Metallic Nanoclusters for Cancer Imaging and Therapy
Background: Nanoclusters are made of a few to tens of atoms with a size below 2 nm. Compared with nanoparticles, they exhibited excellent properties, such as tunable fluorescence, ease of conjugation, high quantum yield and biocompatibility, which are highly desired in the development of cancer nanotheranostics. Hence, the metallic nanoclusters have emerged as a newcomer in cancer nanomedicines. This review aims to summarize recently developed approaches to preparing metallic nanoclusters, highlight their applications in cancer theranostics, and provide a brief outlook for the future developments of nanoclusters in nanomedicine. Method: We carried out a thorough literature search using online databases. The search was focused on a centered question. Irrelevant articles were excluded after further examination and directly relevant articles were included. The relevant articles were classified by the subjects and the information from these articles was synthesized. Results: One hundred and forty-three articles were included in this review. About eighty articles outlined the development in the synthetic methods of nanoclusters. The synthesis approaches include chemical reduction, photoreduction and so on. The progress in the application of gold and silver nanoclusters to cancer theranostics was described in fifteen and eight articles, respectively. The rest articles were about the advancements in the use of other metal nanoclusters and nanocluster nanocomposites as cancer theranostic agents. Conclusion: This review summarizes the synthesis and use of metallic nanoclusters or their nanocomposites as cancer theranostic agents. It confirms their importance, advantages and potentials in serving as a new generation of cancer theranostics in clinics.
INTRODUCTION<!>THE SYNTHESIS OF NCs<!>Chemical Etching<!>Photoreduction Method<!>Electrochemical Synthesis<!>Sonochemical Synthesis<!>Microwave-assisted Synthesis<!>Chemical Reduction Approach<!>Thiols Used in Chemical Reduction Approach<!>Other Small Molecules Used in Chemical Reduction Approach<!>Proteins Used in Chemical Reduction Approach<!>Polymers Used in Chemical Reduction Approach<!>DNA Used in Chemical Reduction Approach<!>APPLICATION OF NCs IN CANCER THERAPY AND IMAGING<!>Au NCs as an Agent in Cancer Theranostics<!>Ag NCs as an Agent in Cancer Theranostics<!>Other Metal NCs as an Agent in Cancer Therapy and Imaging<!>NCs Nanocomposites as an Agent in Cancer Therapy and Imaging<!>SUMMARY AND PERSPECTIVE
<p>Cancer is the major mortal sickness worldwide and has attracted much
attention of the public. The stage of the tumor at which it is discovered is
strongly associated with the survival of cancer patients. The identification and
detection of cancer at an early stage before metastasis is very important for the
cure of cancer. Hence, there is a pressing need in the development of rapid,
specific, and sensitive methods for cancer diagnostics. Cancer nanomedicine, which
integrates molecular diagnosis, medical imaging, and cancer cell-targeted therapy in
a single platform, is a typical and desired method in the application of
nanotechnology in cancer theranostics [1,
2]. All kinds of inorganic nanoparticles
(e.g., metallic nanoparticles, quantum dots,
Fe3O4 nanoparticles) and polymeric nanoparticles
(e.g., dendrimers and micelles) are used in cancer nanomedicine
because they can serve as agents with specific structures and functions suitable for
cancer imaging, diagnosis, and therapy [3,
4].</p><p>In contrast to nanoparticles, nanoclusters (NCs) are a relatively newcomer in
nanomedicine. NCs are made of only a few to tens of atoms and the size of their
cores is usually below 2 nm [5]. Since the
ultra-small size of the NCs is close to the Fermi wavelength of electrons, the
continuous band turns to discontinuous and becomes molecule-like discrete energy
levels. Consequently, NCs exhibit a multitude of special optical and electronic
characteristics, such as highest occupied molecule orbital-lowest unoccupied
molecule orbital (HOMO-LUMO) transition, tunable fluorescence, large Stokes shift,
molecular chirality, magnetism, and quantized charging. Nanoclusters (NCs) as a type
of emerging nanomaterials have received much attention in catalysis [6], detection of biomolecules [7] and heavy metals [8], biosensors [9], and solar
cells [10]. Recently, much work has been done
to explore the applications of NCs in cancer imaging and therapy due to their good
stability, facile synthesis, excellent biocompatibility, ease of conjugation, and
renal clearance [11, 12].</p><p>NCs display excellent fluorescence due to their sturdy photostability, huge
two-photon absorption cross section, and strong two-photon luminescence compared
with other fluorescent agents [13]. For
example, organic dyes, commercially available fluorophores, are usually used as
photon imaging agents in several clinical fields ranging from cancer imaging, and
radiotherapy to targeted drug delivery. But they are limited by a few inherent
drawbacks including photobleaching, native function perturbation by the labeled
biomolecules, and biotoxicity [14].
Semiconductor quantum dots are attractive candidates as fluorescent labeling agents
for biological processes because of their photostability, small size, narrow
emission profile, and high quantum yield. But they contain heavy metals that usually
show potential dangers for body [15]. In
addition to acting as a fluorescent (FL) imaging agent, NCs can serve as probes for
computed tomography (CT) and positron emission tomography (PET) imaging with their
inherent properties or conjugated with other imaging probes [16]. For example, Au NCs can serve as FL and CT imaging
probes thanks to their fluorescence, large atomic number, and electron density
[17]. Thus, NCs are a good candidate for
multimodal cancer imaging platforms.</p><p>The inherent properties of NCs including ultrasmall size with narrow size
distribution and ease of conjugation not only offer the tunable fluorescence to NCs,
but also make them very suitable for the application in cancer therapy. Traditional
nanoparticles cannot escape from the kidney barrier and result in severe side
effects owing to their aggregation in the liver and spleen, but NCs can be
effectively removed from the body via renal clearance [18, 19].
