Nano Res

1

Harnessing the collective properties of nanoparticle

ensembles for cancer theranostics

Yi Liu1,2

, Jun-Jie Yin2, Zhihong Nie

1()

Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-014-0541-9

http://www.thenanoresearch.com on July 10, 2014

© Tsinghua University Press 2014

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Harnessing the Collective

Properties of Nanoparticle

Ensembles for Cancer

Theranostics

Yi Liu1,2, Jun-Jie Yin2, Zhihong Nie1*

1University of Maryland, United States 2U.S. Food and Drug Administration,

United States

The self-assembly of NP ensembles from NP building blocks and their application in

cancer imaging and therapy are summarized. Because of the new and advanced collective

properties, the NP ensembles show many advantages over existing individual NP-based

theranostic systems.

Provide the authors’ webside if possible.

Author 1, webside 1

Author 2, webside 2

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Nano Res.

Harnessing the collective properties of nanoparticle

ensembles for cancer theranostics

Yi Liu1,2

, Jun-Jie Yin2, Zhihong Nie

1()

Received: day month year

Revised: day month year

Accepted: day month year

(automatically inserted by

the publisher)

© Tsinghua University Press

and Springer-Verlag Berlin

Heidelberg 2014

KEYWORDS

vesicle; nanoparticle;

self-assembly; cancer

theranostics

ABSTRACT

Individual inorganic nanoparticles (NPs) have been widely used in fields of

drug delivery, cancer imaging and therapy. There are still many hurdles that

limit the performance of individual NPs for these applications. The utilization

of highly ordered NP ensembles opens a door to resolve these problems, as a

result of their new or advanced collective properties. The assembled NPs show

several advantages over individual NP-based system, such as improved cell

internalization and tumor targeting, enhanced multimodality imaging

capability, superior combination therapy arising from synergistic effect,

possible complete clearance from the whole body by degradation of assemblies

into original small NP building blocks, and so on. In this Perspective, we

discuss the potential of utilizing assembled NP ensembles for cancer imaging

and treatment by taking plasmonic vesicular assemblies of Au NPs as an

example. We first summarize the recent development on the self-assembly of

plasmonic vesicular structures of NPs from amphiphilic polymer-tethered NP

building blocks. We further review the utilization of plasmonic vesicles of NPs

for cancer imaging (e.g., multi-photon induced luminescence, photothermal,

and photoacoustic imaging), and cancer therapy (e.g., photothermal therapy,

and chemotherapy). Finally, we outline current challenges and our perspectives

along this line.

1 Introduction

Although the past few years have witnessed an

unprecedented revolution in cancer diagnostic and

therapeutic, the clinical outcomes for cancer patients

have largely remained disappointing. The extremely

dismal statistics arises, mainly due to the lack of

suitable tools for early detection of cancer (in terms

of type, classification, and location) and ineffective

therapeutic strategies [1]. Many conventional

imaging techniques are available for the detection

and characterization of tumors, such as X-ray,

positron emission tomography, and magnetic

resonance imaging. These techniques, however, do

not provide contrast, sensitivity, dynamic range, and

Nano Research

DOI (automatically inserted by the publisher)

Address correspondence to znie@umd.edu

Review Article

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Nano Res.

spatiotemporal resolution sufficient enough in many

situations [1]. As to therapeutic strategies, tumors at

initial stages are usually removed by surgery if

possible at the present time [2]. Unfortunately, the

surgical treatment has severe limitations and show

relatively poor long-term clinic outcomes.

Chemotherapy and radiotherapy (or following

surgery sometimes) are considered as other effective

alternative strategies. However, systemic drug

delivery is often failed to deliver sufficient amount of

chemotherapeutic drugs specifically to tumor sites to

suppress cancer metastases [3, 4]. The severe side

effect arising from non-specificity often makes the

patient extremely weak and even results in death.

Moreover, because of the poor efficacy of

non-specific chemotherapy, drug resistance will

develop over times (after about one year on average)

for most of anticancer drugs in virtually all patients

[5]. For these reasons, it is necessary to develop more

effective diagnostic and therapeutic strategies for

cancer therapy.

