143 The
AuNP conjugates were effectively taken up by the cancer cells,
and resulted in greater than twice the amount of cell-death
(57%) compared to free Pc (26%), which could probably be
attributed to the 50% enhancement of 1O2 quantum yield
observed for the Pc-AuNP. Subsequently, in vivo PDT efficacy
at 600−700 nm (157 J cm−2), following intravenous injection
of C11Pc (phthalocyanine derivative) conjugated AuNP was
studied in amelanotic melanoma (B78H1 cells) subcutaneously
transplanted on mice.144 Compared to free C11Pc, AuNP-
C11Pc conjugates were found to target cancer tissues more
selectively and to induce more extensive PDT response by
promoting an antiangiogenic response by causing extensive
damage to the blood capillaries and the endothelial cells.
However, the AuNP-C11Pc conjugates were taken up by liver
and spleen, with a prolonged persistence in the liver without
any apparent decrease of the PS, for up to a week. Thus,
suitable strategies to limit the accumulation of the nanoparticle-
associated PS in important organs such as liver and spleen
should be devised. As a subsequent step the same group
reported targeted delivery of PEGylated AuNP-C11Pc
conjugates to breast cancer cells, by attaching anti-HER2
monoclonal antibodies to the PEG chain (HS-PEG-COOH
3000).145 In vitro experiments demonstrated selective targeting
of the 4-component “antibody-C11Pc-PEG-AuNPs” conjugate
to breast cancer cells that overexpress HER2 epidermal growth
factor receptor and its efficacy in PDT applications, although
no in vivo results were presented.
Cheng et al. evaluated covalent and noncovalent attachment
of silicon phthalocyanine 4 (SiPc4) on PEGylated AuNP and
found that, in contrast to slow intracellular release and reduced
drug efficacy of covalently attached PS, noncovalent adsorption
showed efficient drug release into HeLa cancer cells by
membrane-mediated diffusion.147 Subsequently, they explored
the drug delivery mechanism and pharmacokinetics following
intravenous administration of noncovalently bound PEG-SiPc4-
AuNP conjugates over a period of 7 days.146 In vivo
experiments revealed enhanced accumulation of SiPc4
molecules in tumor sites of cancer-bearing mice and dramatic
decrease of the drug delivery time from 2 days to 2 h (Figure
11). Moreover, noncovalent attachment of PS to AuNP
resulted in efficient release and penetration of the PEG-
SiPc4-AuNP conjugate fast and deep into the tumors. Although
Au concentration in most tissues reached its maximum by 4 h
followed by its gradual decrease over time, Au concentration in
liver and spleen continued to accumulate over time to reach a
maximum at the latest time point measured, 7 days.
Biodistribution results suggested that the PEG-SiPc4-AuNP
conjugates were removed from both the cardio−pulmonary and
S DOI: 10.1021/cr5004198
Review
immune systems and were channeled into the excretory organs
where it was finally removed from the body by renal and
hepatobiliary routes. As seen with other nanocarriers, develop-
ment of multifunctional AuNP offering synergistic therapeutic
approaches such a photothermal and PDT, has been the new
trend in this field. The earlier reports however used two
different excitation wavelengths to accomplish PTT and PDT.
For example, Jang et al. demonstrated a 95% reduction in
tumor growth in vivo using a gold nanorod AlPcS4 complex
excited by 810 and 670 nm lasers.148 Khlebtsov et al. developed
composite nanoparticles consisting of a gold−silver nanocage
core and a mesoporous silica shell functionalized with the PS
(Yb-2,4-dimethoxyhematoporphyrin) for in vivo PDT applica-
tions.149 The synthesized nanocomposites generated 1O2 under
excitation at 630 nm and produced heat under laser irradiation
at the plasmon resonance wavelength (750−800 nm). In order
to combine and simultaneously activate the two treatment
approach, Gao et al. developed a nanocomplex using a lipid-
loaded HB to coat gold nanocages.150 Such an assembly,
combined with excitation using a 790 nm NIR two-photon
laser, resulted in one-off administration and excitation of both
the gold nanocage, that convert light to heat energy and the
accompanying PS, that generate ROS simultaneously. Similarly,
Seo et al. recently developed MB loaded mesoporous silica
coated gold nanorod for use in cancer imaging and PTT/PDT
dual therapy.151 The PS was physically adsorbed on the pores
of the silica shell. The excellent plasmonic properties of gold
nanorods at NIR, enhanced the NIR light-induced SERS
performance of the embedded PS molecules by a factor of 3.0 ×
1010. Upon irradiation with NIR light at a single wavelength
(780 nm), the viability of CT-26 cells decreased to 31% for
cells transfected with bare gold nanorods, while it dropped to
11% for the MB loaded nanocomposites, clearly demonstrating
the advantage of synergistic combination of the photothermal
and photosensitizing effects. It is to be noted that, unlike PDT
which is dependent on the availability of tissue oxygen, the
PTT effect is oxygen-independent and hence the hypoxia
environment induced by PDT reaction does not affect PTT. In
their excellent review, Liu et al. presented a very detailed and
in-depth account of various nanoparticles used in PTT and
PDT, as well as combination of PDT and PTT.152 Wang et al.
hypothesized that by adjusting irradiation time, an early phase
PDT effect can be coordinated with a late-phase PTT effect to
achieve a better synergistic treatment efficacy.153 They
synthesized a PEGylated gold nanorod, with Ce6 molecules
chemically conjugated to the PEG chains. The utility of the
nanoconjugate was tested both in vitro and in vivo on MDA-
MB-435 tumor bearing mice model. The tumor was exposed to
671 nm laser (1.0 W cm−2) for 6 min, 4 h post intratumoral
administration of the nanoconjugates. A significant reduction in
tumor volume was achieved post laser treatment with Ce6
loaded PEGylated gold nanorods in comparison with free Ce6
or bare PEGylated gold nanorods, demonstrating significantly
improved anticancer effect of the combinatorial approach.
5.1.2.3. Magnetic Nanoparticle. With further improvement
in the field of nanotechnology and nanomedicine, conceptu-
alization and development of multifunctional theranostics
probes became the next big wave in the field of PDT. Now,
the success of PDT depends on choosing the apt time at which
the light irradiation is to be performed when there is the highest
concentration of the PS in the tumor compared to the normal
tissue. This could be realized by using noninvasive visualization
techniques that allow quantification of PS within the tumor.
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Chemical Reviews Review

Figure 12. (A) Schematic illustration for the synthesis and structure of IONC−PEG−Ce6. (B) SEM image of as-synthesized IONCs. Inset: TEM
image of IONCs. (C) T2-weighted MR images and T2 relaxation rates (R2) of IONC−PEG aqueous solutions at different Fe concentrations. (D)
UV/vis/NIR spectra of IONC−PEG, free Ce6, and IONC−PEG−Ce6 at different feeding Ce6 concentrations. (E) Fluorescence images of IONC−
PEG, IONC−PEG−Ce6, and Ce6 aqueous solutions at different excitation wavelengths (661 and 704 nm). The exposure time was 100 ms. (F)
Relative viabilities of 4T1 cells treated with various concentrations of IONC−PEG, Ce6, and IONC−PEG−Ce6 with light irradiation. (G) Tumor
growth curves of different groups of tumors after various treatments indicated. Error bars were based on SD of six tumors per group. MF: magnetic
field; L: light. (H) Representative photos of mice after various treatments. White and red arrows point to tumors without and with magnetic
targeting, respectively. Reproduced with permission from ref 159. 2011 American Chemical Society.

Magnetic resonance imaging (MRI) is one such promising
noninvasive, harmless (no radiation exposure) visualization
technique, in terms of its high spatial resolution and anatomical
definition.154 In order to visualize the tumor and quantitate the
amount of PS uptake in the tumor, the PS was conjugated to a
MRI contrast agent and encapsulated in a nanocarrier. Among
the broad spectrum of nanoscale materials available, iron oxides
exhibiting superparamagnetism, have been most commonly
used in conjunction with PDT. Generally, the magnetic
nanoparticles in the core are shielded from the surrounding
environment with polymers like PEG, dextran, chitosan, PEI, or
T DOI: 10.1021/cr5004198
phospholipids. The polymeric shell also serves as a suitable
surface to further conjugate it with various biomolecules for
targeted delivery. In 2005, Kopelman et al. combined MRI
contrast enhancement with targeted PDT, by constructing PAA
core nanoparticles encapsulating clinical PS, Photofrin, and iron
oxide.155 The nanoconstruct was further surface functionalized
with PEG and integrin-targeting RGD peptide to enable
simultaneous cancer targeting, detection, therapy, and monitor-
ing. A similar technique was adopted by Reddy et al. in which a
vascular homing peptide, F3 was conjugated to a PAA core
nanoparticle encapsulating Photofrin and iron-oxide/fluores-
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Chemical Reviews Review

Figure 13. Multifuntional theranosomes (A) Left: MRI scans of TC-1 tumors before and 48 h after PBS intratumoral injection (control group).
Right: MRI scans of tumors before and 48 h after theranosome intratumoral injection. (B) Normalized tumor growth curves in mice intratumorally
injected with theranosomes (25 μM m-THPC) or m-THPC (25 μM) followed by light exposure (λ = 630 nm, 30 J cm−2, 77 mW cm−2 for 390 s) 20
h later. All error bars reflect the SEM (n = 5). * and ** indicate p < 0.05 and p < 0.01, respectively, versus untreated control at day 7. (C) False-color
images of fluorescence emission from nude mice bearing TC-1 tumor before and after theranosome or m-THPC intratumoral injection, as a function
of time. Reproduced with permission from ref 163. 2011 American Chemical Society.

cent imaging agent.156 In both studies, the presence of targeting
agent enhanced the ability of the nanoconstruct to specifically
target the tumors as revealed by MRI, thus increasing the
efficiency of tumor killing post PDT. The ease of synthesis of
silica nanoparticles with desired shape, size, and porosity, as
well as its permeability to 1O2 has allowed incorporation of
magnetite nanoparticles and PS into silica-based nanoparticles.
Chen et al. described the synthesis of a multifunctional silica
nanoparticles (20−30 nm) encapsulating a porphyrin derivative
and iron oxide nanoparticles by microemulsion and sol−gel
methods, with a PS encapsulation efficiency of 20.8%.157 The
nanoconstruct possessed good biocompatibility and 1O 2
generating ability, to cause remarkable photodynamic anti-
tumor effects. However, its ability as a MRI contrast agent was
not proven in vivo. Benachour et al. utilized active targeting of
small silica-based nanoparticles with gadolinium oxide core for
MRI and a chlorin based PS, as well as a peptide targeting
neuropilin-1 (NRP-1) overexpressed in tumor vasculature.158
In vivo studies on rats bearing an orthotopic human brain
tumor (U87) presented tumoritropic accumulation of the
nanoparticles, following intravenous administration due to the
presence of tumor vasculature targeting peptides, as revealed by
real-time MRI analysis.
Around this time, use of physical forces such as an external
magnetic field (MF) for enhancing the tumor localization
U DOI: 10.1021/cr5004198
emerged as a new tumor-targeting strategy.160 In this technique,
systemically applied magnetic nanoparticles carrying therapeu-
tic agents would be attracted by the MF applied on the tumor,
resulting in enhanced enrichment of therapeutic agents in
targeted tumor region, consequently resulting in a significantly
improved therapeutic effect compared to passive tumor
targeting that is simply based on the EPR effect. Additionally,
magnetic targeting may be applicable to a wide range of solid
tumors regardless of the expression of cancer linked receptors/
markers, unlike active tumor targeting strategies that is based
on the ligand−receptor binding. Along these lines, Sun et al.
synthesized porphyrin derivative encapsulated chitosan shell
surrounding an iron oxide nanoparticle via chemical copreci-
pitation method.161 The core−shell structured nanoparticles
with a size range of 20.6 ± 5.1 nm and PS encapsulation
efficiency of ∼65.4%, were administered intravenously to
xenograft mouse tumor model. The magnetic nanoparticles
were targeted to the tumor by externally localized magnets and
PDT was performed when a typical low intensity T2 weighted
image was observed in tumor area, thereby achieving excellent
tumor targeting, imaging and therapeutic efficiency in vivo.
Huang et al. prepared magnetic nanoparticles covalently
conjugated with Ce6 for simultaneous targeted PDT and in
vivo dual-mode imaging (NIR fluorescence imaging and
MRI).162 The synthesized nanoparticles with a size range of
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10 ± 2 nm and PS loading efficiency of ∼88.68%, had good