Besides, NCs usually are stabilized with surface ligand such as proteins [20], thiols [21], and polymers [22], allowing
them to be facilely conjugated with other imaging agents, anticancer drugs, or
physiotherapy agents in the preparation of multimodal theranostic platforms.
Therefore, NCs rise as a burgeoning nanomaterial in developing cancer
nanomedicine.</p><p>In this mini-review, we first summarize the synthesis and properties of NCs.
Then we discuss some representative applications of NCs in cancer imaging and
therapy reported in recent years. Finally, we point out the challenges the field of
NC-based cancer nanomedicine before NCs can be used in clinic. For detailed
information on the functionalization, cytotoxicity, biodistribution and clearance of
NCs, the readers are suggested to refer to another recent review [18].</p><!><p>In 1969, Mooradian [23] et
al. discovered the photoluminescence of bulk, single crystal wafer and
thin film of Au and Cu. Though the Au NCs capped with phosphine were studied in
1960s [24], the work was delayed by their
limited stability. Then the photoluminescence of Ag NCs in zeolite [25–27],
inert gases [28, 29] and inorganic glass [30, 31] was reported. The
Henglein group [32] first synthesized Ag NCs
and studied their properties [33–38] except for photoluminescence due to their
limited stability. In 1994, Brust and Schiffrin [39] developed a chemical reduction approach to prepare
alkanethiol-stabilized Au NCs. When these thiolated Au NCs were dried and
re-dispersed, their properties were not changed. A series of thiolate- protected Au
NCs were prepared by using the chemical reduction method. Though Ag was easy to be
oxidized, Dickson [40] et
al. first synthesized stable hydrosoluble Ag NCs by using dendritic
polymer (Polyamidoamine, PAMAM) in 2002 and triggered the use of Ag NCs as probes.
Then, Baskakov [41] et al.
first synthesized water-soluble fluorescence Ag NCs with peptides as templates and
applied them in biological imaging successfully. In 2009, the Xie group [42] synthesized red fluorescence Au NCs by
using bovine serum albumin (BSA) and propelled the application of metal NCs in
biomedicine.</p><p>The unique optical properties, good biocompatibility and easy conjugation of
NCs have attracted the interest of physicists, chemists, material scientists and
biologists. Dramatic advances have been achieved in developing new synthetic
methods, understanding their properties and achieving biocompatibility in the last
two decades. In general, there are two strategies to produce NCs:
"bottom-up" method and "top-down" method. In the
"top-down" process, a ligand (e.g. thiol compounds) is used to etch
pre-formed nanoparticles into ultra-small clusters. The "bottom-up"
approach means to assemble a few metal atoms into a cluster in the presence of a
ligand or template, which could in turn stabilize the resultant NCs. The
"bottom-up" method includes chemical reduction, photoreduction,
microwave-assisted synthesis, sonochemical synthesis and electrochemical synthesis.
We will discuss these methods and highlight some examples in the following
sections.</p><!><p>Nanoparticles with a core size of 2–4 nm can be etched into NCs
by excess ligands. For example, Au NCs were prepared from the etching of Au NPs
by thiol ligands (e.g. 11-mercaptoundecanoic acid); this is pivotal because
their size and optical properties depended on the thiol compounds [43]. Two possible routes for etching are
proposed by researchers. In the first approach, atoms are detached from the
nanoparticles surface by the ligand and then form NCs via
strong atom-atom interactions [44]. In
the second approach, the nanoparticles are etched by the ligand and their sizes
are reduced in steps till proper NCs have formed [45].</p><p>Stable nanoparticles are generally tailored into NCs by ligands such as
octanethiol or bidentates [46, 47]. The etching process usually occur at
the interface between water and oil [48,
49]. To avoid using poisonous organic
solvents such as toluene and tetrachloro- methane, Liu et al.
[44] used a gelatin framework to
stabilize the precursor Ag nanoparticles in water solution, and prepared Ag NCs
by etching Ag nanoparticles with dihydrolipoic acid (DHLA) in an aqueous
solution. In addition to the ligands, the precursor can also induce
nanoparticles etching. AuNPs stabilized by di- dodecyldimethylammonium bromide
were etched by Au precursors (HAuCl4 or AuCl3) into
smaller NCs (AuNC@DDAB) (3.17±0.35 nm) [47]. The hydrophobic AuNC@DDAB could be further etched by exchange
of dihydrolipoic acid (DHLA) to AuNC@DHLA (1.56±0.3 nm) and became water
soluble.</p><p>Using ligands to etch nanoparticles, the weak nucleophilic ligands on
the surface of nanoparticles are generally replaced by strong ones [50, 51]. The strong nucleophilic ligands are rarely exchanged by weak
ones [52]. Jin et al.