The utilization of inorganic nanoparticles (NPs)

as theranostic agents holds the potential to offer

unique solutions to current issues in combating

cancers [6, 7]. Inorganic NPs have shown intrinsic

optical, electronic, and magnetic properties that are

dependent on their size, shape, and composition.

This enables their biomedical application by

integrating effective imaging, diagnosis, and therapy

in one system [8-10]. Generally, NPs in an individual

or simple clustered form are used as delivery

vehicles for this application. For example, Au NP

based theranostic platform uniquely combines

targeting, multimodal imaging including

multi-photon induced luminescence (MAIL),

photothermal (PT) [11], and photoacoustic (PA)

imaging, and combination therapy including

chemotherapy and PT ablation (PTA) of cancer cells

[6, 12, 13]. Despite promising future, significant

hurdles still remain at this frontier [6, 14]. Firstly, the

efficiency with which individual NPs absorb and

scatter light requires further improvement, in order

to achieve optimal imaging. Particularly, our inability

to tune the interactions of light with a collection of

NPs for theranostic severely limits our capability of

further improving current individual NP-based

theranostic systems. Secondly, the loading capacity of

therapeutic and imaging agents by individual

vehicles (mostly through surface immobilization) is

relatively low, which limits the delivery efficiency of

these active agents to specific tumor areas. Last but

not least, long-term safety in terms of toxicity,

degradation, and clearance of NPs has to be

improved and fully understood before the translation

of this technology to the clinical realm [15]. Clinical

translation of inorganic NPs is fundamentally limited

because of the inherent difference in their uptake and

clearance from the body. Smaller NPs are eliminated

quickly from the body, but they are either less likely

to be taken up by passive targeting via such as

enhanced permeable retention (EPR) or do not offer

satisfactory physical properties for diagnosis.

Scheme 1. Schematic illustration of biodegradable Au vesicles

assembled from amphiphilic Au NPs for cancer theranostics. The

absorption of the plasmonic vesicles can be tuned within NIR

wavelength range. The biodegradable Au vesicles loaded with

therapeutic agents will accumulate at cancer cells via improved

EPR effect and passive targeting. The release of payloads can be

triggered by NIR light. After effective detection and treatment of

cancer cells, the vesicles will degrade into original small NP

building blocks for ease elimination from the body. The strong

plasmon coupling between Au NPs within membranes will

endow biodegradable Au vesicles significantly enhanced imaging

capability and combination therapy.

Although individual NPs are no doubt exciting,

ensemble of interacting NPs can exhibit a rich variety

of novel and extremely useful collective properties

that can be radically different from their individuals

[16-20]. These new synergistic properties arise from

the coupling interactions between metallic,

semiconductor or magnetic NPs within the ensemble.

The successful use of NP ensembles has been

demonstrated in the field of such as energy,

biosensing, metamaterials, and optoelectronics

[21-23]. It is expected that harvesting the collective

properties of NP ensembles would enable full

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Nano Res.

realization of the enormous potential of inorganic

NPs in cancer imaging and treatment. Such

expectation is also originated from at least the

following factors:

i) Harvesting the collective properties of NP

ensembles will maximize our capability of

manipulating the interaction of a collection of NPs

with light, thus enabling superior imaging (i.e, PT

and PA imaging) and combination cancer therapy

[24-27].

ii) The ease in fine tailoring the

physicochemical properties of NP ensembles by

controlling individual building blocks will dictate

their enhanced blood stability, biodistribution, and

specificity of targeted delivery, as the superstructures

of NP ensembles interact with cells and tissues as a

whole [28].

iii) After treatment, the degradation of NP

ensembles into original building blocks, which can be

eliminated quickly from the whole-body, will

guarantee their long-term safety in terms of clearance

and toxicity, when the surface of NPs is proper. This

strategy naturally resolve current challenge in

achieving high specificity and efficiency of tumor

targeting via passive targeting (which is generally

more effective for NPs with a diameter of ~30-50 nm),

while promoting the clearance of NPs from the body

(which is more favorable for small NPs) [28, 29].

iv) The synergistic cytotoxic effect (e.g., the

combination of PTA and chemotherapy) may arise

from a collection of NPs carrying imaging or

therapeutic agents, thus leading to better tumor

control for patients [24].