biocompatibility and remarkable photodynamic efficacy upon
irradiation. Furthermore, the nanoparticles were found to target
the tumor with an externally applied MF and their
biodistribution could be optically followed by NIR fluorescence
imaging. Similarly, Li et al. reported of 100 nm sized iron oxide
nanoclusters (IONC) exhibiting superparamagnetic property,
functionalized with PEG (Figure 12).159 Further loading of Ce6
was found to red shift the absorption peak of Ce6 at 650 to 700
nm. In vitro PDT at under 704 nm light irradiation (5 mW
cm−2 for 30 min, 9 J cm−2) showed remarkable concentration
dependent phototoxicity, owing to the enhanced uptake of
IONC-PEG-Ce6 compared to free Ce6. Additionally, enhanced
targeting of IONC-PEG-Ce6 post IV administration in a 4T1
mouse tumor model was achieved by subjecting the tumor to
MF, as confirmed by the reduction in the averaged T2 signal
intensity by 94.4% as compared to only 11% in the tumor that
was not subjected to MF. In vivo PDT of MF controlled tumor
at 704 nm (5 mW cm−2 for 1.5 h, 27 J cm−2) exhibited
significant tumor growth delay compared to the tumor that was
not subjected to MF, revealing highly effective tumor homing
ability of the multifunctional magnetic PDT agent.
Recently Silva et al. was the first to report proof of principle
of designing a natural, cell-derived, submicrometer vesicle as
multifunctional theranostic nanovector, named therano-
somes.163 Their strategy involved engineering of the parent
cells to enclose both iron oxide nanoparticles and a PS (m-
THPC), which were then stimulated to generate nanovesicles
(mean vesicle size of 332 ± 94 nm derived from transmission
electron microscopy (TEM)) laden with both agents conferring
magnetic and optical responsiveness, allowing therapeutic and
imaging functions. The MR scans following intratumoral
injection of theranosome at a density of 3 × 108 per μL
containing m-THPC concentration of 25 μM and iron
concentration of 5 mM exhibited large zone of hypointense
signal within the tumor, confirming its intratumoral loacaliza-
tion (Figure 13). At the same time, fluorescence imaging Figure 14. Schematic illustration of photodynamic reaction upon light
revealed an increase in fluorescence signal 20 h after injection, irradiation on (A) fullerene and (B) TiO2 nanoparticle. (•OH,
for both theranosomes and free m-THPC, perhaps due to m- hydroxyl radical, O2•−, superoxide anion, HO2•−, hydroperoxyl radical
and H2O2, hydrogen peroxide).
THPC diffusion toward the tumor surface. However, signals
from theranosomes were restricted to the tumor site while
extratumoral extravasation was observed for the free drug. In their extended π-conjugation, fullerenes absorb light in the
vivo PDT following intratumoral administration of therano- ultraviolet (UV) or blue regions of electromagnetic spectrum to
somes in TC-1 tumor bearing mice revealed significant form long-lived triplet state and generate ROS upon
reduction in tumor growth compared to nontreated control illumination, allowing them to act as PSs. In the recent years
at day 7. However, systemic administration of theranosomes its potential as PS has been widely studied due to several
needs to be addressed to gain insight in the translational advantages. Fullerenes are comparatively more photostable and
potential of this approach. not easily susceptible to photobleaching.164 They undergo both
Type I and Type II reactions generating both free radicals and
5.2. Nanoparticles as Downconverting Photosensitizers 1
O2. Furthermore, they do not breakdown and are excreted
Certain nanoscale materials have the ability to generate ROS, intact. However, the chief disadvantage of fullerenes is their
due its unique optical absorption properties, thus allowing them optical absorption properties; as they absorb light where the
to behave as PSs by itself. In the following section, the tissue penetration depth of light is shortest due to a
properties and application of three such nanomaterials are combination of light absorption by endogenous chromophores
discussed. and light scattering by cellular structures. Second, they are
5.2.1. Fullerenes. Buckminsterfullerene (or bucky-ball) is a innately insoluble in water, due to which they severely
carbon allotrope discovered in 1985, with a spherical shape and aggregate in aqueous conditions, hampering its biological
usually composed of 60 (C60) or 70 (C70) carbon atoms application. This shortcoming of the fullerenes have been
(Figure 14A). It has a hollow cage-like fused structure with sp2 overcome by surface modification or supramolecular ap-
carbon atoms arranged in hexagons and pentagons. The proaches such as PEGylation,165 encapsulating in liposomes,166
diameter of the fullerene cage is approximately 7−10 Å, thus micelles,167 or chitosan.168
making it less likely to be taken up by the macrophage cells As with other nanoparticles used in PS delivery, fullerenes
compared to other larger conventional nanoparticles. Due to have also been modified to carry imaging agents to bestow
V DOI: 10.1021/cr5004198
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Chemical Reviews
multifunctionality. Liu and others developed a theranostic
hybrid system in which diethylenetriaminepentaacetic acid
(DTPA) was attached to the terminal group of PEGylated C60
(C60-PEG-DTPA) and subsequently mixed with gadolinium
acetate solution to obtain Gd3+-chelated C60−PEG−DTPA−
Gd.165c Upon light irradiation (400−500 nm, 53.5 J cm−2)
following intravenous administration of C60−PEG−DTPA−
Gd into tumor-bearing mice, there was significant antitumor
PDT effect, which correlated with tumor accumulation as
detected by the enhanced intensity of MRI signal. Recently,
functionalized fullerenes targeted to cancer cells have been
developed and its potential in PDT is being investigated. In
2013, Liu et al. developed trimalonic acid-modified C70
fullerenes (TF70) conjugated with aptamer R13, specifically
targeting epithelial growth factor receptors (EGFR) overex-
pressed in certain cancer cells.169 Incubation of R13 conjugated
TF70 with A549 lung cancer cells, displayed good binding
ability to the EGFR expressing A549 cells. Further studies also
revealed preferential localization of the fullerene conjugate in
the lysosome and facilitated efficient cell-kill under illumination
with white light. In a yet another approach, malonic acid C60
derivative (DMA-C60) modified with DSPE-PEG2000-malei-
mide was conjugated to Asn-Gly-Arg (NGR) peptide, that
selectively targets CD13 isoform overexpressed in tumor
vasculature and certain tumor cells.170 2-Methoxyestradiol
(2ME), a nonpolar endogenous metabolite of 17β-estradiol
exhibiting antiproliferative and antiangiogenic activities, was
then attached to DMA-C60-NGR through physisorption, to
form a novel tumor-targeting drug delivery system DMA-C60−
2ME-NGR for synergistic enhancement of cancer therapy. The
presence of targeting peptide enhanced cell uptake of DMA-
C60−2ME-NGR that localized in the cytoplasm and also
promoted a faster 2ME transfer into cells, exerting a stronger
inhibition on MCF-7 breast cancer cells. The results revealed a
synergistic enhancement of breast cancer therapy using PDT
induced by DMA-C60-NGR and 2ME.
5.2.2. Titanium Dioxide Nanoparticle. Titanium dioxide
(TiO2), also known as titania, is the naturally occurring oxide of
titanium. Ever since the discovery of photoinduced decom-
position of water on TiO2 electrodes under UV light was
made,171 it has found multifunctional applications which
underpin many important energy-conversion processes, includ-
ing photocatalysis, photodegradation, environmental remedia-
tion, water splitting for hydrogen fuel, CO2 reduction, self-
cleaning coatings, electrochromic devices, sensors, and low-cost
solar cells.
In recent years, TiO2 has been regarded as potential
photosensitizing agent for PDT due to its low toxicity,
physiological inertness, excellent biocompatibility, and unique
photocatalytic activity.172 Under irradiation with UV light with
energy greater than the band gap energy of TiO2 (i.e., shorter
than 385 and 400 nm corresponding to a band gap energy of
3.23 and 3.06 eV for anatase and rutile polymorph of TiO2
respectively), the electrons in the valence band of TiO2 are
excited to the conduction band, thus resulting in the formation
of photoinduced hole−electron pairs (Figure 14B). These
photoinduced electrons and holes possess strong reduction and
oxidation properties that can interact with surrounding O2 and
H2O molecules to generate various ROS, such as hydroxyl
radical (OH•),173 superoxide anion radical (O2•−)174 and
hydrogen peroxide (H2O2),175 which react with biological
molecules, such as lipids, proteins, and DNA, and eventually kill
cancer cells.176 Fujishima et al. was the first to report
W DOI: 10.1021/cr5004198
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photokilling of malignant HeLa cells grown on a polarized
illuminated TiO2 film electrode.177 Although the initial
experiments showed successful photocatalytic cell killing, the
use of a TiO2 electrode system was not considered to be
feasible in vivo. Hence, a series of in vitro studies on HeLa cells
followed using colloidal TiO2 particles in suspensions, as it
could be incorporated by the living cells and had extremely
large surface area, thus resulting in higher photocatalytic
reaction rates.172c,178 Subsequently, the detailed mechanism of
UV induced phototoxic effect of TiO2 nanoparticles was
reported on a series of human cancer cells such as bladder
cancer cells (T24),179 monocytic leukemia cells (U937),180
adenocarcinoma cells (SPC-A1),181 colon carcinoma cells (Ls-
174-t),182 breast epithelial cancer cells (MCF-7, MDA-MB-
468),176b and glioma cells (U87).183
Only a few studies have reported the in vivo application of
TiO2 nanoparticles for PDT in animal models. Generally, the
TiO2 particles were either administered intratumorally or
subcutaneously over the tumor tissue, rather than intravenous
delivery due to its insolubility and aggregation issues in
physiological environment, which means that it could be easily
recognized and removed from the circulation by the cells of the
MPS and ultimately reduce its accumulation in the tumor.
Following administration of TiO2 nanoparticles, the tumors
were irradiated with UV light after the skin was surgically
removed to expose the tumor. This is because the penetration
depth of UV light (300−400 nm) into the tissue is limited to a
few fractions of a millimeter.184 Cai et al. reported significant
suppression of HeLa cells implanted in nude mice for up to 30
days, compared with those receiving TiO2 alone or UV
irradiation alone.172c Furthermore, when tumors received a
second repeat treatment of TiO2 and UV light irradiation on
day 13, the tumor growth was inhibited more dramatically.
Similarly, Kubota et al. also reported a drastic delay in T-24
bladder cancer tumor implanted in nude mice up to 30 days
post-PDT using TiO2 particles under UV light illumination.179
Likewise, Wang et al. found that the combination of TiO2 and
UV irradiation was more effective not only in reducing tumor
growth but also in promoting survival.183 However, it should be
noted that this technique was not effective in retarding a tumor
that had grown beyond a certain size.
5.2.3. Zinc Oxide Nanoparticle. Nanosized zinc oxide
(ZnO), a well-known photocatalyst comparable to TiO2, is
frequently found in industrial products including cosmetics,
paints, and medical materials. ZnO has almost the same band
gap energy (3.2 eV) as TiO2 and has a similar photocatalytic
activity. Li et al. compared the phototoxic effect of ZnO
nanoparticles of three different sizes (20, 60, and 100 nm)
following irradiation with the UVC (254 nm; 0.1 mW cm−2 for
180 s) and studied the synergetic cytotoxicity of the anticancer
agent daunorubicin (DNR) with ZnO nanoparticles against
hepatocellular carcinoma cells (SMMC-7721) in vitro.185 The
phototoxic effect was found to be size dependent. Meanwhile,
significantly greater cell-kill was achieved when SMMC-7721
cancer cells were treated with a combination of ZnO
nanoparticles and daunorubicin. In 2010, Hackenberg and co-
workers studied the photocatalytic effects of ZnO nanoparticles
on human head and neck squamous cell carcinoma (HNSCC)
cell lines and nonmalignant cells in vitro.186 Studies revealed
that ZnO nanoparticles at concentrations of 0.2 and 2 μg mL−1
in the presence of UVA (20 mW cm−2 for 15 min) presented
the optimal conditions for cancer cell-kill, mainly via a
combination of late apoptosis and necrosis. In addition to its
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photosensitizing capabilities, ZnO was also used as a delivery