[53] reported a similar process, as
shown in (Fig. 1A). The Au12 shell was
peeled off from phenylethanethiolated core-shell
Au25(SC2H4Ph)18 to prepare
Au13(PPh3)8(SC2H4Ph)32+
by etching PPh3. The
Au13(PPh3)8(SC2H4Ph)32+
could be further etched into Au11(PPh3)
8(SC2H4Ph)2+ and finally into biicoso-
hedral
Au25(PPh3)10(SC2H4Ph)5Cl22+.</p><!><p>In 2001, Dickson et al. [54] first demonstrated that NCs could be produced by
photoreduction without the addition of reduction agents. Metal ions encapsulated
in microgel could efficiently and spontaneously form NCs under sunlight. Aqueous
microgel dispersions can produce H•,OH•,and perhaps organic
radicals by the irradiation of UV. Metal ions were reduced into metal atoms by
these radicals [55]. Banerjee et
al. [56] prepared a stable
hydrogel with silver ions encapsulated using N-terminally Fmoc-protected
dipeptide and then formed silver NCs upon the sunlight irradiation at a
physiological pH value (7.46). Later, the same group used an amino acid that was
Fmoc-protected at the N- terminal to form a hydrogel and then prepare silver NCs
under a similar condition [57]. In
addition to hydrogels, polymer is the other excellent template when the
photoreduction method is used to synthesize NCs. For example, Ras and coworkers
[58] mixed polystyrene-block-poly
(methacrylic acid) block copolymer (PS-b-PMAA) and silver salts
in selected organic solvents to prepare NCs with the irradiation of a visible
light. When poly(methacrylic acid) was used as a template, copper, silver and
gold NCs functionalized with pentaerythritol tetrakis 3-mercaptopropionate were
synthesized by a soft photoreduction of their inorganic precursors [59]. Small molecules can also stabilize
NCs. With the use of small molecules (D-penicillium and L-penicillium), Sun and
coworkers [60] synthesized Ag NCs though
a photoreduction process.</p><p>Solid templates can be used to facilitate the formation of NCs by
photoreduction. Takagi and coworkers [61]
reported a photosensitized template reduction method to prepare gold NCs.
Porphyrin molecules were assembled on the clay surface and formed a unique
pattern. Then gold NCs were deposited on its surface via the UV
photoreduction of gold precursors. The gold NCs were assembled into a pattern
defined by the pattern of porphyrin molecules. The deposition density and
aggregation of NCs could be precisely controlled by this method without
protective agents. The authors proposed a mechanism for the photosensitized
template reduction method. Specifically, under the UV irradiation, Tetrakis( 1
-methyl-4-pyridiniumyl)porphyrin (TMPyP) on the clay surface was first reduced
to TMPyPred though the electron transferring from trimethylamine
(TEA), then TMPyPred reduced the gold precursors located nearby into
gold NCs.</p><!><p>Electrochemical synthesis is another method for preparing metal NCs.
Reetz and co-workers [63] first developed
this method in 1994. During the progress of electrochemical synthesis, metal
ions are produced from a sacrificial anode and reduced into metal atoms at the
cathode. These metal atoms further aggregated into NCs in the presence of
surfactants or ligands. It is easy to manipulate the NCs size though controlling
the current, voltage, concentration of stabilizers, and electrolyte
etc. Based on this mechanism, copper NCs (Cun,
n<≈14) were synthesized by a simple electrochemical technique with
tetrabutylammonium nitrate as a stabilizer [64]. These copper NCs displayed photoluminescence in the visible
range. Both apolar and polar solvent could be employed to disperse these Cu NCs,
and these dispersed Cu NCs were very stable for years. Lo- pez-Quintela and
coworkers [65] produced well defined Ag5
and Ag6 NCs by one-pot electrochemical method. These Ag5 and Ag6 NCs protected
by dode- canethiol/tetrabutyl ammonium (DDT/TBA) were soluble in both organic
and aqueous solvents and monothiol Ag NCs were remarkably stable.</p><p>A solid template can be used in an electrochemical reduction to prepare
metal atomic clusters. The metal deposition method was introduced on ordered
alumina pores with pulsed electrodeposition by Gosele et al.
[66]. Gonzalez et
al. [62] later modified this
process and used porous alumina to produce NCs (Fig. 1B). Briefly, a hexagonally ordered porous alumina substrate
was generated. The diameter, depth and interpore spacing of the nanoporous
structure were 10 nm, 1 Î¼m and 35 nm, respectively. After being immersed
into a metal plating bath, the system was subjected to a pulsed
electrodeposition program. Gold and nickel NCs were generated at the bottom of
pore in nanoporous alumina. This strategy showed many advantages including
simplicity, high stability against aggregation, and cluster size control. In
addition, the metal NCs provide a non-blocked active surface because of the
absence of stabilizers.</p><!><p>Sonochemical synthesis is an important synthetic method in materials
chemistry. Ultrasound is irradiated into a liquid and triggers the nucleation,
development, and break-up of bubbles (acoustic cavitation) [67]. The acoustic cavitation is the source of the
chemical effects resulted from a high intensity ultrasound. For example, the
acoustic cavitation can generate localized high pressures and hot spots with
temperatures up to ~5000 K [68].