In this Perspective article, we will convey the

concept of utilizing NP ensembles for cancer

theranostics by taking plasmonic vesicular

assemblies of noble metal NPs as an example

(Scheme 1). Unless otherwise specified, vesicular

assemblies of NPs will be written as NP vesicles

briefly herein. First, we will highlight current

advances in assembling NPs into vesicular ensembles

with programmable coupling between NPs with the

assemblies. Second, we will discuss the utilization of

NP vesicles in cancer imaging and treatment. Finally,

we will outline current challenges and our

perspectives along this line.

2 Construction of NP vesicles

Amphiphilicity-driven self-assembly of lipids

gives rise to liposomes with a bilayer structure which

is analogous to the structure of cell membrane [30-33].

Similarly, block copolymers (BCPs) can assemble into

polymersomes with tailored mechanical and

structural properties through self-aggregation of

their hydrophobic tails in an aqueous medium [34,

35]. Inspired by the well-established self-assembly of

these natural and synthetic molecular amphiphiles,

the concept of amphiphilic colloidal NPs has been

recently proposed. Amphiphilic NPs are generally

made by decorating the surface of NPs with

hydrophilic and/or hydrophobic molecules. Driven

by the directional interactions induced by molecular

tethers, amphiphilic NPs can spontaneously organize

into the desired entities.

The amphiphilicity of NPs can be achieved by

modifying a hydrophilic (or hydrophobic) NP with

hydrophobic (or hydrophilic) polymer brushes, while

selectively exposing part of the NP surface to

surrounding media. For instance, selective end

functionalization of hydrophilic cetyl

trimethylammonium bromide-coated Au nanorods

with hydrophobic polystyrene (PS) brushes generates

a new class of fascinating one-dimensional NP

amphiphiles [36]. These amphiphilic nanorods can

assemble into a wide range of nanostructures (e.g.,

nanochains, rings, bundles, and vesicles) with

tunable optical properties. Weller and Förster studied

the self-assembly of CdSe/CdS core-shell NPs

modified with a brush-like layer of poly(ethylene

oxide) (PEO) chains into spherical, cylindrical, and

vesicular structures in dilute solution [37].

A common feature of the amphiphilic NPs

described above is that, unlike true block copolymers,

only one of the chemical constituents is polymeric;

hence the comparable rigidity of the other section

may limit the inherent complexity of assemblies due

to packing constraints. A closer block copolymer

analogue can be produced by functionalizing NPs

with mixed brushes of hydrophilic and hydrophobic

chains, such that both of the incompatible chemical

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Nano Res.

sections exhibit conformational flexibility. For

example, metallic NPs covalently attached with

amphiphilic V-shaped PS-b-PEO molecules on their

surface can assemble into one-dimensional tubular

structures [38]. Ultra-small Au NPs asymmetrically

functionalized with a single amphiphilic triblock

copolymer chain per NP assembled into micelles,

vesicles, rods, and large compound micelles [39]. In

these cases, the amphiphilic NPs could be viewed as

one type of Janus NPs, where NP core served as the

junctions of hydrophobic and hydrophilic

homopolymers.

Figure 1 (a) Schematic illustration of self-assembled NP vesicles

and tubules from amphiphilic NPs. (b), (c) Representative SEM

images of NP vesicles (b) and tubules (c) respectively. Inset in (b)

is the FFT pattern of SEM images. Scale bars: 200 nm in (b), (c).

Copyright 2012, American Chemical Society. (d)-(g)

Representative SEM images of Janus-like vesicles with spherical

(d), hemispherical (e), and disk-like (f), (g) shapes. SEM images

of patchy vesicles (h) and heterogeneous vesicles (i). Scale bars:

400 nm in (d), 150 nm in (e), 200 nm in (f), (h), (i), and 600 nm

in (g). Copyright 2014, American Chemical Society.

Other than NPs with asymmetrical

functionalization, NPs tethered by a mixture of

hydrophilic/hydrophobic polymer chains can also

self-assemble into NP vesicles. Recently, Duan et al.

demonstrated the assembly of plasmonic vesicular

nanostructures from Au NPs carrying a mixture of

hydrophilic poly(ethylene glycol) (PEG) and

hydrophobic poly(methyl methacrylate) (PMMA)

polymer brushes [40]. Moffitt and co-workers

showed the synthesis and self-assembly of CdS NPs

decorated with a mixture of hydrophobic PS and

ionizable hydrophilic (polyacid) polymer chains [41].