agent of anticancer drugs for combination therapy. Hariharan et
al. fabricated PEGylated ZnO (ZnO/PEG) nanospheres loaded
with anticancer drug of DOX and studied both its antibacterial
and antitumor activity.187 The DOX loading and encapsulation
efficiency on ZnO/PEG nanospheres were found to be 70.35 ±
6.20% and 21.34 ± 2.4%, showing its potential as drug delivery
agents. DOX-ZnO/PEG nanocomposites proved to be capable
of photodynamically inactivating of Gram-positive micro-
organisms under visible light. Furthermore, the nanocomposites
exhibited dose-dependent toxicity toward HeLa cell lines, as
well as demonstrated improved therapeutic efficacy upon UV
irradiation, thus minimizing the side-effects.
5.3. Nanoparticles as Energy Transducers
Certain nanoparticles are not just carriers of PS but they also
actively participate in energy transfer to the attached PS.
Generally, the energy harnessed from light is then transferred
to the PS associated with it, thereby offering the possibility of
indirectly activating the PS using light at wavelengths that it
normally does not absorb.
5.3.1. X-ray Activatable Nanoparticle. The first report
on X-ray activatable nanoparticles was back in 2006 by Chen et
al., where they described a new approach to treat cancer
through a combination of radiation therapy and PDT, termed
as self-lighting PDT.188 The rationale behind this technique was
to utilize the stronger absorption of the porphyrins at around
400 nm (Soret band) rather than the weak absorption at
around 600−800 nm (Q-band). This is because excitation of
the PS at the Soret band can be much more efficient in the
generation of ROS. Unfortunately, attempting to activate PS
through absorption at the Soret band will compromise the
Figure 15. Schematic illustration of energy transfer from X-ray
clinical usefulness of PDT, as UV/blue light has minimal activatable scintillation nanoparticle to the attached PS.
penetration in tissues. Hence, red light (>600 nm) is often
utilized to activate the weak Q-band of the PS. In an effort to
utilize the strongly absorbing Soret band of PS at the UV/blue emitting strong luminescence.192 Again, nanoparticles with
region, scintillation or persistent luminescence (“after glow”) small diameters are preferred as they have a weak electron−
nanoparticles (e.g., BaFBr:Eu 2+ , Mn 2+ , LaF 3 :Ce 3+ , and phonon coupling and consequently exhibit greater lumines-
LaF3:Tb3+), with attached porphyrin PS were used as in vitro cence efficiency due to a smaller Stokes shift.193 And last, in
PDT agent. These nanoparticles upon exposure to X-rays emit order to have efficient energy transfer, the distance between the
luminescence in the visible region (peaking at 400, 500, and donor and the acceptor should be less than 10 nm.
650 nm) of the spectrum activating the attached PS at both the The highlight of this approach is that as there is no limitation
Soret band and Q-band, resulting in the efficient generation of to the penetration depth achievable by X-rays in tissue,
1
O2 (Figure 15).188,189 In a pilot study, the energy transfer theoretically any deep-seated or solid tumors can be treated
following X-ray irradiation of water-soluble LaF3:Tb3+ nano- effectively by photoactivation of the attached PS by the X-ray
particles to the attached meso-tetra(4-carboxyphenyl)porphyrin activatable nanoparticles. Moreover, the synergistic effect of low
(mTCP) was demonstrated by fluorescence quenching dose radiation therapy and PDT is expected to enhance the
techniques.190 In a very recent mechanistic study by the same cancer cell killing effect. Lastly, as the nanoparticles are self-
group, Ce3+-doped lanthanum(III) fluoride (LaF3:Ce3+) nano- illuminating especially if it emits an “after glow”, there is no
particles with strong green emission at approximately 520 nm need of an external light source, making this therapy highly site
upon excitation with X-rays, were encapsulated in poly(D,L- specific. Afterglow is the persistent luminescence that remains
lactide-co-glycolide) microspheres along with PpIX.191 Upon X- even after the X-ray irradiation is turned off. Generally, an
rays irradiation (90 kV), energy transfer from afterglow nanoparticle also has scintillation luminescence, but a
LaF3:Ce3+nanoparticles to PPIX resulted in 1O2 generation scintillation nanoparticle might not necessarily emit an
and cancer cell kill, due to oxidative stress, mitochondrial afterglow. Use of nanoparticles with such an “afterglow” and
damage and DNA fragmentation. Thus, the energy transfer long decay lifetimes could greatly reduce the amount of
from the X-ray scintillation nanoparticles to the PS is very radiation dose required for initial excitation of the scintillation
critical and this requires very good overlap between the nanoparticle and prolong the PS excitation period. However, in
emission peaks of the nanoparticles and the absorption or the earlier studies X-rays with energy in the kilovoltage range
excitation peaks of the PS. Second, such nanoparticles must were used, while beams with energy in the megavoltage range
have strong luminescence efficiency. Generally, higher atomic are normally used in the clinics for conventional radiotherapy.
number phosphors usually have greater radiation stopping It was not reported whether the scintillation nanoparticles
power and higher absorption coefficient for radiation, thus could be excited by megavolatage (MV) X-rays, and if then,
X DOI: 10.1021/cr5004198
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Chemical Reviews
Figure 16. Schematic illustration of energy transfer from nanoparticles to PS and corresponding simplified energy diagrams. (A) QD transferring
energy to PS via FRET, (B) gold nanorod as a two-photon absorbing nanoparticle.
there will be enough energy transfer to the conjugated PS for
significant cell killing. To address these critical questions, Yang
et al. developed a amine-functionalized PEGylated quantum dot
(QD-CdSe core with ZnS shell) conjugated with porphyrin and
studied the energy transfer and cell killing effect following
irradiation with 6-MV X-rays.194 A significant cell killing was
observed with combined radiation and PDT treatment
compared to radiation alone. The linear relationship between
the radiation dose rate and QD excitation allows prediction of
light dosimetry, which would be useful in the translation of the
novel conjugates for clinical application.
5.3.2. Quantum Dots. Semiconductor QDs holds great
promise in PDT applications. Depending on the size (ranging
from 1 to 6 nm) and composition, the QDs can have unique
optical and emission properties that can be precisely tuned
from the UV to the infrared region. The surface of QDs can be
modified and functionalized to make them both water-soluble
and biocompatible. Furthermore, they possess a large transition
dipole moment, making them strong light absorbers, ideal for
PDT applications. QDs could act as energy donors and transfer
the energy to cell molecules like triplet oxygen, reducing
equivalents or pigments, potentially inducing generation of
ROS leading to cell kill. Although Samia et al. reported that
cadmium selenide (CdSe) QDs could be a potential PS on its
own and generate 1O2, the quantum yield of CdSe-generated
1
O2 was rather low (5% versus 40−60% by conventional
PSs).195 However, the authors also reported that a more
efficient (77%) energy transfer via FRET mechanism occurs
between a QD and PS (Pc4) when excited at 488 nm, due to
the close steric proximity between the two structures (Figure
16A). Here, Pc4 was excited by CdSe-QD emission at 568 nm
and thus enabling the use of an excitation wavelength where the
PS alone does not absorb in a normal scenario. However,
insolubility of the conjugate in water hampered its application
in biological systems. Later Shi et al. fabricated water-soluble
QDs with porphyrin PS, meso-tetra (4-sulfunatophenyl)
porphyrin (TSPP) electrostatically attached to the QD.196
Upon excitation at 355 nm, at which the PS will otherwise not
absorb, the QD-PS hybrid nanocomposite was found to
generate 1O2. However, potential toxicity of synthesized
uncapped QDs was the major drawback. Tsay et al. coupled
two different PS, Rose Bengal (RB) and Ce6 to a peptide,
which was in turn attached to CdSe/CdS/ZnS QDs.197
Y DOI: 10.1021/cr5004198
Review
Although the QD by itself did not generate 1O2 and the
quantum yield of 1O2 both via direct excitation of PS or
indirectly through the FRET mechanism was relatively low, the
advantage of this technique was that several PS molecules could
be coupled to QD to improve the FRET efficiency. Recently,
Hsu and co-workers demonstrated that self-illuminating QDs
can serve as a versatile light source for activation of PS and may
overcome the limitation of light penetration for PDT.198 They
fabricated a Renilla luciferase-immobilized QDs (QD-RLuc8)
conjugate. After the addition of coelenterazine (the substrate of
RLuc8), energy is released from RLuc8 and transferred to QDs
via bioluminescence resonance energy transfer (BRET), leading
to self-illumination of QD-RLuc8 conjugate at 655 nm. This
bioluminescent photon emitted from QD-RLuc8 conjugate, in
turn excites the Foscan-loaded micelles and successfully
generated ROS achieving ∼50% cell kill in vitro and delayed
tumor growth in vivo.
5.3.3. Two-Photon Absorbing Nanoparticles. Certain
molecules with two photon absorption (TPA) cross-section can
simultaneously absorb two low-energy photons and emit higher
energy photons (Figure 16B). Such dyes offer new perspectives
for the treatment of solid tumors using PDT, as they can be
excited in the NIR region allowing deeper light penetration in
tissues. Two-photon PDT (TP-PDT) allows 3-dimensional
selectivity to precisely target tumor cells, thus preventing
damage to adjacent healthy tissues. Conventional PSs usually
have low TPA cross sections in the biological spectral window
(i.e., 700−1000 nm), requiring high excitation powers that are
close to the threshold of tissue photodamage. Thus, even
though the activation of some PSs such as ICG falls within the
NIR range (∼800 nm),199 their quantum yield of 1O2 remains
low due to insufficient triplet formation.200 Thus, most of the
initial studies combined TPA dyes with PS to convert the
energy emitted from TPA dyes to activate PS for PDT.
However, dark-toxicity of these dyes was often a major concern.
As a solution to this problem, Kopelman and co-workers
entrapped a TP dye 5,10,15,20-tetrakis(1-methyl 4-pyridinio)
porphyrin tetra(p-toluenesulfonate) (TMPyP) in PAA-based
nanoparticles.201 A different approach was adopted by Prasad
and co-workers, where they coencapsulated a TP dye, 9,10-
bis[4′-(4″-aminostyryl)styryl]anthracene (BDSA) and HPPH
in a ORMOSIL nanoparticles.202 Here, the BDSA acted as a
two-photon absorbing dye unit (energy donor) that indirectly
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Chemical Reviews
Figure 17. Schematic of energy transfer from UCN phosphor to the attached PS and corresponding energy diagram.
excited HPPH (energy acceptor), via FRET and cytotoxic effect
under two-photon irradiation (850 nm) was demonstrated.
Velusamy et al. synthesized quadrupolar type TPA chromo-
phores exhibiting remarkably large TPA cross section values of
about 7000 Goeppert−Mayer units (GM) at 800 nm in
toluene.203 To further improve its biocompatibility and
solubility in aqueous solution, the compound was encapsulated
in silica nanoparticles. In vitro PDT experiments at 800 nm
NIR irradiation for 3 min at 468 J cm−2 using macrophage cells
(Raw 264.7) killed about 40% of cells at a concentration of 100
μg mL−1. Gary-Bobo and co-workers demonstrated efficacy of
TP-absorbing porphyrin derivative covalently conjugated in
mannose functionalized biocompatible MSNs in an in vivo
model.204 TPA properties of the PS were retained in the MSNs,
with giant TPA cross sections (up to 8 MGM) for a single
MSN and induced significantly more cell death in vitro
following irradiation at 760 nm by three scans of 1s duration
each delivering a power of 80 mW. Intravenous administration
of 16 mg kg−1 mannose-functionalized porphyrin loaded MSNs
in HCT-116 xenograft mouse model, revealed that a single
injection was sufficient to target these nanoparticles to the
tumor area, while TP-PDT treatment at 760 nm induced a
major reduction of the tumor size. Thus, the initial studies
utilized nanoparticles as a carrier of TP absorbing dyes/PS,
rather than actively participating in TPA. Thereafter, nano-
particles having large TPA cross sections, were utilized for TP
excitation in a donor−acceptor system for indirect excitation of
PS and one such example is QD-PS conjugate. Dayal et al.
conjugated Pc4 with CdSe QDs and demonstrated successful
energy transfer to PS following two-photon excitation of the
QDs at 1100 nm.205 Wen et al. fabricated water-soluble CdTe
QD as an energy donor to indirectly excite electrostatically
attached mesotetra-4-sulfonatophenyl-porphine dihydrochlor-
ide (TSPP), with an energy transfer efficiency of 82%,
demonstrating its potential in TP-PDT.206 Qi et al. covalently
attached water-soluble porphyrin on the biocompatible
amphiphilic polymeric micelles encapsulating CdSe QDs, to
enable FRET to porphyrin upon two-photon excitation of QD
at 800 nm.207 However, covalent conjugation of QD−PS
conjugates required additional chemical procedures which are
not always feasible, while electrostatic interaction might be less
stable in biological media. Recently, Skripka et al. exploited the
hydrophobic interaction to design QD−PS complex, consisting
of QD coated with PEGylated phospholipid layer which serves
as a binding sites for nonpolar moiety of Ce6.208 The PS was
Z DOI: 10.1021/cr5004198
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effectively excited via TP irradiation at 1030 nm and FRET
efficiency of up to 82% was obtained when multiple Ce6
molecules were bound to QD. However, none of these reports
backed up their results with in vitro or in vivo PDT studies.
Conjugated polymers have large delocalized π-conjugated
backbones and are known to have large TPA coefficients, high
fluorescence quantum, and good biocompatibility, and nano-
particles made of such conjugated polymers were found to be
promising materials for TP-PDT applications. Xu et al.
encapsulated hydrophobic tetraphenylporphyrin (TPP) in
poly[9,9-dibromohexylfluorene-2,7-ylenethylene-alt-1,4-(2,5-
dimethoxy)phenylene] (PFEMO) nanoparticles (∼40 repeat
units).209 Here, PFEMO nanoparticles (∼50 nm) served as
both the hosting material and two-photon light harvesting
complex and the TPA cross section of PFEMO was measured
to be 2160 GM per molecule (54 GM per repeat unit), while
the TPA cross section of TPP was 12 GM. The two-photon
emission of TPP was found to be significantly enhanced by up
to 21-fold in the nanoparticles, and there was a significant
enhancement of 1O2 generation with 1% TPP-doped PFEMO
nanoparticles under TP excitation at 800 nm. This confirmed
energy transfer from PFEMO to TPP, thus demonstrating its
potential as photosensitizing agents for two photon-PDT and
related applications. Although the PFEMO nanoparticles
displayed good stability in water, they aggregated in high
ionic strength medium like PBS. To circumvent this issue of
nanoparticle aggregation, the same group incorporated a
nonionic surfactant, polyoxyethylene nonylphenylether (CO-
520), in the polymeric nanoparticle, to confer hydrophilic
polyoxyethylene chains on the nanoparticle surface.210 In vitro
studies revealed negligible dark-toxicity in HepG2 cells.
However, under 800 nm laser excitation (630 J cm−2), the
cell viability decreased to about 58%, demonstrating for the first
time the application of TP excitable PS-doped conjugated
polymer nanoparticles for cancer cell killing in vitro.
Gold nanorods are notable for their large two-photon
absorption cross-section and tunable longitudinal plasmon
band (Figure 15B). Very recently, Zhao et al. developed
composite nanoparticles by covalently binding porphyrin
molecules (T790) to silica shell coated gold nanorods.211
The silica shell acted as a spacer to control the distance
between gold nanorod and the optimum two-photon excitation
fluorescence enhancement of these core−shell nanoparticles
was achieved with a shell thickness 20 nm, by a factor of 11.8.
1
O2 generation capability and PDT effect of these core−shell
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Chemical Reviews
nanoparticles under two-photon excitation was evaluated in
HepG2 cells and showed a 50% reduction in cancer cells with 8
min irradiation using femtosecond laser at 800 nm. Despite
being a highly spatially focused technique, the requirement of
high excitation intensities and simultaneous absorption of two
coherent NIR photons, reduces the probability and, thus,
efficiency of this approach. Furthermore, it excites a tiny tissue
volume usually resctricted to a few femtolitres,212 often
resulting in the need of prolonged treatment time, thus limiting
its clinical use especially in the treatment of large tumors. This
underscores the need for a “nano-transducer” that can convert
deep penetrating low-energy NIR light to a higher-energy light
that matches the absorption spectrum of PS associated with it.
In the following and final section of this review, we will discuss
in detail the concept and principle behind developing UCNs
that has the ability to harness energy from the NIR light and
convert it to higher energy−visible or UV light, which can then
excite the PS associated within its close proximity. Although
UCNs have many applications in biomedicine like bioimaging,
biosensing, and drug or gene delivery, this article essentially
focuses on reviewing its application in the field of PDT. We will
then present an overview of the recent reports, summarize its
current limitations, and further discuss ways to augment PDT
efficacy in order to take this technology ahead from bench to
bed-side.