As a consequence, highly reactive species, such as radical
HO2•, H•, OH•, and possibly free electrons are
generated during the irradiation of ultrasound [69, 70]. These highly
reactive species can reduce metal ions into metal atoms. Suslick and coworker
[71] prepared light-emitting, stable,
and water-soluble silver NCs by a handy sonochemical process with
polymethy-lacrylic acid as a ligand. The properties of the Ag NCs could be
regulated by varying the time of sonication, ratio between two species
(carboxylate groups and Ag ions), and the molecular weight of polymer.</p><!><p>Microwave irradiation is able to rapidly generate homogeneous heating
and is widely used in the synthesis of organic compounds and inorganic
nanocrystals. Homogeneous and rapid heating in a solution induced by the
microwave irradiation can offer homogeneous nucleation and shorter
crystallization times. Obviously, microwave irradiation is also very suitable
for the synthesis of uniform and monodisperse NCs. Zhu and coworkers [72] prepared highly fluorescent
water-soluble Ag NCs in by means of microwave irradiation using polymethacrylic
acid sodium salt as templates. The reaction was fast and the reaction time was
reduced to seconds. The resultant Ag NCs are highly stable, monodisperse, highly
fluorescent under visible light.</p><!><p>The chemical reduction approach is a widely used method in preparing
NCs. Different from the other methods described above, reducing agents are
needed in chemical reduction process. Brust-Schiffrin method and their variants
are the representative chemical reduction synthetic strategies. Whyman and
coworkers [39] first developed the
Brust-Shiffrin method in 1994. They synthesized gold NCs by using
BH4- as a reducing agent and
C12H25SH as a protecting ligand. This synthesis can be
described by the phase transfer of metal precursors (reaction 1) followed by the reduction of metal ions
(reaction 2): (1)AuCl4−(aq)+N(C8H17)4+(toluene)→N(C8H17)4+AuCl4−(toluene)
(2)mAuCl4−(toluene)+nC12H25SH(toluene)+3me−→4mCl−(aq)+(Aum)(C12H25SH)n(toluene)</p><p>A modified Brust-Schiffrin method directly reduced metal ions in
solution by using one-phase system without the phase transfer of metal
precursors. There are many elements that can affect the core size and the
surface properties of metal NCs, such as the ratio of ligand to metal, reducing
agents, the stabilizing ligands, time and temperature of reaction, and the pH
value. The nature of the protecting ligands is very important for the
application of NCs in biomedicine. Phosphine, thio-lates, polymers, proteins,
and DNA have been widely used as templates and/or protecting ligands in the
chemical reduction process.</p><!><p>Thiols are often used as self-assembled monolayer (SAM) agents to
modify the gold surface. The interaction between the thiolate ion and gold
atom is similar to that between two gold atoms, making it possible for
thiolate ligands to break Au-Au bond and consequently form S-Au bond. Thus,
thiols as protecting ligands are widely used to prepare and functionalize Au
NCs. Jin and coworkers [73]
synthesized barrel-shaped chiral
Au130(p-MBT)50 NCs, in which
p-MBT represents 4- methylbenzenethiolate, and
determined its structure by X-ray crystallography (Fig. 2A). AuI(p-MBT) complexes were
first reduced by NaBH4 into a mixture of
Aux(p-MBT)y NCs, then
Au130(p-MBT)50 as the most stable
form of NCs was selected by the treatment of excess p-MBT
thiol at 80°C. In addition to NaBH4, the other inorganic
reduction agents were applied to prepare metal NCs. For instance, Xie
et al. [77]
developed a two-step reduction approach using carbon monoxide (CO) as a
reducing agent to synthesize highly luminescent gold NCs
(Au22(SR)18, where SR represents a thiolate
ligand). First, Au-glutathione complexes were reduced into
Au18(SR)14 by CO. Then the final production of
Au22(SR)18 was achieved by a pH-induced
aggregation of small Au-glutathione complexes onto
Au18(SR)14. Polymers can also be used as a
reducing agent and template to synthesize NCs. For example, Ren and
coworkers [78] synthesized Au:P3HT
(P3HT denotes poly(3-hexylthiophene-2,5-diyl)) nanohybrids by using
polystyrene-b-poly(4-vinylpyridine) (termed
PS-b-P4VP) as a reducing agent. The nanohybrids
displayed unique thermally responsive optical behaviors, optoelectronic
properties and charge-transfer controlled magnetism. To date, a number of
thiolate-protected gold NCs (e.g. Au18 [79], Au30 [80], Au133 [81], etc) have been prepared and
their structures have been confirmed</p><p>Compared with Au NCs, Ag NCs are unstable because their atom cores
have a higher tendency of being oxidized. However, they are cheaper than Au
NCs and show excellent photoluminescent properties as same as Au NCs. A lot
of Ag and Ag-rich alloy NCs protected by thiolate ions have been prepared.
Recently, Bakr's group [82]
synthesized monodisperse Ag25 NCs protected with
2,4-sdaxsdazdimethylbenzenethiol ligands. A series of
[Au12+nCu32(SR)30+n]4−
(n= 0, 2, 4, 6) NCs were prepared and the evolution process of their
structures was investigated by Yang and coworkers [83]. The novel photoluminescent properties of
metal NCs stabilized by thiol are generally based on the aggregation-induced
emission (AIE) phenomenon [84]. The
Jin group [85] reviewed almost all Ag
and Ag-rich alloy NCs protected by thiolate and their unique physicochemical
properties.</p><!><p>Inspired by the success of the thiolate-stabilized NCs preparation,
other small molecules such as phosphines and alkynyl were applied to protect
NCs. Wang and coworkers [74] prepared
a novel Au19 NCs composition:
[Au19(PhC≡C)9(Hdppa)3](SbF6)2.
They were composed of a centered icosahedral Au13 core and coated
by three V-shaped PhC≡C−Au−
C≡C(Ph)−Au−C≡CPh motifs (Fig. 2B). More recently, Zheng et
al. [76] prepared an
intermetallic Au24Ag20 superatom nanocluster
Au24Ag20(2-SPy)4(PhC≡C)20Cl2
which displayed three kinds of anionic ligands, including phenylalkynyl,
2-pyridylthiolate, and chloride, on its surface at the same time as a
concentric three-shell (Fig. 2D).
Thanks to the co-presence of three different surface ligands, these NCs
exhibited a site-specific surface and could be easily functionalized though
these surface ligands.</p><!><p>Proteins are biological macromolecules and are widely used to
produce NCs with improved biocom-patiblity. Xie et al.