Tethering a mixture of hydrophilic/hydrophobic

polymer brushes on the surface of NPs is an effective

strategy for driving the self-assembly of NPs.

However, the intrinsic difference in bonding

strengths and absorption kinetics of different

polymers onto NPs makes it relatively difficult to

quantitatively control or predict the relative density

of each type of polymer, and hence their assembly

structures [42-44]. In contrast, BCP tethers offer

greater control over the chemical functionality and

composition (i.e., relative volume of

hydrophilic/hydrophobic moieties) as well as

architectural complexity of polymer chains on NP

surface. The chemical incompatibility and

conformational flexibility of isotropically grafted

BCPs gives rise to spontaneous anisotropy and

directional interactions to the colloidal building

blocks, their unique assembly behaviors. Based on

this concept, Nie et al. presented a new class of

amphiphilic NPs composed of inorganic NPs

tethered with amphiphilic linear BCPs (e.g.,

poly(2-(2-methoxyethoxy)ethyl

methacrylate)-b-polystyrene (PMEO2MA-b-PS) or

PEO-b-PS) [26]. As shown in Fig. 1(a)-(c), driven by

the conformational changes of tethered BCP chains,

such amphiphilic NPs can self-assemble into

well-defined nanostructures in selective solvents.

They include unimolecular micelles, clusters with

controlled number of NPs, tubular and vesicular

nanostructures comprising a monolayer shell of

highly ordered, hexagonally packed NPs [25]. This

strategy is applicable to the assembly of NPs with

various sizes, shapes (i.e., nanorods), and

compositions. More importantly, the interparticle

distance between Au NPs in the assemblies can be

tuned to achieve control over the plasmonic

properties of assembled structures by varying the

molecular length of hydrophobic blocks. This opens a

door to the utilization of the collective properties of

NP ensembles for biomedical applications.

Most recently, Nie et al. achieved the assembly of

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NP vesicles with well-defined shape, morphology,

and surface pattern. The concurrent assembly of

amphiphilic BCP and NP building blocks produced

patchy vesicles with multiple small amphiphilic NP

domains surrounded by BCP phase, Janus-like

vesicles with distinguished BCP and NP halves, and

heterogeneous vesicles with uniform distribution of

NPs [45] (Fig. 1(d)-(i)). The formation of

nanostructures with various morphologies arises

from the delicate interplay between the dimension

mismatch of the two types of amphiphiles, the

entanglement of polymer chains, and the mobility of

amphiphilic NPs. It is interesting to note that the

entropic attraction between amphiphilic NPs, as a

result of the maximization of the conformational

entropy of BCP chains, plays a dominant role in

controlling the phase-separation of the two types

amphiphiles in the membranes.

3 NP vesicles for cancer theranostics

Vesicles of Au NP ensembles will not only

preserve the intrinsic properties of polymeric and Au

components, but also acquire new or advanced

functionalities, such as encapsulation, local release,

tunable absorption, enhanced PT conversion

efficiency, and biodegradation. These unique features,

which are mostly not attainable by individual NPs,

facilitate their performance in cancer theranostics.

Cellular Internalization and Colocalization. The

vesicular assemblies of NP ensembles interact with

cells or tissues as a whole system. The

physicochemical properties (i.e., size, surface

chemistry, surface topology, and mechanical property)

of NP ensembles can be precisely engineered by

controlling the physical and chemical features of

individual NP building blocks. The unprecedented

control over the trait of NP assemblies offers us new

opportunities of enhancing the targeting and

biodistribution of theranostic vehicles. Recent

preliminary in vitro studies by Nie et al. showed that

the NP vesicles interact with cells as one system, and

colocalized within the lysosomes of MDA-MB-435

breast cancer cells [24]. LysoTracker was used to stain

lysosome and the subcellular localization of NP

vesicles encapsulated with photosensitizer Ce6 was

traced by confocal laser scanning microscopy (CLSM).

The fluorescent signals from LysoTracker and

vesicles matched very well, which suggests that

majority of vesicles colocalized within lysosomes.