5.3.4. Upconversion Nanoparticles. UCNs are nano-
meter-sized materials that convert low energy light to high
energy light through sequential excitation with multiple
photons, via an anti-Stokes emission process. Materials
exhibiting such photon upconversion are usually comprised of
host lattices of ceramic materials, embedded with transition
metal, actinide or lanthanide ions like Yb3+, Er3+, and Tm3+.213
Such materials possess multiple “real” metastable electronic
states which can principally be used to generate upconversion
luminescence (UCL; Figure 17).214 In general, the lowest
metastable state acts as an energy store and absorbs in the NIR
region to promote electrons to the first metastable state S1.214
A subsequent absorption of a second NIR photon promotes the
electrons from metastable state S1 to a much higher metastable
state S3. Although, some internal conversion and relaxation of
electrons back from S3 to S2 is possible, the electrons return to
the ground state by emitting a higher energy photon. Due to
the uniqueness of this material it has found numerous biological
applications by virtue of its tunable emission, large anti-Stokes
shift, sharp emission bandwidth, and high photostability.215
Recently, it has become increasingly necessary to develop new
PSs that could be excited at NIR range, to augment the scope
and applicability of PDT especially for the treatment of solid
and large tumors. This is where the UCN technology becomes
very useful, because UCNs could act both as delivery agent of
the PS as well as convert the NIR light to visible light, required
for the activation of associated PS at much deeper levels in the
tissues. While, two-photon excitation requires simultaneous
absorption of two photons using a virtual state with a lifetime
scale of femtosecond, upconversion process requires sequential
absorption using a real metastable state with a lifetime in the
order of microseconds. Again, the typical power densities for
upconversion is usually in the range of 1−103 W cm−2
achievable by continuous wave lasers, where as that of two-
photon absorption is 106−109 W cm−2. Hence, the efficiency of
upconversion process is several orders of magnitude higher
than that of two-photon emission. In the following sections, we
will discuss in detail the different strategies adopted to design a
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UCN-PS system for PDT as well as methods utilized to
incorporate multiple functionalities to the construct, in order to
develop it into a multifunctional theranostic probe for
simultaneous imaging and therapy for a “see and treat”
approach.
5.3.4.1. Selection of UCNs and Photosensitizers. The UCN
constructs used in PDT generally consists of three components
namely host, dopants, and PS. In the following section we will
review the selection criteria of these three components, to
generate an efficient UCN based PDT system.
Host. The host material provides a crystalline lattice in which
the dopant ions are accommodated. Hence, there should be a
close match between their lattices, to achieve high doping
levels. Inorganic materials such as trivalent rare earth ions (La3+,
Gd3+, and Y3+), alkaline earth ions (Ca2+, Ba2+, and Sr2+), or
transition metals (Ti4+ and Zr4+) can be used as host
materials.216 The optical properties of the UCNs such as
emission profile and upconversion efficiency depends on the
host, and hence selection of host material is critical for efficient
PDT. In order to reduce energy losses during upconversion via
multiphonon relaxation and to maximize radiative emission, a
low phonon energy lattice is essential.217 The crystal structure
of the host material also influences the crystal field around the
dopant ions, resulting in different optical properties of the
UCNs.218 The densely packed hexagonal (β-phase) materials
allows efficient energy transfer between the dopant ions, thus
exhibiting higher upconversion efficiencies than their loosely
packed cubic (α-phase) counterparts.219 Thus, Jin et al. induced
a cubic to hexagonal phase transition of the NaYbF4 crystal by
Gd3+ ion (24%) doping, thereby enhancing the emitted blue
UCL by 30%.220 Another important factor is the phase-purity
of the host where phase-pure hexagonal host materials with an
even symmetry exert uniform crystal-field around its dopant
ions and minimize the energy loss arising from crystal defects,
resulting in enhanced upconversion efficiency.219a Furthermore,
the cationic size of the host also influences the upconversion
efficiency such that a host with a smaller unit-cell volume has
greater crystal-field strength around its dopant ions and hence a
higher upconversion efficiency.216 Despite the vast knowledge
available on various host materials, NaYF4 has been the host
material of choice for UCN constructs in PDT. Perhaps, this
could be due to the low phonon energies (∼360 cm−1),221
smaller size and high chemical stability of this fluoride based
host material. A few studies have also reported the use of other
host materials containing Gd222 (NaGdF4) and Yb ions220
(NaYbF4) that can also function as MR and CT contrast agents
respectively, thus imparting additional imaging capabilities to
the UCN construct. Recently, Wang et al. synthesized
tetragonal-phase LiYF4 hosts with good crystallinity and high
upconversion quantum yield and further demonstrated an
improved upconversion efficiency with the increase in size, due
to reduced surface quenching effects of the UCNs.222c To
minimize the surface defects and resultant luminescence
quenching effects, the UCN core may further be coated with
an undoped matrix forming a shell such as NaGdF4220,223 or
CaF2.222b
Dopants. The dopant ion is the key player in the process of
upconversion that actually performs the absorption and
emission of photons. The emitted UCL largely depends on
the type and the amount of ions doped. Rare earth metals, such
as lanthanides, are suitable candidates as dopants, as most of
them (except La, Ce, Yb, and Lu) have appropriately spaced
multiple ladder-like metastable states, necessary for the
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Figure 18. Schematic illustration of various PS loading strategies in UCNs.
phenomenon of upconversion.214,217b This class of materials
have 4f electrons spatially buried by lower energy 5s and 5p
electrons resulting in a weak electron−phonon coupling.224
Thus, multiphonon relaxation becomes less efficient due to
sufficiently large gap to the next lower energy level and thereby
resulting in long-lived excited states (up to 0.1 s).214 The
UCNs used in PDT usually consists of at least two dopant ions
namely, sensitizer and activator. Such a codoped system enables
us to enhance the upconversion efficiency. A commonly used
sensitizer ion is Yb3+ that has a relatively large absorption cross-
section and a single metastable state.225 Furthermore, it has a
reduced tendency of concentration-dependent quenching,
hence increasing the concentration of Yb3+ could increase the
excitation probability of the commonly used activator ions like
Er3+/Tm3+ via an energy transfer process. This efficient energy
transfer occurs because the energy levels of Yb3+ and Er3+ are
perfectly in sync that they do not undergo phonon relaxation.
Nevertheless, to minimize the loss of the excitation energy by
cross-relaxation process, the doping level of the activator is kept
below 2 mol %, whereas that of the sensitizer is around 20 mol
% in such a codoped system.226 Such Yb−Er based UCNs
usually give a bright green emission (around 550 nm) and a
weaker red emission (around 660 nm), the latter which is often
utilized to activate the attached PS. In order to obtain a single
band, dark red emission for a higher FRET efficiency to red
light absorbing PS, Mn2+ ions could be doped into the host
matrix.227 Another strategy involved increasing the Yb3+ ion
concentration from the usual 20% to 25%. This was shown to
enhance the emission intensity of red UCL band at 660 nm to
about 2.69 folds higher than the green UCL band.228 Strong
multicolour UCL could be obtained by separately doping
NaYF4 core and shell with Er3+ and Tm3+ (NaYF4:Yb3+, Er3+/
NaYF4:Yb3+,Tm3+) instead of homogeneous codoping.229 Such
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a system can be utilized for efficient energy transfer when PS
with a broad absorption bands such a fullerenes are used.
Photosensitizers. Zhang et al. was the first to report the use
of NaYF4:Yb3+,Er3+ UCNs with a thin layer of silica doped with
a lipophilic PS merocyanine-540 (MC-540), for PDT.230 Since
then many PSs, mostly hydrophobic (zinc phthalocyanine
(ZnPC), Ce6, HP, TPP, hypocrellin A (HA), Ppa, silicon
phthalocyanine dihydroxide (SPCD) etc.) have been used in
combination with UCNs for PDT. A few water-soluble PS like
MB and RB have also been chosen to be used in conjunction
with UCN technology. The selection of PS to be used in
combination with a particular UCN type is critical and there
must be a very close match between the UCN emission and
absorption maxima of the PS for efficient upconversion to
occur. Another important factor to be considered is the method
of PS loading and the proximity of PS molecule to the UCN
core. As PS absorbs the upconverted light emitted by the UCN
core, it has to be placed in close proximity to the UCN core for
efficient generation of ROS for PDT. PS can be entrapped,
noncovalently adsorbed, and chemically or electrostatically
attached on the UCN surface. The different strategies adopted
to attach PS to UCN are discussed in detail elsewhere in this
review.
5.3.4.2. Surface Modification of UCN and Attachment of
Photosensitizer. Following the synthesis of hydrophobic UCNs
that usually involves preparation in organic solvents, it is
transferred to aqueous phase before further utilizing it for
biological applications. This is often done by surface coating or
modifying the core to impart hydrophilicity by addition of
stabilizers. The surface chemistry of UCN ultimately
determines its solubility, stability, biocompatibility, pharmaco-
kinetic properties, and capability of anchoring different
functional groups, targeting ligands as well as PS or other
drugs. To date the UCN surface chemistry has been improved
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by either polyethylenimine (PEI),231 PEGylation (addition of
PEG),232 surface coating with amphiphilic PEG block
copolymers,232a,233 PEG-phospho-lipid,223 chitosan,234 BSA or
coating with silica.222a,b,226b,230,235
PEI coating has the advantage of being a one-step “one pot”
hydrothermal process, whereby amine functionalized UCNs are
synthesized that can be utilized for covalent conjugation of
other ligands via 1-ethyl-3(3-(dimethylamino)propyl)-
carbodiimide hydrochloride (EDC)/N-hydroxysulpho succini-
mide (NHS) reactions.67b However, PEI is an inherently toxic
polymer. PEGylation, a very often used technique to improve
water dispersibility of nanoparticle and shield it from being
removed from the circulation by the immune cells, has been
frequently used to modify UCNs.234a,b But one drawback of
PEGylation is that it could be a multistep time-consuming
process. Several PEG block copolymers such as PEG-b-PCL232a
and PEG-b-PLA236 block copolymer have also been employed
as polymeric stabilizer. The assembly process is via flash
nanoprecipitation technique, where all the constituents
including the UCNs and the hydrophobic PS like TPP are
dissolved or suspended in the organic phase, which is then
mixed at high speed against a large volume of water, yielding
the composite nanoparticle.232a,236 This technique requires
thorough optimization of the constituents as inadequate PEG
coating will lead to UCN aggregation in physiological solution;
whereas higher amount of the polymer will lead to formation of
void polymeric micelles. Furthermore, higher polymer concen-
tration may lead to attenuation of the UCL. Wang et al. used a
PEG-grafted poly(maleic anhydride-alt-1-octadecene)
(C18PMH−PEG) amphiphilic polymer to transfer hydro-
phobic UCNs to aqueous phase, yielding PEGylated UCNs
with excellent water solubility.232b Recently, an outer layer of
charge-reversible polymer containing dimethylmaleic acid
(DMMA) groups and PEG chains were utilized to coat the
UCNs via a LbL technique involving electrostatic interactions
in which the outer PEG coated surface is negatively charged at
normal physiological pH 7.4 and switches to a positively
charged naked surface at pH 6.8, demonstrating enhanced
tumor-homing properties.227b In a different approach, poly-
meric liposomes consisting of amphiphilic polymers of
octadecyl-quaternized lysine modified chitosan (OQLCS)
functionalized with FA and transactivating-transduction protein
(TAT) were utilized, which displayed multiple functions as
carriers of UCN and PS, as well as tumor targeting agents while
imparting good water dispersibility.237 Among biodegradable
polymers, wrapping of UCNs with positively charged, hydro-
philic, and nonantigenic polymer chitosan has shown to be very
effective in accumulating within the negatively charged tumor
cell membrane due to electrostatic interactions.234 The
amphiphilic nature of chitosan was exploited to harbor
hydrophobic PSs like Ce6 and PpA, and at the same time
they can carry various functional groups on the outside for
further chemical modifications with other targeting moieties.
Surface coating of UCNs with a thin layer of silica was found
to be stable as silica was resistant to enzymatic or pH
dependent degradation.230 Silica encapsulation (Figure 18A),
which involves incorporation of PS in the silica shell, is a widely
employed strategy to load PS to UCN.226b,230,235 The porosity/
pore size of the silica layer can be tuned such that it can host a
wide range of differently sized PS molecules. The presence of
pores/channels is advantageous to such PDT system, which
requires the oxygen and ROS to diffuse in and out of the
construct to promote effective cell killing during PDT. The
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silica coating first described by Zhang et al.230 lacked this
permeability, which could have resulted in decreased PDT
efficiency. Second, surface modification by silica coating often
leads to particle agglomeration in physiological conditions.
Mesoporous silica coating of UCN was found to have superior
properties than a simple amorphous silica coating, such as
better solubility and greater efficiency of energy transfer, due to
its porous nature.235 Furthermore, it had large surface area that
could be tailored to carry multiple PS and additional tumor
homing motifs for targeted PDT. It is worth noting that this
technique is more suitable for loading cationic hydrophilic PSs
due to the negatively charged nature of the silica matrix.
However, with hydrophilic PSs, there is a high chance of its
leakage out of the uncapped pores during systemic circulation.
This could probably lead to phototoxicity and reduction in the
amount of PS ultimately reaching the tumor. Despite its merits,
this method is a slow multistep process and the amount of PS
loaded was highly variable from batch to batch. Thus, accurate
dosing of the PS becomes a problem, when employing this
technique.
Chemical linkage or covalent conjugation of PS (Figure 18B)
to the surface of UCN offers superior stability, as the PS is
chemically attached to UCN surface, and thus could prevent
leakage of PS during systemic circulation.222a,b,229,234b,238 This
method is widely adopted to trap PS to the UCNs as unlike
chemotherapy drugs, PS need not be released from the UCN
construct to exert its cell killing effect. Often PS with a free
carboxyl group (−COOH) is utilized to attach to an amino
(−NH2) functionalized UCN surface. Qiao et al. utilized a
slightly different method and grafted HP and SPCD on
mesoporous silica shell through the hydrolysis and condensa-
tion of the silanol groups to achieve stable loading of the PS in
the pores of silica layer.222b In addition to achieving stable
binding of the PS, this method also allows attachment of
additional functional moieties like targeting ligands to the
−NH2 group on the UCN surface, enabling targeted delivery to
cancer cells expressing the receptors to these ligands. PS
loading by covalent linkage further offers the advantage of
having a short distance between the UCN surface and attached
PS molecule, thus maximizing the FRET efficiency between the
two. However, despite of the stable binding ability, the PS
payload achieved by covalent linkage was comparatively low,
limiting the application of such constructs to in vitro studies.
Physical adsorption (Figure 18C) of PS to the UCN surface
utilizes noncovalent hydrophobic forces. Usually a hydrophobic
PS is adsorbed on to the hydrophobic layer on the UCN. While
transferring the bare UCNs capped with a hydrophobic layer
(such as an oleic layer) into an aqueous phase by wrapping it
with polymers like PEI,67b PEG,232,236 chitosan,234a,c α-
cyclodextrin,227a or Tween-20,220 a thin hydrophobic layer is
created beneath the polymer on the surface of the UCN. The
hydrophobic PS attaches to this thin hydrophobic layer
sandwiched between the UCN and polymer, via hydrophobic
forces. This also allowed close binding of the PS to the UCN
core, and hence a better energy transfer from the UCN core to
the adsorbed PS was achieved. In addition to the superior
energy transfer, this method of PS loading was found to attain
the highest PS payload among the different strategies proposed
to date. However, a higher PS loading does not always
guarantee a better PDT efficacy, as at high concentrations the
PS molecules could aggregate or form dimers239 which could
inversely affect its performance as PDT agents. Therefore, it
becomes essential to determine the optimal amount of PS that
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Table 3. List of Various UCN Nanoconstructs Synthesized to Date for PDT Applications