[42] first applied bovine serum
albumin (BSA) as both a protecting agent and a reducing reagent and prepared
fluorescent gold NCs. Motivated by Xie's research, Irudayaraj
et al. [86] used
denatured bovine serum albumin (dBSA) and synthesized highly stable
fluorescent Ag NCs. So far, a number of proteins have been successfully
adopted to prepare NCs such as lysozyme [87], insulin [88],
trypsin [89], ovalbumin [90]. Eggshell membrane is a solid
protein. It was found that the membrane could serve as a unique platform to
generate fluorescent silver and gold NCs [91]. Moreover, peptides and amino acids are excellent templates
and/or reducing agents. Ogawa et al. [92] designed and synthesized α-helical
coiled coils in the forms of peptide trimers, tetramers, and hexamers; these
peptide polymers could be specifically combined with 6, 8, and 12
Ag+ ions. When treated by the NaBH4, a set of
peptide-capped Ag NCs were produced. They further demonstrated that these
NCs exhibited a strong visible fluorescence, and that their emission
energies were associated with the number of metal atoms included by the NCs.
Glutathione (GSH) includes a γ-amido bond and a thiol and can serve
as a ligand and reducing agent. Luo et al. [93] mixed aqueous solutions of HAuCl4
and GSH to synthesize strongly orange-emitting Au NCs. They reported that
the strong luminescent emission was resulted from aggregation-induced
emission (AIE). Bertorelle and coworkers [94] reported a simple cyclic reduction method to synthesize
silver:glutathione (Ag:SG) NCs. In particular,
Ag15(SG)11 NCs stably emitted a strong light. Roy
et al. [95]
synthesized blue, green, and red emitting silver NCs by using reduced
glutathione as a stabilizer in the presence of NaBH4 at a pH
value of 7.46 and a temperature of 140°C. These NCs displayed very
exciting fluorescence characteristics with large Stokes shifts and a high
quantum yield.</p><!><p>The other categorized ligands for preparing NCs are polymers, such
as dendrimers, polyelectrolytes. Mat- toussi et al. [96] synthesized bidentate ligands by
conjugating a poly (ethylene glycol) short chain or a zwitterion group on a
lipoic acide (LA), i.e. LA-PEG and LA-Zwitterin. Then they successfully
prepared a series of intense fluorescent Au NCs employing these ligands in
the presence of NaBH4. Dispersions of these Au NCs showed
excellent long-term stability and fluorescent lifetimes. In addition, due to
the functionalization with reactive radicals (for instance, carboxylic acid
or amine), these NCs were suitable for common coupling strategies. More
recently, Pal et al. [97] reported a convenient and eco-friendly approach for the
preparation of Ag NCs. In this process, a poly(N-vinylpyrrolidone)
homopolymer acted as a stabilizer and acetonitrile or N,N-dimethylformamide
(DMF) was used as both a solvent and a reducing agent. On the other hand,
polymers can also form micelles which can assemble metal atoms into NCs.
Zhang et al. [98]
prepared an interfa-cially cross-linked reverse micelle by cross-linking a
cationic surfactant with a hydrophilic dithiol. The cationic surfactant was
capped with a triallylammonium headgroup. The interfacially cross-linked
reverse micelle could extract AuCl4- into the
hydrophilic core, followed by the reduction of the captured
AuCl4- into gold NCs without the presence of extra
reducing agent.</p><!><p>DNA oligonucleotides are usually used in the synthesis of NCs. In
2004, Dickson et al. [99] used DNA as a template and synthesized DNA-capped Ag NCs.
Thereafter, various DNA sequences have been applied to synthesize NCs, and
to reveal the mechanism based on which DNA interacts with NCs [100, 101]. Han et al. [100] reported that duplex, hairpin, i-motif and
G-quadruples DNA could stabilize NCs. Besides, the binding affinities of
Ag+ by polymorphic DNA were correlated with the secondary
structure of the DNA templates, with an increasing order (the G-quad-ruplex
< the duplex < the i-motif < the coiled C-rich strand)
[102]. Fluorescence stability of
Ag NCs capped by these polymorphic DNA is related to their binding
affinities and the C-rich strand could stabilize Ag NCs for over 300 h. Wang
and coworkers [101] used DNA
monomers (deoxycytidine, deoxyadenosine, de-oxythymidine and deoxyguanosine
monomers) as the scaffolds to synthesize silver NCs. They demonstrated that
only the silver NCs stabilized by deoxycytidine monomers displayed
fluorescent properties. More recently, Teng et al. [103] synthesized dark silver NCs
protected by DNA and these NCs showed emission fluorescence through DNA
hybridization. A 70-fold increase in the strength of fluorescence was
observed when the two templates (5'-AAAAAAAAAAAAAAA
ACCCCCCCCCCCC-3', 5'-CCCTTAATCCCCAATC ATCTCTTCC-3')
were located on the opposing end of the duplexes. DNA was also used to
prepare other metal NCs such as DNA-capped gold and copper NCs. Yang
et al. [75] used
poly(thymine) (with 20 monomers) as a template to synthesize fluorescent
copper NCs with the reduction of sodium ascorbate (Fig. 2C). Simultaneously, their results indicated
that random single-stranded DNA, including poly adenine (A), poly cytosine
(C), and poly guanine (G) but excluding poly thymine (T), failed to guide
the formation of Cu NCs. Martinez et al. [104] synthesized gold NCs of ~1 nm in
diameter using a hybrid DNA and a dimethylamine borane, which functioned as
a template and a reducing agent, respectively. These gold NCs showed
phosphorescence characteristic of large Stokes shift and microsecond
lifetime.</p><!><p>Metal NCs possess non-continuous energy levels like molecules due to their
ultra-small size being close to the Fermi-wavelength of electrons. They also exhibit
a multitude of excellent properties such as tunable luminescence, large Stokes
shift, ease of conjugation, high quantum yield, extended photostability,
biocom-patiblility. Moreover, they demonstrated a high renal evacuation efficacy.