Furthermore, the uptake of vesicles is internalized

via an energy-dependent endocytosis mechanism. In

another study, Duan et al. also revealed that

Herceptin conjugated NP vesicles could quickly

bound to HER2-positive SKBR-3 breast cancer cells

and be uptake by the cells through the endocytic

pathway [46]. In contrast, after incubating with NP

vesicles, only sparsely distributed vesicles can be

found in HER2-negative MCF-7 breast cancer cells. In

addition, Ijiro confirmed that NP vesicles showed

twice the level of cellular uptake compared with that

of dispersed NPs (same surface chemistry), which

indicates that NP vesicles can be efficiently

internalized into cells as their size is suitable for

endocytosis [47]. All these results together

demonstrate the potential of using bioconjugated

plasmonic NP vesicles to recognize and target

specific types of cancer cells.

Enhanced in vitro and in vivo cancer imaging

and treatment. The manipulation of the interactions

between light and a collection of NPs offers us a

powerful tool to achieve optimal design and physical

properties of nanostructures. This enables the

superior performance, which is beyond the capability

of individual NP-based theranostic platform, of NP

ensembles in cancer imaging and treatment. The

coupling between Au NPs within the vesicular

membranes resulted in a drastic red-shift of the

localized surface plasmon resonance (LSPR) peak

and a significant enhancement of LSPR absorption in

the near-infrared (NIR) range. These features enable

their superior performance in biomedical application.

Recent studies showed that Au NP vesicles exhibit

enhanced imaging capability originated from the

coupling between Au NPs within vesicular

membranes [25, 29]. First, NP vesicles show a ~7-fold

enhancement in MAIL imaging of 4T1 breast cancer

cells, compared to individual Au NPs with the same

quantity and diameter [25]. MAIL imaging of cells

internalized with vesicles was achieved by exciting

with 800 nm light and recording in a wide spectra of

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470-600 nm. It is reasonable to believe that

optimization of the system can further improve

MAIL signal.

Figure 2 (a) Thermal images of MDA-MB-435 tumor-bearing

mice exposed to 808 nm laser for 5 min at post-injection of PBS

or biodegradable Au vesicles (BGVs). (b) Heating curves of

tumors upon laser irradiation as a function of irradiation time. (c)

PA signals of BGVs and Au nanorods (GNRs) as a function of

optical density. (d) In vivo 2D and 3D ultrasonic (US) and PA

images of tumor tissues at pre-injection and post-injection of

BGVs. Arrows indicate the location of BGVs. (e) PA intensities

of tumor tissues with intratumoral administration of the same

amount of regular Au vesicles (GVs) or BGVs. Copyright 2013,

Wiley-VCH.

Second, the coupling between Au NPs within

vesicular membranes leads to a strong NIR

absorption and a significant increase in PT

conversion efficiency (η) of the assemblies, hence

improving their performance in PT/PA imaging and

PTA (Fig. 2) [48]. For example, biodegradable Au

vesicles composed of poly(ethylene

glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) tethered

Au NPs showed an ultra-strong plasmonic coupling

effect, due to the small interparticle distance of NP

building blocks [29]. The η of biodegradable Au

vesicles (~150 nm in diameter, ~800 nm absorption)

with strong-coupling 25-nm NPs is 37%, which is

much higher than 18% and 22% for that of regular Au

vesicles and Au nanorods (~10 nm in diameter and

~42 nm in length), at equal optical density (OD) at

808 nm (Fig. 2(a), (b)). The increase in η of NP

vesicles significantly enhanced their performance in

PT and PA imaging in vitro and in vivo (Fig. 2(c), (d)).

In vivo study showed that the PA signals in the

tumor region was ~10-fold stronger than control

group without Au vesicles injection. Even compared

with Au nanorods, PA signals from biodegradable

Au vesicles are almost 7-fold stronger when the OD

at 808 nm is 1.0. Furthermore, the η and resulting

imaging capability of NP vesicles is strongly

dependent on the coupling strength between Au NPs

within membranes (Fig. 2(e)). The vivo PA signal

doubled when the distance between Au NPs within

assemblies decreased.