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Table 3. continued

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Table 3. continued
can be loaded on to the UCNs to achieve maximum PDT effect
while fabricating UCNs for PDT.
Although not very common, electrostatic interactions
between the counter charged UCN surface and PS were also
utilized to load PS close to the UCN surface (Figure 18D).
Wang et al. synthesized oleate ligand free UCN surface by acid
treatment, which not only imparted hydrophilicity and
biocompatibility but also exposed the positively charged Ln3+
ions on the surface of the UCNs.240 These bare Ln3+ ions were
then utilized for attaching electronegative groups of mono-
substituted-β carboxyphthalocyanine zinc (ZnPC−COOH) via
electrostatic interactions. Such interactions allowed very close
binding of the PS to the UCN surface owing to the short
distance between the donor and acceptor, resulting in a very
efficient energy transfer (96.3%, the highest to date), although
the drug loading was low (1.1 wt %).
It is to be noted that none of the above-mentioned strategies
guarantee controllable loading or attachment of PS to the
UCN, often compromising the repeatability of PDT results. It
is essential to design robust UCN constructs with controllable
and stable PS loading, for optimal and reproducible therapeutic
efficiency. Recently, our group reported a facile method of
stably and uniformly surface coating PS or photocatalyst TiO2
on the surface of silica coated UCN and their application in
PDT (Figure 18E).241 We demonstrated stable coating of a thin
layer of TiO2 on individual UCNs, to form a well-defined core−
shell structure with uniform size of about 50 nm. Such a design
allowed controllable and uniform PS loading on individual
UCNs and completely eliminates the possibility of PS leakage,
thus ensuring significant ROS generation for effective and
consistent PDT results in vitro and in vivo.242 Studies are still
underway to optimize the surface properties of UCNs for
efficient and stable PS loading to achieve maximum 1O2 output.
5.3.4.3. Application of UCNs in PDT. This section
summarizes the recent developments in the application of
UCNs for PDT (Table 3).
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5.3.4.3.1. In Vitro PDT. The very first study reported the use of
NaYF4:Yb3+, Er3+ UCNs coated with a thin layer of silica, as
delivery agents of hydrophobic PS MC-540.230 The UCN
construct was also functionalized with a tumor targeting anti-
MUC1/episialin antibody and thus demonstrated superior
targeting toward MCF-7/AZ breast cancer cells. In vitro PDT
was performed by irradiating MCF-7/AZ cells with a NIR light
source at a wavelength of 974 nm, with an output power of 60
mW for 36 min, following which the cells displayed shrinkage
and membrane damage. However, ROS producing ability of
this nanoconstruct could have been relatively hindered as the
PS was physically encapsulated with in the nonporous silica
layer. Since the success of PDT is attributed to the efficient
production and diffusion of the ROS, it is important to modify
the UCN in such a way that the ROS diffused out of the
nanoparticles effectively. Chatterjee et al. demonstrated the
ability of PEI-modified NaYF4 nanocrystals loaded with ZnPC,
for the destruction of HT29 human colonic adenocarcinoma
cells.231 In this work, high PS encapsulation efficiency of 97%
was reported by physical adsorption of nonpolar ZnPC on to
the highly nonpolar UCN core. Furthermore, the effectiveness
of these FA conjugated nanoconstructs in selective targeting
and destruction of folate receptor (FR) overexpressing HT29
cells following NIR irradiation at 980 nm for 5 min was
demonstrated in vitro. However, in addition to being toxic, the
positively charged PEI could stick to any cells in vivo regardless
of whether it overexpresses FR. It was then assumed that
perhaps the addition of a biocompatible PEG coat could
decrease the toxicity and increase the availability of the UCNs
in circulation. A higher 1O2 production using TPP coencapsu-
lated PEGylated UCNs was demonstrated by Ungun et al.232a
Here, the PEG coating improved the biocompatibility and also
allowed penetration of oxygen and diffusion of the 1O2
produced. Yet, this study did not validate the PDT efficacy of
these particles in cells as well as in vivo models. To enhance the
diffusion of generated ROS, our group synthesized UCN
construct modified with a mesoporous silica shell onto which
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Figure 19. (A) Schematic diagram of synthesis of chitosan modified ZnPC loaded UCN. (B) TEM images of ZnPC loaded nanoparticles. (C)
Viability of MCF-7 cells incubated with void and ZnPc-loaded UCNs for different irradiation time intervals (0, 5, 10, and 15 min). Reproduced with
permission from ref 234a. 2012 The Royal Society of Chemistry.
the PS (ZnPC) was loaded by physical entrapment.235c In vitro
studies on MB49-PSA cells confirmed that a concentration of
100 μg mL−1 of ZnPC loaded UCN could cause about 70% cell
death when irradiated at 980 nm using a laser output power of
0.5 W for 5 min. In a subsequent paper the molecular effects of
PDT induced cell death was also discussed and the major mode
of cell death was confirmed to be apoptosis.235a Despite its
advantages, it is to be noted that PS loading in such a system is
highly variable, leading to batch variations due to difference in
pore size that cannot be controlled. Second, due to the
presence of a double layer of silica (first layer of nonporous
silica to reduce surface defects of UCN core, and a second
mesoporous layer), the size of the particles were relatively large
(∼90 nm). Furthermore, there is an increased chance of
particle growth and aggregation due to the higher diffusion rate
at higher temperatures during calcination process. Another
group also employed a similar technique to load HP and SPCD
onto the mesoporous silica coating, over a CaF2 shell coated
UCN.222b However, this time the PS was covalently grafted on
the surface of the UCN. The CaF2 shell reduced surface defects
of the core improving the UCL as well as its biocompatibility.
In vitro PDT under 980 nm NIR light demonstrated that a
strong irradiation dose (2.5 W cm−2) was required to transfer
energy from UCN core to the PS molecule, to generate enough
1
O2 in order to promote efficient cell death. The PDT effect
was found to be proportional to both concentration and light
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dose, where the first 5 min of irradiation generated most of the
1
O2 due to the consumption of dissolved oxygen in the
medium. Apart from its application in PDT, the construct also
exhibited potential for fluorescence and magnetic resonance
imaging (MRI) due to the presence of gadolinium ion (Gd3+)
in the UCN core.
Recently, chitosan wrapped UCN loaded with PS showed
promising results as PDT agents.234a,b Cui et al. reported
loading of ZnPC via hydrophobic interactions achieving one of
the highest PS payloads of 10.8% (Figure 19).234a In vitro PDT
at 980 nm (output power of 0.6 W for 10 min) using 400 mg
mL−1 chitosan wrapped ZnPC loaded UCN following 24 h
incubation killed about 80% of MCF-7 breast cancer cells, while
cells incubated with void-chitosan wrapped UCN demonstrated
more than 80% viability following NIR irradiation. The mode of
cell death was predominantly found to be via apoptosis. In a
different study, Zhou et al. demonstrated PS loading via
chemical linkage of PS−pyropheophorbide A to the amino
group of chitosan that was also tagged with a cyclic RGD
peptide, c(RGDyK).234b Such a covalent conjugation rendered
stable PS loading to the UCN as well as retained the
spectroscopic and functional properties of the PS. The presence
of c(RGDyK) demonstrated targeting abilities to integrin αVβ3
expressing U87-MG glioblastoma cancer cells and selective
destruction of U87-MG cells upon NIR irradiation. In vitro
PDT was performed using 980 nm at 0.5 W cm−2 and different
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Figure 20. (A) Schematic illustration of the mechanism of ROS generation upon irraiation of TiO2−UCN with NIR light for PDT. (B) TEM image
of TiO2−UCN (scale-bar: 50 nm). (C) Fluorescence emission spectrum of 1 mg of TiO2−UCN suspended in 1.5 mL of water, under 980 nm
excitation. (D) ROS production from 0, 2, 4, and 8 mg of TiO2−UCN suspended in 2 mL of water, under 980 nm irradiation (2.16 W cm−2). (E)
Dark-toxicity of TiO2−UCN incubated with cancer cells, OSCC and normal cells, NHF for 6 h.Values are means (n = 3) ± SD *P < 0.05 compared
to control cells without exposure to TiO2−UCN. (c) In vitro PDT using TiO2−UCN on OSCC and NHF cells. Values are means (n = 3) ± SD *P
< 0.05 compared to control cells treated with the respective concentration of TiO2−UCN but without exposure to 980 nm laser (i.e., dark-toxicity
values). Reproduced with permission from ref 241. 2014 The Royal Society of Chemistry.

radiation doses ranging between 75 and 300 J cm−2. It was
shown that for optimal PDT cell kill, a concentration of 200 ug
mL−1 and photoirradiation energy greater than 225 J cm−2 was
necessary.
Thereafter, Xu et al. reported synthesis of an anti-cAngptl4
antibody conjugated nanocomposite consisting of thioglycolic
acid functionalized nitrogen-doped-titanium dioxide (N-doped-
TiO2) nanoparticles, covalently attached on a NaYF4:Yb3+,
Tm3+ UCN core.238a Upon NIR irradiation the UCN core
emitted visible light which excited the attached photocatalyst
N-doped TiO2 to generate highly reactive electron−hole pair. It
further reacts with water and oxygen to produce ROS leading
to cancer cell death. However, the synthesized nanocomposites
did not seem to have a uniform size, as controlling the number
of TiO2 nanoparticles attaching to the UCN surface is not
practically possible. The study demonstrated in vitro NIR-
triggered drug release and targeted cancer cell killing of
malignant A-5RT3 cells predominantly via apoptosis. Realizing
the need for controllable and stable loading of PS, very recently
our group developed a method for uniformly surface coating
photocatalyst TiO2 on individual UCNs, to achieve well-
defined core−shell structured nanoparticles (Figure 20).241 Our
method has the advantage of stable PS attachment with
minimal batch variations, preventing any possibility of PS
leakage, thus ensuring consistent PDT results. Here, the
photocatalyst TiO2 was stably and uniformly coated using a
modified Stöber method, following an electrostatically guided
attachment of a Ti precursor on to the silica coated UCNs
bearing positively charged amino groups on its surface.
AH
Following 980 nm NIR irradiation, the photoinduced TiO2
resulted in the generation of more than one type of ROS
including hydroxyl radicals, superoxide anions, and hydrogen
peroxide, and their potential in inducing cell death was also
successfully demonstrated in vitro. However, we observed
saturation of its cell killing ability, such that the photocatalytic
killing of nanoparticles did not increase much in a dose-
dependent manner. This could perhaps have been due to the
formation of large aggregates in the complex biological
environment, hampering its effective uptake into the cells.
Hence in a recent and subsequent study, we further surface
modified the core−shell TiO2−UCNs with polyethylene glycol
(PEG) to impart stealth properties and stability in terms of its
size, toward making it more conducive for biological
applications.242 PEGylated TiO2−UCNs were found to be
both biocompatible and hemocompatible, and demonstrated
dose dependent ROS production and corresponding cell killing
abilities in vitro.
A few other groups also reported synthesis of multifunctional
theranostic UCNs for both imaging and therapy. Zhao et al.
synthesized small (∼38 nm), size-controllable silica coated
UCN with PS (tetra substituted carboxy aluminum phthalo-
cyanine, AlC4Pc) covalently linked to the silica shell.222a This
allowed close proximity of the PS to UCN core, demonstrating
a better PDT efficiency where nearly 40% of the cells treated
with 100 ug mL−1 where killed after 5 min of irradiation using a
980 nm laser at a power density of 0.5 W cm−2. Solution based
studies revealed that the synthesized particle had good water
dispersibility, 100% entrapment efficiency and no PS leakage.
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Figure 21. (A) Schematic illustration of α-CD surface functionalization procedure and the preparation of PS@UCN complexes and (B) 980 nm NIR
induced PDT using PS@UCNs. SEM image of (C) unmodified UCNs and (D) α-CD-UCNs. (D) Room temperature upconversion emission of
oleic acid capped (OA-UCNs) dispersed in cyclohexane (1 mg mL−1, solid line) and α-CD-UCNs dispersed in water (1 mg mL−1, dashed line),
respectively (inset: their corresponding luminescent photographs under 980 nm NIR excitation, power density: 0.5 W cm−2). (D) Cell viability of A-
549 cells treated with Ce6/DOX@UCN complex (980 nm laser density: 1 W cm−2; irradiation time: 5 min). Error bars were based on triplicated
samples. Reproduced with permission from ref 227a. 2013 Wiley-VCH Verlag GmbH & Co. KGaA.