Therefore, noble metal NCs have been widely studied in the fields of biosensing,
biomedicine as well as biodetection. Besides, functionalized NCs as potential
theranostic agents provide a new chance to clarify drug resistance and enhance cure
effects. According to clinical applications, metal NCs are considered one of the
best cancer therapy and imaging platforms. Much work has been done to explore the
application of NCs in cancer theranostic. In what follows, we will give some
examples to detail the progress made in applying the diversified metal NCs in cancer
therapy and imaging.</p><!><p>Au NCs are an important part of the theranostic approach when dealing
with cancer, they exhibit excellent properties such as stability, chemical
inertness, and a higher atomic number and electron density. Qing et
al. [105] first
investigated the fluorescence properties of Au NCs in a live mice. High
accumulation of Au NCs in tumor areas because of their extremely small
hydrodynamic diameters (~2.7 nm) and the intensive permeability and
retention (EPR) effects indicated that they could act as a tumor probe. The high
photostability of AuNCs provided the possibility of continuous fluorescence
tumor imaging in vivo. Thereafter, Wang and coworkers [106] reported fluorescent gold NCs
biosynthesized in situ. Specifically, micromolar chloroauric acid solutions were
reduced and used to form fluorescent NCs in cell's cytoplasm; ultimately
the NCs were concentrated around nucleoli when they were incubated with only
cancer cells. The process of biosynthesis could proceed in
vivo: injection of a millimolar chloroauric acid though the
subcutaneous region could form Au NCs in xenograft tumors without any obvious
spreading to the surrounding normal tissues. The novel biosynthesis method holds
promise for the use of unique fluorescent NCs as the self-bio-marking of tumors.
In addition to the passively targeting to tumor by the EPR, the folic acid as a
targeting ligand was conjugated with ovalbumin-protected Au NCs by using a
homopolymer N-acryloxysuccinimide as a linker to positively target toward cancer
[107]. The novel nanoconjugate could
be used as a specific fluorescent stain agent for Hela cells.</p><p>In the preclinical and clinical application and research, positron
emission tomography (PET) is a very important radionuclide imaging technology.
Radioactive elements are easily integrated in metal NCs and synthesized as a
two-in-one imaging platform. Liu et al. [108] prepared 64Cu-Au alloy NCs, which
were stabilized with polyethylene glycol (64Cu-AuNCs- PEG350). They
evaluated the pharmacokinetics and systemic clearance in a mouse prostate cancer
model. The integrated 64Cu-Au alloy NCs were rapidly cleared
via renal and hepatobiliary pathway and exhibited low
non-specific tumor retention [108].
Almost at the same time, Chen and coworkers [109] synthesized 64Cu- doped chelator-free gold NCs with
human serum albumin (HSA). The near-infrared (NIR) fluorescence imaging of these
NCs was due to Cerenkov resonance energy transfer (CRET). 64Cu with
radioactivity acted as both the energy donor for NIR fluorescence and the probe
for PET imaging. Au NCs accepted the energy and played a role as NIR
fluorescence imaging agents. In a glioblastoma xenograft model,
64Cu-doped Au NCs displayed efficient PET and CRET-NIR imaging (Fig. 3).</p><p>Au NCs are also a suitable drug delivery carrier because their surface
features are tunable and their excellent capability in loading unique cell
targeting molecules. Supramolecular conjugates of polysaccharide and gold NCs
were prepared by conjugating the cyclo- dextrin-grafted hyaluronic acid and
adamantane moie- ties-caped gold NCs via the highly specific
interaction of adamantane moieties between β-cyclodextrin cavities [110]. The supramolecular conjugates were
used to load and deliver different cancer therapeutics (such as doxorubicin,
hydrochloride, paclitaxel, camptothecin etc.) to cancer cells
by utilizing the efficient cancertargeting of hyaluronic acid. Further, a
multifunctional nanocarrier with a core-shell structure was constructed for
targeted anticancer drug delivery [111].
The core of the multifunctional nanocarrier was gold NCs and its shell was a
folate-grafted amphiphilic hyperbranched block copolymer. These nanocarriers
showed red fluo-rescence emission and high anticancer efficiency by testing with
HeLa cells when camptothecin, an anticancer drug, was loaded in them. Zhou and
coworkers [112] synthesized gold NCs and
combined a cisplatin prodrug (Pt) and folic acid (FA) with them to synthesize
FA-GNC-Pt conjugates. Their result showed that FA-GNC-Pt could target tumor and
emit bright fluorescence at the tumor site. In order to more accurately target
tumors, Chen and coworkers [113]
developed dual targeting luminescent gold NCs for the application of tumor
theranostics (Fig. 4a,b). In this novel
nano-platform, cyclic RGD and aptamer AS1411 were used to target
Î±vÎ²3 integrins and nucleolin,
respectively, both of which were over-expressed in tumor tissues. A NIR
fluorescence dye was conjugated on this nano-platform to enhance the
signal-to-noise contrast ratio. Furthermore, Doxorubicin (DOX) was integrated in
this platform to improve the antitumor capabilities. The AuNC-DOX-cRGD-Apt could
effectively target the tumor and develop significant morphological changes to
the apoptosis of cells in the mice tumor model (Fig. 4c,d,e).</p><p>Au NCs can serve as radiosensitizers because they can strongly absorb a
radiotherapy ray and effectively produce secondary electrons upon the
irradiation of gamma ray or X-ray. For instance, Zhang et al.