Because of the higher η, the biodegradable Au

vesicles also exhibited significantly enhanced PT

therapeutic efficacy as compared with Au nanorods

and regular Au vesicles [29]. After laser irradiation,

all the tumors injected with biodegradable Au

vesicles were effectively ablated, leaving black scars

at their original sites without showing reoccurrence

within ~4 weeks. The mice were tumor-free and

survived over 30 days without a single death or

tumor reoccurrence. In contrast, Au nanorods and

regular Au vesicles administration/irradiation groups

showed slower delay in tumor growth or tumor

regression, all mice showed average life spans of

14~20 days since treatment started. Hematoxylin and

eosin (H&E) staining of tumor slices was also carried

out for tumors collected immediately after laser

irradiation. Significant cancer cell damage was

observed in the Au vesicle (with strong

coupling)-treated group, but not in groups treated

with Au nanorods or Au vesicles (with weak

coupling).

Improved Delivery of Therapeutic Agents for

efficient therapy. Organic vesicles (i.e., liposomes)

have made the greatest clinical impact in drug

delivery, because of their unique ability to

encapsulate and deliver hydrophilic and/or

hydrophobic compounds simultaneously [30-35]. NP

vesicles mimic the function of organic vesicles for

encapsulation, while showing significantly improved

ability of retaining drugs in the cavity. The loading of

therapeutic or imaging agents within vesicular

cavities overcomes the surface-area-limited loading

of drugs on Au NPs by conventional approaches

[49-52]. The kinetically trapping of Au NPs in the

members possibly minimizes the leakage of mostly

toxic drugs during circulation. Moreover, the

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spatiotemporal release of drugs from NP vesicles can

be activated specifically at target sites by NIR light or

chemical triggered breakup of the vesicular

membranes [27, 53, 54]. These unique features of NP

vesicles may drastically improve chemotherapy (or

other therapies such as photodynamic therapy)

results and minimize side-effects of non-specific drug

delivery in cancer treatment.

Figure 3 (a) UV-vis spectra of Au vesicles (GVs) (black), Ce6

(blue) and Au vesicle-Ce6 (GV-Ce6) (red). The arrows indicate

characteristic Q-bands of Ce6; (b) Ce6 loading efficiency of

GV-Ce6 as a function of Ce6 concentration; (c) the changes of

fluorescence intensity at the characteristic peaks of SOSG and

Ce6 (528 and 662 nm) as a function of laser irradiation time. (d)

Tumor growth curves of different groups of tumor-bearing mice

after treatment. Tumor volumes were normalized to their initial

sizes. Error bars represent the standard deviations of 4-6 mice per

group. Asterisk indicates P < 0.05. Copyright 2013, American

Chemical Society.

Most recently, Nie and co-workers reported the

design of multifunctional photosensitizer Ce6-loaded

plasmonic Au vesicles for trimodality

fluorescence/thermal/PA imaging guided synergistic

photothermal/photodynamic therapy (PTT/PDT)

cancer treatment [24] (Fig. 3). The Au vesicles

showed a strong absorbance in the NIR range of

650-800 nm, as a result of the plasmonic coupling

between neighboring Au NPs in the vesicular

membranes. This enables the use of 671 nm laser

irradiation to simultaneously excite both Au vesicles

and Ce6 to produce heat and singlet oxygen, ablating

cancer cells (Fig. 3(a)). When the weight ratio of Ce6

to Au vesicles (mCe6/mv) is 60%, the Ce6 loading

efficiency is up to 18.4 wt %; and this value can be

further increased with increasing mCe6/mv (Fig. 3(b)).

In contrast, the loading efficiency of Ce6 onto Au

nanorods through electrostatic interaction saturated

at ~9 wt%. The efficient loading of Ce6 in Au vesicles

significantly increases the accumulation of Ce6 in

cancer cells. The heating effect upon laser irradiation

dissociates the Ce6-loaded Au vesicles to release

payloads (Fig. 3(c)). The tumor tissues visualized by

the fluorescence, thermal and PA signals from

Ce6-loaded Au vesicles can be selectively destroyed

in a noninvasive manner by the illumination of 671

nm laser. Both in vitro and in vivo therapeutic

efficacies of Ce6-loaded Au vesicles were enhanced

compared to either individual PTT or PDT alone, or

the simple summation of PTT/PDT due to the

synergistic effect (Fig. 3(d)). In another example,

Doxorubicin (DOX) was encapsulated in Au NP

vesicles with surface-conjugated with targeting

moiety such as monoclonal antibody and folate. The

platform can selectively enter various cancer cells

including SKBR-3 breast cancer cells and

MDA-MB-435 breast cancer cells, and the release of

payloads was achieved by pH or photo-stimulation

[46, 55]. Moreover, the dramatic change in scattering

properties and SERS signals upon the dissociation of

the entity of vesicles allows one to trace the

intracellular drug delivery by plasmonic imaging

and SERS spectroscopy.