The presence of Gd3+ ions in the matrix further facilitated both
T1- and T2-weighted MRI. Chen et al. demonstrated water in
oil strategy to trap positively charged hydrophilic PS MB into
the silica shell.226b No leakage of MB was detected from the
UCN construct due to strong electrostatic attraction between
positively charged MB and the silica shell. The construct also
had potential for MRI due to the presence of Gd3+ ions in the
core. It is to be noted that majority of the hexagonal-phase
NaYF4:Yb/Er UCNs developed for PDT typically emitted a
strong green and a weak red fluorescence. However, most of
the PSs that were associated with such β−NaYF4:Yb/Er
absorbed at the red region of the spectrum. Apparently, the
dominant green emission has no contribution in the excitation
of PS, which resulted in relative low efficiency in the FRET
process. Recognizing this fact, Tian and co-workers synthesized
strong dark red-light emitting α-cyclodextrin (α-CD) modified
manganese ions (Mn2+) doped UCNs (Figure 21).227a The
cube shaped UCNs with a single-band red UCL emission
peaking at 650−670 nm were further loaded with lipophilic
PSs, Ce6 or ZnPc, and hydrophilic MB. As the loading of the
PS on the UCNs employed hydrophobic interactions, the more
hydrophobic PSs (Ce6 and ZnPC) were more efficiently loaded
compared to MB. In vitro PDT with Mn2+-doped UCNs
AI
coloaded with a chemotherapeutic agent DOX and Ce6,
induced significantly greater cell-kill compared to treatment
using a single modality. Similarly, Wang et al. also utilized Mn
doping to synthesize UCNs emitting single-band intense red
light (650−670 nm) UCL.227b The Mn2+-doped UCN were
further made water-soluble, by replacing the hydrophobic
surface ligands through a ligand exchange reaction using PAA.
They, then utilized LbL self-assembly approach to load the
negatively charged Ce6 conjugated polymer (PAH-Ce6-
succinic anhydride (SA); Figure 22). The UCN/PAA was
first mixed with oppositely charged PAH to form positively
charged UCN/PAA/PAH, on which the negatively charged
PAH-Ce6-SA was attached via electrostatic interaction, to load
up to 3 layers of the Ce6 conjugated polymer. This technique
resulted in achieving a very high loading of PS (up to 11.0 wt %
for 3 layered Ce6 polymer-UCN composites), however 7.7 wt
% (two-layer) was found to be optimal. The tight coupling
between PS molecules and UCNs prevented Ce6 leakage from
UCNs up to 2 d under physiological conditions. To further
impart UCNs with pH-responsive charge reversibility, PAH was
cografted with PEG and DMMA to obtain negatively charged
PAH-DMMA-PEG, which was then used to coat UCNs by
electrostatic interactions. On incubating the pH-sensitive
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Figure 22. (A) Schematic showing the fabrication process of pH-sensitive charge-reversible UCNs with multilayers of Ce6 loading. (B) TEM
micrograph of Mn2+-doped UCNs; inset: high-resolution TEM image of a UCN. (C) UCL emission spectrum of Mn2+-doped UCNs under 980 nm
laser excitation; inset: photo of a UCN solution under ambient light and under 980 nm laser excitation. (D) T1-weighted MR images of UCN
solutions at different concentrations. (E) T1 relaxation rates (r1) of UCN solutions at different Mn2+ concentrations. (F) Cell viability of HeLa cells
after various treatments indicated with and without the 980 nm laser irradiation as evaluated by the standard MTT assay. Error bars are based on four
parallel samples. Reproduced with permission from ref 227b. 2013 Wiley-VCH Verlag GmbH & Co. KGaA.

UCNs with HeLa cells grown at pH 6.8 and 7.4, significantly
greater UCL signals were obtained from HeLa cell at pH 6.8,
indicating significantly enhanced cellular internalization at
slightly acidic pH due to their charge conversion from negative
to positive. This also led to a correspondingly high cell death
following irradiation at 980 nm light. In addition to the
enhanced therapeutic effect, the Mn2+ doped nanoconstruct
also demonstrated potential as T1-weighted contrast agent in
MR imaging. In order to develop a T2-weighted MRI contrast
agent, Zeng et al. reported the synthesis of multifunctional
MRI/UCL/PDT nanocomplexes composed of 10 nm Fe3O4
core, coated with a tetragonal NaYF4:Yb/Er shell.252 The
nanoconstruct also exhibited good therapeutic performance
which resulted in nearly 70% MCF-7 cell death following
irradiation at 980 nm at a power density of 0.02 W cm−2 for 5
min. A different approach was adopted by Liu and co-workers,
where they fabricated UCNs by separately doping core and
shell with Er3+ and Tm3+ instead of homogeneous codoping, to
achieve strong simultaneous multicolour UCL around 450, 475,
540, 650, and 808 nm upon 980 nm NIR excitation.229 They
then utilized monomalonic fullerenes (C60MA), covalently
conjugated to the core−shell structured UCNs as energy
acceptors and ROS generating agents. The nanocomposites
were additionally modified with PEG-succinimidyl carbonate
(PEG-SC), to improve biocompatibility and stability in
biological solvents and further conjugated with FA to bestow
targeting abilities to folate receptor expressing HeLa cells. A
AJ
dose-dependent decline in the viability of HeLa cells upon NIR
irradiation at an intensity of 1.37 W cm−2 confirmed the
capabilities of this multifunctional UCN−fullerene nanoplat-
form in NIR imaging guided cancer therapy. Recently, Chen
and co-workers demonstrated for the first time synergistic
PDT/PTT photokilling effects of bovine serum albumin (BSA)
modified NaGdY4-based UCNs.256 Here, two types of dye
molecules, including a PS, RB, and an NIR-absorbing dye,
IR825, were simultaneously loaded into the BSA layer via
hydrophobic interaction, so that under 980 nm excitation RB
absorbs green light emitted from UCNs and induced
photodynamic cancer cell kill, while IR825 under 808 nm
laser irradiation offers a strong photothermal effect. Although
the PTT effect (808 nm excitation, 0.5 W cm−2 for 5 min)
caused by the hyperthermia induced by IR825 loaded
nanocomposites was slightly more effective in killing 4TI cells
in vitro than the PDT effect (980 nm excitation, 0.4 W cm−2 for
10 min), the combination of both therapies induced a
synergistic effect resulting in significantly greater cell kill
compared to the monotherapy.
5.3.4.3.2. In Vivo PDT. The biggest challenge in the application
of UCNs for PDT in vivo has been the delivery of the particles
such that it selectively accumulates in the tumor, preventing
systemic toxicity. Most of the earlier studies reported delivery
of UCNs via intratumoral injections. This route of admin-
istration was chosen as the particles synthesized initially were
large and aggregated in the physiological milieu. Furthermore, it
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Table 4. Summary of Various PDT Parameters for in Vivo Application of UCNs

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Figure 23. (A) Schematic drawing of NIR-induced PDT using UCN-Ce6. (B) TEM image of PEGylated UCNs. (C) Growth of 4T1 tumors on
different groups of mice after various treatments indicated; relative tumor volumes were normalized to their initial size (n = 10). (D) The survival
curves of mice in 60 days after various treatments indicated; UCN-Ce6 PDT treatment drastically prolonged survival time of mice. (E) Schematic of
in vivo PDT experiment where tumors were hidden below a slice of pork tissue (8 mm) during light treatment. (F) The growth of 4T1 tumors on
different groups of mice after various treatments indicated. The relative tumor volumes were normalized to their initial sizes. (n = 10) *p < 0.05.
Error bars represent standard errors of the mean. Reproduced with permission from ref 232b. 2011 Elsevier.

had limited specificity and selectivity to the tumor that most of
them accumulated in the organs of the MPS leading to toxicity.
Thus, to obtain higher PDT efficiency and fewer side effects,
UCNs were administered intratumorally. The various in vivo
studies and the parameters used in PDT are summarized in
Table 4.
Wang et al. demonstrated in vivo PDT in murine breast
tumor using Ce6 loaded PEGylated UCNs (Ce6-PEG-UCN;
Figure 23).232b PEGylation improved the stability as well as
decreased the toxicity of Ce6-PEG-UCN, where the hydro-
phobic Ce6 molecule was simply adsorbed onto the oleic layer
of the PEGylated UCN, to achieve a very close binding of Ce6
on the UCN. This strategy had an added advantage of offering
a higher resonance energy transfer between the photon donor
and acceptor molecule, by virtue of its adjacency. The size of
this particle (∼30 nm) was the smallest compared to all the
previously reported UCNs for PDT. Yet, particles were
administered intratumorally, rather than intravenously into
AL
the animals. Furthermore, there was an enhanced retention of
the UCNs in the organs such as spleen and the liver 15 days
post injection, indicating apparent translocation of the UCNs
from the injected site via the lymphatic circulation. In vivo PDT
using 980 nm laser irradiated at a power density of 0.5 W cm−2
for 30 min (1 min interval after each min of irradiation)
appeared quite promising with 70% of the tumors disappearing
without regrowth in 2 weeks, exhibiting longer morbidity free
survival. It was clearly shown that NIR light penetrated the 8
mm pork tissue placed over the tumor, triggering Ce6-UCN in
the tumor leading to significant reduction in tumor growth rate.
However, direct application of NIR light to the tumor did not
generate as much 1O2 as that generated by free-Ce6 triggered
by visible 665 nm light, thus suggesting that NIR-induced PDT
using UCN has the potential to be developed for the ablation of
solid, deep seated tumors.
Cui et al. reported in vivo PDT using chitosan modified
UCN loaded with ZnPC.234a PDT at 980 nm NIR laser
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Figure 24. (A) Schematic diagram of ZnPC and MC540 coloaded with in the mesoporous silica shell coated UCN. (B) TEM image of the
mesoporous-silica-coated UCNs (scale bar, 50 nm). (C) Schematic diagram showing UCN-based targeted PDT in a mouse melanoma model
intravenously injected with UCNs surface modified with FA and PEG. Scale bar, 10 mm. (D) Change in tumor size as a function of time following
intravenous injection of UCNs with and without targeting agent (FA) to assess the effectiveness of UCN-based targeted PDT in tumor-bearing mice.
Values are means ± s.e.m. (n = 7 mice per group). *P < 0.05 compared to control group 3 by Kruskal−Wallis ANOVA. (E) Representative images of
mouse intravenously injected with FA-PEG-UCNs, unmodified UCNs or PBS showing the change in tumor size (highlighted by dashed white
circles) before (0 d) and 7 d after PDT treatment. Scale bars, 10 mm. Reproduced with permission from ref 235b. 2012 Nature Publishing Group.

irradiation at 0.4 W cm−2 for 15 min, following intratumoral
administration of these particles produced significant reduction
in tumor via necrotic cell death, within 14 days compared to the
untreated group. In addition, there was no sign of adverse side-
effects and lethality among PDT treated animals due to the
presence of a nontoxic and biocompatible chitosan coat.
The first study to demonstrate systemic administration of a
42 nm UCN carrying Ce6 was by Park et al.223 Here, Ce6 was
attached both via physical adsorption to phospholipid layer as
well as covalent conjugation to the amino group on UCN,
achieving stable and highest PS payload (>1000 Ce6
molecules/UCN). Due to the presence of the biocompatible
polymer PEG, these UCNs carrying Ce6 had better circulation
half-life than free-Ce6. Particles mainly accumulated in the liver
and spleen; however the amount dropped considerably 7-days
post injection, reducing long-term toxic effects. Following in
vivo PDT at 980 nm, there was a 6-fold decrease in tumor size
among the treated group, thus clearly demonstrating its
potential as PDT agent. Thereafter, our team reported
therapeutic efficiency of mesoporous silica coated UCNs
coloaded with two different PS, ZnPC and MC-540 (Figure
24), using mouse melanoma model in a stepwise fashion.235b
First, the melanoma cells were prelabeled with PS loaded
UCNs and then injected subcutaneously to develop UCN
loaded tumors in mice. This was followed by assessment of
PDT efficacy by injecting PS loaded UCNs intratumorally into
tumor bearing mice and subsequently intravenous admin-
istration of FA conjugated- PEGylated- PS loaded UCNs was
performed. This was the first time that two different PSs
absorbing at two different wavelengths (660 and 540 nm) of
AM
the emitted upconverted light was being utilized for maximum
PDT efficacy. The results were quite promising in that all the
three modes of UCN administration produced significant
reduction in tumor volume compared to the untreated group.
However, a very long irradiation time of 1−2 h (at 980 nm,
power density of 415 mW cm−2), was employed for in vivo
PDT. This could have been due to the inefficient energy
transfer from UCNs to the PS probably due to the limited PS
loading capacity (<0.6 wt %) and poor stability of the PS that
was physically adsorbed onto the mesoporous silica. Perhaps,
covalent binding of the PS to the surface of UCNs could
enhance the upconversion efficiency and produce a more
pronounced PDT effect in vivo.
Cui et al. again demonstrated remarkable therapeutic efficacy
with tumor inhibition ratio of up to 50% using FA conjugated
chitosan modified ZnPC loaded UCNs in comparison with a
tumor inhibition ratio of 18% achieved by conventional visible
light-activated PDT.234c Here, PS was loaded via hydrophobic
interactions to achieve high drug loading capacity of about 10%.
The FA conjugated UCNs were administered intravenously and
exhibited high tumor targeting efficiencies in vivo, where it
accumulated as early as 4 h post administration, whereas the
unconjugated UCNs became visible only after 12 h. The
subcutaneous tumor developed on the mouse were covered
with 1 cm pork tissue to simulate tumors inside the body and
was irradiated with either 660 or 980 nm light following
administration of PS loaded UCNs. Survival rates of mice in
this deep penetrating PDT experiments reached 88% for 980
nm induced PDT compared to 75% using 660 nm light
irradiation demonstrating the ability of the nanoconstructs in
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the ablation of deep-seated tumors. The results indicated that