[114] synthesized ultra-small Au NCs
coated by glutathione (GSH-Au25 NCs). They demonstrated that the GSH-
Au25 NCs effectively accumulated in tumor though the EPR effect and enhanced the
cancer radiotherapy efficacy. Most of all, the GSH-Au25 NCs could be
metabolized through kidney. Parallel to this research, the same group [115] prepared ultrasmall
Au29–43(SG)27–37 NCs, which displayed
bright orange luminescence, high tumor uptake, low toxicity and strong
sensitizing enhancement for radiation. They also demonstrated that these
Au29–43(SG)27–37 NCs could act as an
X-ray computed tomography imaging agent. Their research implied that these
Au29–43(SG)27–37 NCs were good
candidates for the cancer theranostics.</p><p>On the other hand, classical photosensitizer and targeting agents can be
combined with Au NCs to synthesize a multifunctional agent for the cancer
theranostics. For example, Cui et al. [117] synthesized highly fluo-rescent
glutathione-capped Au NCs with folic acid and polyethylene glycol (PEG)
conjugated on their surface by covalent coupling. A photosensitizer (chlorin e6,
Ce6) was trapped into PEG to form nanoprobes (Ce6@GNCs-PEG2k-FA).
These nanoprobes could selectively target tumors and effectively restrain the
growth of tumor by photodynamic therapy. Actually, Au NCs can produce singlet
oxygen when they are excited by visible light or short NIR light. According to
the phenomenon, Hwang et al. [116] synthesized alkyl thiolated Au NCs (RS-Au NCs) which sensitized
the production of singlet oxygen when excited by NIR light. Additionally, these
RS-Au NCs could emit visible light with a peak at 610 nm when they were excited
by 365 nm light. To make the RS-Au NCs more effective in killing cancer cells, a
TAT peptide (peptide sequence: N-GRKKRRQRRR-C) was integrated with the RS-Au NCs
together to achieve the highly efficient nucleus-targeting (Fig. 5a). Their results demonstrated that the TAT
peptide modified RS-Au NCs could be internalized by HeLa cells and enter their
nuclei (Fig. 5b). The cellular viabilities
of HeLa cells with internalized TAT peptide-RS-Au NCs dropped sharply with the
irradiation of 980 nm continuous-wave laser (Fig.
5c). The TAT peptide-modified Au NCs exhibited an ultrahigh gene
transfection efficiency in HeLa cells when they served as DNA carriers. Besides,
the TAT peptide-modified Au NCs loaded with plasmid DNA (pDNA) molecules could
be effectively internalized by cells and transferred gene when they were
microinjected into zebrafish (Fig. 5d).</p><!><p>The synthesis difficulty and instability of silver NCs delay their
development. Bigioni et al. [118] prepared ultra-stable silver NCs protected by
p-mercaptobenzoic acid (p-MBA) and made a
breakthrough on the Ag44 NC structure. Recently, obvious progress has
been made in the application of Ag NCs in cancer theranostics. Sheikhnejad
et al. [119]
inserted a cytosine loop (C6 loop) into a double stranded DNA and used it to
synthesize Ag NCs. The Ag NCs were pink in visible light and could emit strong
fluorescence when they met unmethylated DNA, but they lost these properties when
mixed with methylated DNA. According to these results, the C6-Ag NCs could be
employed to directly detect the methylated DNA for early cancer diagnosis.</p><p>Wang et al. [120] used a metallothionein (MT) to synthesize Ag NCs (MT-Ag NCs).
The MT was a cysteine-rich protein with low molecular weight. The MT- Ag NCs
possessed a good antioxidant capacity and showed intense fluorescence when they
were uptaken by HeLa cells. The color of fluorescent emission can be regulated
by the ligand conjugated with Au NCs. Xu et al. developed Ag
NCs which were tagged by AS1411 aptamer or MUC 1 aptamer [121]. The Ag NCs tagged with AS1411 aptamer emitted
green fluorescence whereas the Ag NCs tagged with MUC 1 aptamer emitted yellow
fluorescence. These NCs could be employed in multicolor imaging of cancer cells.
Wang and coworkers [122] synthesized
thiolpolyethyleneimine stabilized silver NCs (SH-PEI-AgNCs) and conjugated
folate with them to target the tumor. These NCs showed ultra-small size, low
toxicity, intense NIR fluorescence, excellent photostability and chemical
stability. Hence, they hold promise for in vitro and in
vivo targeted cancer imaging. Luo and coworkers [123] synthesized small-molecule-protected silver NCs
(Ag14(SG)11) (SG denotes glutathione), which possessed
a smaller hydrodynamic diameter than DNA- protected silver NCs. They
successfully used these NCs to label cancer cells (A549) for fluorescent
imaging. Similar to the biosynthesis of Au NCs in situ, the Wang group [124] reported a specific biosynthesis of
silver NCs. Innocuous silver salts could be reduced to form highly NIR
fluorescent Ag NCs by the cancer cells and tumor themselves. This novel strategy
could be applied in selective and accurate imaging of cancer cells and tumors.
Additionally, Ag NCs synthesized in situ in tumors resulted in
a sharp reduction of tumor size, which even led to complete remission. This
phenomenon suggested these NCs prepared in situ in tumors are
promising new cancer therapeutics.</p><p>In addition to serve as an imaging agent, Ag NCs can sensitize the
generation of singlet oxygen and serve as a valid PDT agent. Tan et
al. [125] synthesized BSA
protein-templated Ag NCs (BSA-Ag13 NCs), which displayed a stronger
singlet oxygen generation capacity than their Au analogue. MCF-7 breast cancer
cells possessed good uptake for BSA-Ag13 NCs and were efficiently
killed due to the irradiation of white light via PDT. Wang
et al. [126]
prepared water-soluble Ag NCs encapsulated by a DNA scaffold, which displayed
NIR fluorescence and photothermal effect. Moreover, AS1411, a nucleoli-targeting
agent, was connected with the DNA scaffold and the protoporphyrin IX (PPIX) was
captured by AS1411. The multifunctional nanoconjugates (PPIX-AS1411-Ag NCs)
showed photodynamic and photothermal efficiency to the colon cancer cells and
could serve as NIR fluorescent probes for nuclei simultaneously.</p><!><p>Copper (Cu) is one of the most significant trace element in the human
body and thus is relatively biofriendly for human beings compared with gold and
silver. Highly fluorescent Cu NCs were fabricated when hydrazine served as a
reducing agent in the presence of lysozyme [127]. These Cu NCs possessed photo- and chemical- stability and were
employed to successfully label HeLa cells. 64Cu is a general PET
imaging agent and has been doped in other metal NCs or introduced into
biomaterials via macrocytic chelators in clinical application.