Pharmacokinetics and clearance of NP vesicles.

As mentioned earlier, the realization of inherent

contradictory processes ─ the improved passive

targeting (e.g., through ERP effect) and the clearance

of NP from the body ─ is very much fundamentally

limited. One fascinating concept to solve the

challenge is to construct NP ensembles with desired

physicochemical properties for delivering the

vehicles efficiently to targeted regions, while the NP

ensembles can dissociate into much smaller original

NP building blocks which can be effectively

eliminated from kidney and the whole body. In a

recent fascinating example, DNA was used to initiate

the assembly of NPs into larger core-satellite

structure with controlled biological delivery and

elimination properties [28]. Through burying DNA

inside the inner and using NPs as scaffolds, the

assembled structure decrease their accessibility to

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cellular interactions but increase the density of PEG

coverage above the DNAs. This design can reduce

assembled structure’s uptake and sequestration by

macrophages, improve their accumulation into

tumors. More importantly, the complex variety of

hydrolytic enzymes in cells will quickly hydrolyse

the DNA linkages that connect the NPs, facilitate the

elimination of NPs from the body. In a word, the

elaborate design of the core-satellite structure not

only improves the tumor-targeting efficiency,

avoiding uptake in the reticuloendothelial system,

but also maintains the clearance of injected NPs in

living animals.

With respects to vesicular assemblies of NP

ensembles, the system will not only enable efficient

delivery of imaging and therapeutic agents, but also

can degrade into small building blocks and hence

potentially clear out from the body. The

biodistribution and pharmacokinetics of

intratumorally injected NP vesicles was studied [29].

After the utilization of NP vesicles assembled from 25

nm Au NPs for theranostics, the degradation and

redistribution of Au NPs in different organs was

quantified using inductively coupled plasma mass

spectrometer (ICP-MS) analysis. The concentration of

Au element in different organs was measured at 1, 2

and 8 day (n=3/group). Their studies indicates that

after the laser irradiation, the biodegradable Au

vesicles using PEG-b-PCL as polymer tethers

would degrade into original NP building blocks and

leak into circulation and prominently accumulate in

the reticuloendothelial system (RES) including liver

and spleen at 2 day. After 8 days, vesicles are partly

cleared from the RES. The biodistribution of

biodegradable Au vesicles after intratumoral

injection showed that biodegradable Au vesicles

would leak into circulation and prominently

accumulate in the RES including the liver and spleen

on day 2 and cleared from the RES on day 8. It is

worthy to note that the laser irradiation speeds up

the degradable of NP vesicles into individual NPs.

However, if without laser irradiation, the

biodegradable Au vesicles always stayed in the

tumor tissues. Together these results suggest that it

is promising to improve the clearance of NP vesicles

by breaking down the assemblies into individual NPs

with proper surface, although more systematic study

is required.

4 Conclusions and outlook

Harvesting the new or advanced physical and

chemical properties of NP ensembles holds the

promise to offer new solutions to existing problems

in the field of cancer theranostics. This is reflected by

current exciting advances in the construction and

biological application of plasmonic NP vesicles.

Assemblies made from other types of NPs (e.g.,

quantum dots and magnetic NPs) or the combination

of multiple types of NPs are also attractive for a

range of biomedical applications, due to the exciton

coupling, plasmon-exciton coupling, and

magnetic-magnetic interactions between NP subunits.

These NPs have also been integrated in vesicular

membranes of polymers or lipids for cancer

diagnosis, imaging and treatment, although more

efforts should be made to utilize their collective

properties [56-58].