PDT treatment based on FASOC-UCN-ZnPc could effectively
improve the survival quality of mice and prolong their lifetime.
Using the pH-sensitive charge reversible nanoconstruct, Wang
et al. demonstrated significantly delay in tumor growth
following 980 nm NIR PDT at 0.5 W cm−2 for 30 min (900
J cm−2), 1 day after intratumoral injection of 0.4 mg UCNs per
4T1 tumor-bearing Balb/c mouse.227b At the same time,
significantly higher uptake of UCNs (6.4 folds) were observed
in the tumor, 2 h following intravenous administration of same
dose of UCNs. Despite an increased tumor accumulation of
UCNs, the majority of injected dose were observed in
reticuloendothelial systems (RES), such as the liver and spleen,
following systemic administration. This warrants further
improvement of the surface modification techniques, to
enhance the tumor targeting, and at the same time, reduce
the accumulation and retention of these smart UCNs in the
RES. In their recent publication, Chen et al. demonstrated
synergistic therapeutic efficacies of the combined PTT and
PDT in an in vivo mouse model bearing 4T1 tumors, using a
double dye loaded (RB and IR825) BSA modified UCNs.256
The combination therapy (PDT at 980 nm excitation for 30
min at 0.4 W cm−2 followed by PTT at 808 nm, at 0.5 W cm−2
for 5 min) after intratumoral injection of 10 mg mL−1
RB:IR825 coloaded UCNs significantly inhibited tumor growth
in vivo, while those after monotherapy (PDT only or PTT
only) showed a delayed tumor growth. In addition to the
therapeutic effect, intrinsic paramagnetic and optical properties
of Gd3+-doped UCNs were utilized for in vivo dual modal
imaging, thus proving to be excellent multifunctional Figure 25. (A) Schematic illustration of multifunctional LiYF4:Yb/Er
theranostic agents. UCNs for cancer theranostics. Variation of (B) tumor volumes and
(C) body weights of mice in experiment and control groups,
After successfully developing a positively charged ligand free respectively. Each data point represents the average value of 5 mice.
UCNs on which electronegative PS were tightly coupled via (D) Representative photos of a mouse showing tumors at 14 days after
electrostatic interactions, to obtain a UCN-PS complex with treatment in experiment and control groups, respectively. (E) Images
highest FRET efficiency (96.3%) until date, Wang et al. studied (left) and the corresponding high-resolution images (right) of H&E
the in vivo therapeutic efficiency of the developed PVP coated stained tumor tissues harvested from the experiment and control
UCNs, in a H22 hepatocarcinoma mouse model (Figure groups after 14 days. Reproduced with permission from ref 240. 2014
25).240 NIR PDT of the tumor at 980 nm, 12 h post Royal Society of Chemistry.
intratumoral injection of 20 mg mL−1 ZnPC−COOH loaded
UCNs exhibited significant delay in tumor growth (501 mm3) linkage, minimized premature leakage and improved its
compared to untreated control (1282 mm3) by 14 days. bioavailability. In vivo PDT in mice models of tumors following
Compared to the animals in the untreated control, the body intratumoral delivery of these ALA loaded nanoconstructs
weight of the mice in the treatment group remained unchanged, demonstrated significant tumor growth delay compared to
indicating an overall improvement in their health status. In our controls even in the presence of 12 mm thick pork tissue; the
recent study in which we directly surface coated photocatalyst greatest depth at which significant reduction in tumor growth
TiO2 on individual UCN core and further surface modified was achieved with UCN-PDT.
them with PEG to improve its biocompatibility, we The first ever attempt that combined bimodal imaging and
demonstrated remarkable delay in tumor growth and significant trimodal therapy in a single UCN-based nanotheranostic
improvement in survival of the animals in the treatment groups system was yet recently reported by Fan et al.264 They
compared to the control groups (Figure 26).242 It can be synthesized a sub-80 nm rattle structured nanocomposite with
concluded that stable and controllable loading of PS is essential Gd3+ doped UCN in the core (for simultaneous UCL and MR
for consistent and optimal PDT results. imaging), and a mesoporous silica as the outer shell with a
In order to improve the UCL emission in the red region for cavity in between them (UCMSNs; Figure 28). HP that serves
further improvement in the penetration depth achievable by as both PS and radiosensitizer was covalently grafted inside the
UCN-based PDT, recently Punjabi et al. developed CaF2 silica shell with a loading efficiency of 7.8%, whereas the
coated α-NaYF4:Yb,Er in which the Yb doping was increased chemo-drug docetaxel (Dtxl) was encapsulated with in the
from 20% to 98% (where 80% Yb doping showed optimal inner cavity with a loading efficiency of 4.5 wt %. The
results; Figure 27).266 They reported 15-fold increase in red- nanoconstruct was found to accumulate in the tumor by passive
emissions compared to their hexagonal phased counterparts (β- targeting with a 25% enhancement in T1-weighted MRI signals
NaYF4:Yb(20%),Er(2%)@β-NaYF4), with an absolute quan- within 15 min post intravenous delivery. By simultaneously
tum yield of α-NaYF4:Yb(80%),Er(2%)@CaF2 measured to be imposing NIR excitation and high energy X-ray irradiation
3.2%, the highest reported value for red-emissions. Further- following intratumoral administration of UCMSNs-HP-Dtxl,
more, conjugation of ALA to the UCN surface via a hydrazone the tumors in mouse models were quickly eradicated due to the
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Figure 26. (A) In vivo OSCC tumor growth up to 35 days on different groups of mice after various treatments indicated, n = 5 per group. Error bars
represent standard error of the mean. Representative images of animals with tumors that were (B) not treated (untreated control animals) and (C)
intratumorally injected with 0.1 mg Mal-PEG-TiO2−UCN and irradiated with 980 nm light, at designated time-points. (D) Survival rates of mice in
different treatment groups within 60 days. Reproduced with permission from ref 242. 2014 American Chemical Society.
synergistic effect of PDT/chemo-/radiotherapy. However,
following intravenous delivery and trimodal therapy, the tumors
were found to regrow after an initial drop in the volume. It is to
be noted that most of the in vivo tumor regression studies were
short-term (10−14 days), where majority of the results report a
delayed tumor growth and improved overall survival, rather
than a complete tumor regression. To further improve the
therapeutic efficiency of UCN based nanoconstruct and fine-
tune the PDT regimen to achieve complete or partial tumor
regression, long-term tumor regression studies need to be done.
5.3.4.4. Toxicity and Biodistribution of UCNs. Although
UCNs have been proposed for various bioimaging and
therapeutic applications, numerous unresolved issues with
respect to its interaction with biological system yet remains.
Like many other inorganic nanoparticles, UCNs are not easily
biodegraded in the biological system, causing concerns about its
long-term toxicity and side effects. Although, humans are
exposed to lanthanides based compounds in the form of
therapeutics and diagnostics, they are often considered to be
moderately to highly toxic. Generally, the toxicity depends on
the compound (inorganic versus chelated compounds), route
of administration, and dose.267 Thus, the behavior, biodis-
tribution, degradation, clearance, and toxicology profiles of
UCN in biological systems are the most critical and
fundamental question to be addressed presently. Almost all
studies highlighted in this review paper, have evaluated the in
vitro cytotoxicity of respective UCN constructs by the standard
CCK-8, MTT, MTS, and LDH assays, and have found to be
safe to a broad range of tested cell lines within a certain
concentrations (usually in μg mL−1 range) and a limited
incubation period (usually ranging from 30 min to 24 h). A few
studies utilized Caenorhabditis elegans (C. elegan) as a model
organism for evaluating the toxicity of UCNs and reported that
AO
Review
feeding C. elegans with UCNs did not affect its life span, egg
production, egg viability, or growth rate, indicating that UCNs
were not significantly toxic except at concentrations above 5 mg
mL−1 or higher.268 A list of all the published efforts on
biodistribution, toxicity, and clearance of various UCN
constructs in animals are summarized in Table 5.
To gain insight into the kinetics of these nanocrystals in
rodents, Jalil et al. studied the biodistribution of silica coated
UCNs following intravenous injection in Wistar rats, up to 7
days.269 No obvious acute toxic or adverse health effects were
observed at the dose level of 10 mg kg−1 for up to 1 week post
administration. Quantification of yttrium content in various
organs at specfic time-points revealed that lung and the heart
were the main target organs where the nanocrystals
accumulated immediately (29.2 and 18.0 mg L−1 per g of
tissue at 10 min) post injection. However, the amount dropped
significantly by 24 h. The nanoparticles were also detected in
the spleen, reaching the highest concentration at 30 min (6.3
mg L−1 per g) as well as in the kidneys, where the
concentration of yttrium remained the same up to 24 h.
However, the concentration in blood and liver remained low
throughout. The nanocrystals were mostly cleared from all
major organs by day 7 postinjection indicating that they were
not retained for a long period of time. On studying the long-
term fate of 11.5 nm sized poly(acrylic acid) (PA) coated
UCNs in nude mice, Xiong et al. reported that the major organs
that retained the nanoparticles were the liver and spleen
(Figure 29).270 Although, no major toxic effects were observed,
the study reported a very slow clearance of the nanoconstructs
from the system via hepatobiliary route. However, the study
only included a small number of animals (n = 3) and the blood
biochemical and histology results were collected only at the end
of the experimental period (day 115).
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Figure 27. (A) Emission spectra under 980 nm, 1 W cm−2 excitation of α-NaYF4:Yb,Er@CaF2 UCNs with different Yb-levels. (B) Integrated counts
of red-emission and photographs (inset) of α-NaYF4:Yb,Er@CaF2 UCNs with different Yb-levels. (C) TEM images of PAA−UCNs (a), hydrazide-
functionalized UCNs (b), and ALA−UCNs (c). (D) In vivo volume of tumors exposed to various controls and ALA−UCNs with red and near-
infrared irradiation (0.5 W cm−2) in simulated deep tumors. Statistical significance was determined from one-way t tests; significance (*) was based
on p < 0.05 and p > 0.05 for not significant (**) pairs. Reproduced with permission from ref 266. 2014 American Chemical Society.

Later, Cheng et al. conducted a thorough assessment of the
pharmacokinetics, long-term biodistribution and toxicity of 30
nm sized PEG or PA modified UCNs.271 Although, the blood
circulation curves of UCN-PEG and UCN-PA followed a two-
compartmental model, the blood circulation half-life of UCN-
PEG was significantly greater than UCN-PA, which was
attributed to the biocompatible PEG coating on the UCN
surface and its improved stability in physiological solutions. In
vivo UCL imaging showed that UCN-PA accumulated in liver
within 5 min, but UCN-PEG uptake was slower and observed
in liver at 30 min. Ex vivo UCL imaging revealed accumulation
of UCNs in the liver, spleen, lung and bone at 1 day, but no
signal was detected by day 7. However, the yttrium ion levels in
these organs did not drop much over a course of 90 days,
except in the lung, which showed a rapid decrease of yttrium
ion concentration after 3 days. The presence of nanoparticle
aggregates in the TEM of liver slices from mice 7 days post
injection, indicates partial decomposition of UCNs inside the
macrophage cells after RES uptake and loss of their UCL
emission signals over time. Thus, the study concluded that
nanoparticle residues would stay inside the mouse body for
more than 90 days, which is in stark contrast to the previous
reports. Perhaps the size difference of UCNs in these two
AP
studies could have led to the observed difference in the
excretion routes and clearance of the nanoparticles. But it is to
be noted that, although the nanoparticles remained in the
animal for over 90 days, no appreciable variations were
observed in the blood biochemical, hematological or histo-
pathological analysis.
Presence of targeting ligands were found to channel the
UCNs to the tumor and reduce the percentage of injected dose
that accumulated in other organs such as the liver and spleen.
Cui et al. administered FA conjugated UCNs intravenously,
that exhibited high tumor targeting efficiencies and accumu-
lated in the tumor as early as 4 h post administration, whereas
the unconjugated UCNs became visible only after 12 h.234c The
particles first accumulated in the liver, following which they
were gradually excreted into the intestines by 24 h and the
absence of fluorescence signals from the loaded NIR dye (ICG-
Der-01) at 48 h indicated complete clearance of the UCNs
from the system via enterohepatic route. A significant variation
in the biodistribution was observed in the presence of a
targeting moiety FA at 24 h in a tumor bearing mice, where FA-
conjugated UCN levels were much higher in tumor but lower
in organs like lung, liver, and kidney, compared to the level of
unmodified UCNs. Due to the huge diversity in the nature, size,
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Figure 28. (A) Schematic illustration of the synthetic procedure of UCMSNs. Gd-UCNPs were prepared by epitaxial growth NaGdF4 layer on
NaYF4:Yb/Er/Tm through a typical thermal decomposition process. A dense silica layer was then coated on Gd-UCNPs by a reverse microemulsion
method, designed as Gd-UCNPs@SiO2. Subsequently, a mesoporous silica shell was deposited on Gd-UCNPs@SiO2 via the template of CTAC,
designed as Gd-UCNPs@SiO2@mSiO2. Finally, UCMSNs were successfully fabricated based on a “surface-protected hot water etching” strategy. (B)
Transmission electron microscopic (TEM) images of (a) Gd-UCNPs (NaYF4:Yb/Er/Tm@NaGdF4), (b) Gd-UCNPs@SiO2, (c) Gd-UCNPs@
SiO2@mSiO2, and (d) UCMSNs (scale bar = 50 nm) (C) In vivo T1−MRI images of a 4T1-tumor bearing mouse after intravenous injection of
UCMSNs at designated time points: (a) 0 min; (b) 5 min; (c) 10 min; (d) 15 min. (e) Comparison of MRI signal intensity of the corresponding
4T1-tumor at different time points from 0 to 15 min. (f−h) Ex vivo NIR−NIR upconversion luminescent imaging (UCL) of dissected organs after
the intravenous injection of UCMSNs: 1, heart; 2, liver; 3, spleen; 4, lung; 5, kidney; 6, tumor (i−k). (D) Tumor growth and relative tumor volumes
of different groups post intratumoral injection of UCMSNs-HP-Dtxl and corresponding treatment. Control groups received PBS. (E) Tumor growth
and relative tumor volumes of different groups post intravenous injection of UCMSNs-HP-Dtxl and corresponding treatment. Reproduced with
permission from ref 264. 2014 Elsevier.

composition, charge, surface properties, and presence or UCNs, it becomes very hard to even speculate the
absence of additional functional moieties on the surface of biodistribution and toxicity of these nanomaterials, with the
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Table 5. Summary of the Published Efforts on Biodistribution and Clearance of UCNs in Animal Models

limited knowledge available from the literature to date. Several
questions regarding the ultimate fate of these nanomaterials in
the system, the possibility of their degradation into toxic
byproducts, and their effects following prolonged residence in a
AR
particular organ still remain unanswered. Long-term compara-
tive studies on the biodistribution, metabolism, degradation,
and toxicity profiles of various UCNs of different size, charge,
and surface functionalities are necessary to evaluate the
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Figure 29. (A) Real-time in vivo UCL imaging of athymic nude mice after intravenous injection of PA-UCNs (15 mg kg−1) at different time points.
Column 3: overlays of UCL and brightfield images of mice. Column 6: overlays of UCL and brightfield images of dissected mice. (B) Biodistribution
of particles in organs of mice. Error bars were based on triplet measurements. (C) H&E-stained tissue sections from mice 115 days postinjection
with PA-UCNs (a, c, e, g, i, and k) and mice receiving no injection (b, d, f, h, j, and l). Tissues were harvested from heart (a, b), spleen (c, d), liver (e,
f), lung (g, h), kidney (i, j) and blood smear (k, l). Reproduced with permission from ref 270. 2010 Elsevier.