Recently, Gao et al. [128] synthesized ultra-small radioactive [64Cu]Cu NCs
without chelator by using BSA as a protecting agent. A lung cancertargeting
peptide, termed luteinizing hormone releasing hormone (LHRH), was preconjugated
on BSA to synthesize [64Cu]CuNC@BSA-LHRH. The resultant
product displayed high tumor uptake, high radiolabeling stability, and obvious
renal clearance. Sensitive, deep penetration, and accurate imaging of orthotopic
lung cancer could be achieved by using [64Cu]CuNC@BSA-LHRH
as PET imaging probes in vivo.</p><p>Platinum NCs (Pt NCs) could also be applied in cancer theranostics. Wang
et al. [129]
synthesized fluorescent Pt NCs via incubating cancer cells,
rather than noncancerous cells, with micromolar chloroplatinic acid solutions.
These in situ biosynthesized Pt NCs emitted a strong fluorescence upon the
excitation light at 405 nm and provided a precise bioimaging strategy.
Furthermore, these Pt NCs could effectively kill cancer cells when conjugated
with porphyrin derivatives, a photothermal therapy agent.</p><!><p>NCs have been integrated with other nanomaterials to form attractive
multimodal cancer theranostic agents. Reduced graphene oxide (RGO) display
excellent biocompatibility and can perfectly support various metal NCs,
nanoparticles, and drugs. Gold NCs- reduced graphene oxide (GNC-RGO) were
prepared by simply mixing the RGO with dedecanethiol-CTAB- capped GNCs, which
were synthesized following a conventional Brust-Schiffrin process [130]. GNC-RGO loaded with DOX could result
in growth inhibition of HepG2 cells and enhance the contrast of the edges and
morphology of the cells in the fluorescent imaging.</p><p>In fact, NCs can further be clustered into nanoparticles and lead to
novel and enhanced properties for cancer theranostic application. Akram
Yahia-Ammar and coworkers [131]
clustered Au NCs into nanoparticles of 120 nm in diameter using a cationic
polymer (Fig. 6A). These positively charged
nanoparticles exhibited an enhancement in the already strong fluorescence and
drug delivery compared with free Au NCs.</p><p>NCs also can be integrated with traditional nanoparticles to generate
multimodal nanocomposites for cancer theranostic applications. For example, Li
and co-workers [132] conjugated a
PEG-Gd2O3 nanoparticle with Ag NCs though the bonding
between amino radical and carboxyl radical via the covalent
coupling reaction (Fig. 6B). In the
resultant PEG-Gd2O3/Ag NCs nanocomposite with a suitable
ration of PEG-Gd2O3 to Ag NCs, both the fluorescent
luminescence of Ag NCs and MRI signal of PEG-Gd2O3
increased. Almost at the same time, Croissant et al. [134] coated a mesoporous silica
nanoparticle (MSN) with Au NCs stabilized by BSA (AuNC@BSA) to form MSN-AuNC@BSA
that could serve as a nanocarrier. These MSN-AuNC@BSA nanocarriers could
simultaneously load two drugs (gemcitabine and doxocubicin) and were employed
for nuclear staining and tumor imaging in vivo. NCs and
nanoparticles could be clustered together to form a magical nanocomposite. As an
example, multifunctional clusters (MNCs) Fe3O4-CdSe/ZnS
(Fig. 6C) were synthesized by Kim
et al. [133] and
displayed excellent magnetic and optical properties and biocompatibility.
MNCs-lipid A was prepared by loading lipid A onto MNCs to enhance the migration
of dendritic cells (DC) and antigen-specific T cell responses to tumor
in vivo. That phenomenon suggested that the MNC-lipid A
nanocomposite could act as a candidate for the application in DC-base cancer
immunotherapies.</p><!><p>In short, we have attempted to review the recent progress in the development
of metallic NCs as well as their use in cancer therapy and imaging. In the past two
decades, NCs have been extensively researched and the past studies have
significantly contributed to pushing forward their applications in cancer imaging
and therapy. However, there are still a few problems that remain to be solved.
First, metallic NCs displayed a relatively low quantum yield and short lifetime
compared with quantum dots, and their luminescence wavelength was usually from 500
nm to 800 nm, a window that was greatly absorbed and scattered by tissue. Hence, it
is highly desired to develop metallic NCs with high quantum yield, long lifetime,
and emission in the near- infrared region (1000–1350 nm). Second, some basic
theories need to be developed to understand the controversial issues, such as the
accurate structure of NCs, the relationship between structure and properties, and
the effect of ligands on the characteristics of NCs. Third, though some pioneers
have done some work about the cellular uptake [135–137], cytotoxicity
[138–140], immunological properties [141], bio-distribution and clearance in
vivo [142, 143] of NCs, these reissues still need to be studied in
depth and systematically. Forth, precise targeting and controlled release of drugs
of the NCs are still to be improved in order for us to apply them to achieve precise
targeting and effective cancer therapy. With these problems solved, we believe that
NCs would find eventual clinical applications in the early diagnosis and targeted
therapy of cancer.</p>