usefulness of this class of nanomaterials before its further
development and application in the clinics.
6. CONCLUSION AND FUTURE PERSPECTIVES
PDT is undoubtedly a promising therapeutic option in the
management of cancer. However, it has still not gained
acceptance as a first line treatment option mainly due to the
shortcomings of classical PSs. The application of nanoparticles
in the field of PDT is an extremely promising avenue for future
technological breakthroughs. Nanoparticles can act as carriers
of hydrophobic PSs and transport high PS “payload” to the
tumor site via the EPR effect. Additional surface modification
and functionalization with targeting moieties could further
enhance the selective accumulation of the PS loaded nano-
particles at the target site. In recent years, much effort has been
channelled in the development of multifunctional theranostic
agents where a number of diverse diagnostic and therapeutic
functionalities have been incorporated in a single nanoplatform
for a “see and treat” approach. The versatility of such agents
allows delivery of drugs to the diseased/target site with
enhanced spatial specificity while achieving simultaneous real-
time imaging, monitoring, as well as therapeutic capabilities.
While, an in-depth knowledge and understanding of nanoma-
terial chemistry, nanoscale manufacturing and synthesis has
evolved tremendously in order to develop novel nanoplatform
incorporated with sophisticated functionalities, what is now
AS
lagging behind is a thorough understanding of how they behave
as a collective entity and affect complex biological systems. In
this regard, there is an urgent need for in-depth characterization
of such multifunctional therapeutic agents to check if they
cross-talk, quench, or interfere with each other’s capabilities
impeding the overall outcome. In addition to that, standardized
physicochemical characterization, validation, and safety proto-
cols must be established to achieve pure and homogeneous
nanoparticles that can yield precise and reproducible results.
Furthermore, the fact that none of such multifunctional
theranostic agents have been approved by FDA for clinical
application is undeniably worrying. Again, the major bottleneck
for the translation of these theranostic agents into clinical
practice is the absence of standardized procedures to assess its
in vivo biodistribution, detailed pharmacokinetic pharmacody-
namic (PK−PD) analysis, short-term and long-term effects of
the nanomaterials in vivo, as well as its clearance from the
system. This was exactly the reason behind the establishment of
National Characterization Laboratory by the U.S. National
Cancer Institute (NCI) in 2004, which has developed
standardized evaluation protocols for rigorous characterization
of nanoparticle’s physicochemical properties, in vitro immuno-
logical and cytotoxic characteristics, as well as administration,
distribution, metabolism, and elimination (ADME)/toxicity
profiles in animal models; to facilitate the regulatory review of
nanotechnology-based cancer therapeutics and fast track the
clinical trial process. Today, a decade later, more than 300
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Chemical Reviews
nanoparticle based cancer therapeutics have been characterized
with many more in the pipeline. More such regulatory agencies
that provide a “one-stop” solution for systematic step-by-step
characterization of nanomaterial need to be established, given
the vast number of promising efforts from both academia and
the biopharmaceutical industry.
In this review, we have provided a comprehensive account on
the fascinating development of various nanoparticles and their
application in PDT. As most of the classical PSs have
limitations such as poor solubility under physiological
conditions, undesirable pharmacokinetics, low tumor selectivity,
a suitable nanoplatform carrying the PSs is expected to resolve
these problems to a certain extent. Among the various
nanoconstructs, polymeric (synthetic or natural), liposomal,
or dendrimeric nanoparticles are biocompatible and biodegrad-
able, and hence have the added advantage of being enzymati-
cally hydrolyzed and excreted from the body, minimizing long-
term accumulation.66 These nanoparticles are very versatile due
to the existence of a variety of polymers and manufacturing
methods, and thus the chemical composition and architecture
of the nanoparticles can be customized to accommodate PSs
with varying degrees of hydrophobicity, molecular weight, or
charge.67b,274 Furthermore, the surface properties, morpholo-
gies, and compositions of polymeric matrices can be easily
optimized to achieve controlled degradation of the polymer and
drug release kinetics.275 While these nanoparticles can be
tailored to carry multiple functionalities such as targeting
moieties or additional imaging and therapeutic capabilities,
nonbiodegradable nanoparticles provide in-built multifunc-
tional capabilities due to its intrinsic properties. Some
nanoparticles such as fullerenes, TiO2, and ZnO can also be
excited by light of a specific wavelength to induce generation of
ROS. A third type can transduce energy to the accompanying
PS via FRET, in order to excite PS indirectly at wavelengths
that it does not normally absorb, such as X-ray nanoparticles,
QDs and gold nanorods. Although TP-PDT allows deep-tissue
penetration via simultaneous excitation with two low-energy
NIR photons, the treatment is restricted to a tiny tissue volume
resulting in prolonged treatment time, thus limiting its clinical
use. On the other hand, PDT using UCNs doped with PS is
becoming the new sensation in the field with clear advantages
over most other nanoparticles. UCNs act both as carriers of PS
as well as enables indirect excitation of the accompanying PS
with upconverted light upon excitation with low energy NIR
light. Recently, there has been a growth in the amount of
literature available in this area which clearly depicts the growing
interest, clear advantages, and possibility of further develop-
ments in the field. However, long multistep reaction methods
associated with UCN synthesis and PS loading often leads to
batch to batch differences, making it difficult to compare
results. This is because even subtle changes in composition
arising from minute deviations in the manufacturing process
could substantially affect the PK, biodistribution, and toxicity in
vivo.276 Thus, stringent quality control measures in terms of
uniformity in particle composition, size, surface chemistry, PS
loading, etc. are necessary for commercialization of this
technology. Further simplification of the synthesis steps,
standardization of nanoparticle preparation, testing and
validation protocols, utilization of automated nanoparticle
fabrication processes while implementing good manufacturing
practices would allow greater consistency in the quality of the
UCNs synthesized, besides enabling easy scaling up for
commercialization. Presently, much of the research is still
AT
Review
being carried out at the solution- and cell-based level. Solution-
based studies help to predict nanoparticle toxicity by assessing
the particle size, size distribution, morphology, composition,
dispersion, surface area, surface chemistry, and surface
reactivity. It is to be noted that nanoparticles behave differently
in different solutions and its dispersion in solutions rarely
reflect its distribution at the primary particle size. Nanoparticle
agglomeration could lead to size-, surface area-, and dose-
dependent toxicity.277 Furthermore, it raises concerns about
efficiency of ROS production due to the altered photophysical
characteristics of the agglomerated PS loaded UCNs. Hence, it
is highly essential to study its dispersion characteristics and
stability in physiologically relevant buffers over a period of time,
before its characterization in vitro. However, this is hardly
performed in any study until date, perhaps due to which its
efficacy seem to be undermined under in vitro and in vivo
conditions. A better knowledge of the characteristics of UCNs
at conditions close to the physiological setting will further
streamline its optimization very early during the evolution of
the construct and steer its development as a better PDT agent.
For effective PDT, optimization of the PDT parameters such
as the DLI, laser dosimetry including the fluence, fluence rate,
and time of irradiation is necessary. For PDT using UCNs, a
laser power density ranging between 0.4 W cm−2 to 134 W
cm−2 and fluence >1000J cm−2 have been typically used.
However, in clinical application and PDT experiments using
conventional PS, power density <1 W cm−2 and fluence <1000
J cm−2 will probably be enough for efficient tumor cell death.278
Apparently, UCN based PDT works via anti-Stokes emission
process that is reported to have a quantum yield of less than 1%
in general.279 Hence, the efficiency of this process is less than
direct excitation of PS by visible light source in conventional
PDT. Coating of an undoped shell (such as NaYF4 or CaF2)
around the UCN core have shown to improve quantum yield
by the suppression of surface quenching effects.265 The low
quantum yield also results in unsatisfactory brightness of the
UCNs under low excitation power densities. The brightness of
the UCN is dependent on the 4f-4f optical transition
probabilities of the lanthanide ions, which are remarkably
affected by their local crystal field.280 Consequently, fine-tuning
the local crystal field of the lanthanide ions is an effective
strategy to increase the UCL of lanthanide ion-doped
nanocrystals.281 Doping the UCNs with other elements or
dyes with strong absorption and further optimization of the
doping ion ratios,282 varying the crystal phase of the
nanocrystals,283 fabricating core−shell structures,284 and
utilizing metal-enhanced fluorescence285 are among the
methods being explored to improve the brightness and
quantum yield. Currently, much of the research in this field
uses an NIR excitation wavelength of 980 nm that matches the
absorption of the commonly utilized sensitizer Yb3+. However,
water has strong absorption around 970 nm, which could lead
to heating up of biological tissues when excited around this
wavelength. In addition to the heating effects, a significant
reduction in the penetration depth is anticipated as the 980 nm
light gets attenuated, while traveling through tissues. A shift in
excitation wavelength from 980 nm to a lower NIR wavelength
that does not cause a thermal effect could be more clinically
appropriate. Efforts to excite UCNs using another absorption
peak of Yb3+ at 915 nm were accompanied by a reduction in
excitation efficiency.286 As an alternative, NIR dye sensitized
UCNs were reported to harvest light in the shorter NIR
wavelengths (740 to 850 nm), where the surface bound NIR
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Chem. Rev. XXXX, XXX, XXX−XXX
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dye served as an antenna that absorbed light in the specified field, it is imperative to have frequent facilitations between
spectrum enabling FRET to UCN core to produce an scientists, academicians, engineers, clinicians, and industrial
upconverted light.287 However, such NIR absorbing organic innovators to share and discuss the opportunities and
dyes may succumb to photobleaching, making them unsuitable challenges in the field, so as to bring PDT to the forefront of
for long-term imaging/activation applications. Recently, codop- oncological diagnosis and intervention. The future of PDT lies
ing with a second sensitizer such as neodymium (Nd3+) (that in the development of a single versatile and efficient
has a absorption maxima around 800 nm) was shown to absorb nanoparticle that encompasses its applicability in both
NIR light at 800 nm following which it sensitizes Yb3+, which bioimaging as well as PDT.
then initiates a typical upconversion process with the activator
ions.282a,288 Although, this technique seem quite promising in AUTHOR INFORMATION
alleviating the short penetration depth and overheating effect
associated 980 nm excitation, no in vitro or in vivo work using Corresponding Author
such UCNs in PDT has been reported until date. *E-mail: biezy@nus.edu.sg.
PDT using UCN is definitely a promising treatment Notes
modality; however, currently it faces many limitations apart
from the issues pertaining to the UCN itself. Optimal PS The authors declare no competing financial interest.
loading is one of the critical factors that determine the Biographies
efficiency of this modality. Currently, majority of the in vivo
studies demonstrated administration of the nanoconstructs via
intratumoral route. This was partly due to premature leakage of
the PS resulting in reduction in the amount of PS ultimately
reaching the target site, and partly due to the lack of specificity
of the nanoconstructs to the tumor tissue and accumulation in
other organs leading to acute toxicity. Although several studies
reported modification of the UCN surface with biocompatible
materials and addition of tumor homing motifs for selective
accumulation of the nanoparticles to the tumor, the percentage
of the injected dose that reached the tumor following
intravenous injection was relatively low, resulting in sub-
optimal PDT and tumor recurrence in a majority of the studies.
This indicates the importance of optimizing the molecular
weight, surface structural conformation, and surface coverage
ratio of the biocompatible polymer like PEG that is grafted to Sasidharan S. Lucky graduated with a Master’s degree in
improve stealth properties of UCNs.289 Furthermore, it is well- Biotechnology in 2004. Thereafter, she worked in Tan Chin Tuan
known that the presence of targeting ligands at high densities Laboratory of Optical Imaging and Photodynamic Therapy for over 4
could promote nonspecific interactions with endothelial and years, before embarking on a Ph.D. under the guidance of Prof. Yong
other noncancerous cells and trigger immune response Zhang and Prof. Khee Chee Soo at the NUS Graduate School of
resulting in opsonization-mediated clearance of nanoparticles Integrative Science and Engineering, National University of Singapore.
from the circulation.290 Development of truly tumor “homing” Her research focuses on the application of upconversion nanoparticles
nanoparticles might require stringent optimization of the for photodynamic therapy of cancer.
number of targeting ligands on the surface of nanoparticles,
targeting multiple receptors that are overexpressed in the tumor
as well as tumor vasculature, and inclusion of cleavable
“antibiofouling agents” that mask the targeting ligands until it
reaches the tumor. At the same time, it must be noted that
most of the in vivo toxicology profiling is limited to small
rodents like mice, which may not be translatable to the clinics.
Moreover, often the lengths of such studies that evaluate the
therapeutic and toxicological effects are relatively short (2 week
and 3 months respectively). As UCNs are generally unable to
degrade into biologically benign components, there exists a lot
of uncertainties in terms of their long-term therapeutic effects,
systemic toxicity as well as clearance. Extensive PK−PD
analysis, together with long-term toxicity and clearance studies,
remains unexplored and deserves immediate attention. It is
clear that UCNs have the potential of being developed into a Khee Chee Soo obtained his medical degree in 1975 from University
multimodality theranostic probe, so that it can be simulta- of Singapore’s Medical School and has many years of experience in the
neously used both as a multimodality imaging tool as well as a clinical application of PDT for treatment of head and neck cancers. He
therapeutic agent. Nevertheless, there is still much room for is the Director of National Cancer Centre Singapore, Professor of
improvement in terms of reduction in size of the construct, Surgery at Yong Loo Lin School of Medicine and Vice Dean of
enhancing the upconversion efficiency, stability in PS loading, Clinical and Faculty Affairs at Duke-NUS Graduate Medical School
optimization of PDT parameters, and thereby increasing the Singapore. He continues to champion the use of PDT as an alternative
PDT efficacy. Owing to the multidisciplinary nature of this cancer treatment option by actively recruiting head and neck cancer

AU DOI: 10.1021/cr5004198
Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
patients for pilot clinical trials of fluorescence diagnostic imaging and
photodynamic therapy.
Yong Zhang is a Professor at Department of Biomedical Engineering,
National University of Singapore (NUS), a senior Faculty member of
NUS Nanoscience & Nanotechnology Initiative (NUSNNI/Nano-
Core) and NUS Graduate School for Integrative Sciences and
Engineering (NGS). His current research interests include nano-
biophotonics, nanomedicine, biomedical microdevices, and tissue
engineering.
ACKNOWLEDGMENTS
The authors would like to acknowledge the funding support
from National Medical Research Council (NMRC) CBRG
grant (CBRG13nov052) and Agency for Science, Technology
and Research (A*STAR) Biomedical Engineering Programme:
Grants R-397-000-128-305 (NUS) and BMRNI11133
(NCCS). S.S.L. is a recipient of NGS scholarship from NUS
graduate school (NGS) for Integrative Sciences and Engineer-
ing, National University of Singapore.
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