The utilization of NP vesicles for theranostics

possess at least three advantages over existing

individual NP-based theranostic systems: i) The

physicochemical properties of NP vesicles can be

easily tailored by controlling individual building

blocks; ii) The ensemble structures of NP vesicles

facilitate the uptake by targeted tumor cells, and the

degradable property guarantees their long-term

safety; iii) The fascinating collective properties

enhance their cancer imaging and treatment

capability.

Although these results are encouraging, new

breakthroughs are still needed. First of all, the in vivo

biodistribution and long-term toxicological effects of

these NP vesicles administrated via intravenous

injection must be carefully assessed before their use

in clinical daily practice. Further studies should

systematically evaluate the risks of using NP vesicles

for in vivo imaging and treatment with regards to

organ distribution, accumulation and clearance, and

immunogenicity and inflammation at the whole

organism level, as well as to quantitatively assess

their imaging and therapeutic performance in animal

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Nano Res.

models. Secondly, greater efforts should be made to

understand how light (or magnetic field when

magnetic NPs are used) interacts with a collection of

inorganic NPs. Fundamental understanding of the

system through both experimental and

computational studies will enable us to achieve

optimal design of NP ensembles with desired

properties for these applications. For instance, what

would be ideal size of NP building blocks that will

offer sufficient coupling strength between NPs, while

not sacrificing their elimination from the body?

Finally, challenges remain in precisely engineering

NP ensembles with precisely tunable physical and

chemical properties (e.g., size, size distribution,

interparticle distance, surface), as many factors are

intertwined. For instance, the diameter of NP vesicles

can be reduced down to ~50 nm, when small Au NPs

(~5 nm in diameter) with relatively short ligands are

used [59, 60]. However, the use of smaller Au NPs

significantly reduces the plasmonic coupling

between neighboring NPs, which makes it difficult to

tune the optical property of the NP ensembles. New

advances in both self-assembly and theoretical

prediction of properties are required to achieve better

design of NP ensembles for various biomedical

applications.

Acknowledgements

This work is supported by NSF career award

(DMR-1255377), startup funds from the University of

Maryland, and the Joint Institute for Food Safety and

Nutrition, University of Maryland, College Park, MD

through a cooperative agreement funded by FDA,

Grant No. 5U01FD001418 (Yi Liu). This article is not

an official U.S. FDA guidance or policy statement. No

official support or endorsement by the U.S. FDA is

intended or should be inferred.

References

[1] Wiedmann, T.; Sadhukha, T.; Hammer, B.; Panyam, J.

Image-guided drug delivery in lung cancer. Drug Deliv.

and Transl. Res. 2012, 2, 31-44.

[2] Mitsudomi, T.; Suda, K.; Yatabe, Y. Surgery for NSCLC in

the era of personalized medicine. Nat. Rev. Clin. Oncol.

2013, 10, 235-244.

[3] Devarakonda, S.; Morgensztern, D.; Govindan, R.

Molecularly targeted therapies in locally advanced

non-small-cell lung cancer. Clin. Lung Cancer 2013, 14,

467-472.

[4] Govindan, R.; Bogart, J.; Vokes, E. E. Locally advanced

non-small cell lung cancer: The past, present, and future. J

Thorac Oncol. 2008, 3, 917-928.

[5] Dean, M.; Fojo, T.; Bates, S. Tumour stem cells and drug

resistance. Nat. Rev. Cancer 2005, 5, 275-284.

[6] Sukumar, U.; Bhushan, B.; Dubey, P.; Matai, I.; Sachdev,

A.; Packirisamy, G. Emerging applications of nanoparticles

for lung cancer diagnosis and therapy. Int Nano Lett 2013,

3, 1-17.

[7] Janib, S. M.; Moses, A. S.; MacKay, J. A. Imaging and

drug delivery using theranostic nanoparticles. Adv. Drug

Delivery Rev. 2010, 62, 1052-1063.

[8] Chan, W. C. W.; Nie, S. Quantum dot bioconjugates for

ultrasensitive nonisotopic detection. Science 1998, 281,

2016-2018.

[9] Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.;

Bruchez, M. P.; Wise, F. W.; Webb, W. W. Water-soluble

quantum dots for multiphoton fluorescence imaging in vivo.

Science 2003, 300, 1434-1436.

[10] Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.;

Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S.

S.; Weiss, S. Quantum dots for live cells, in vivo imaging,

and diagnostics. Science 2005, 307, 538-544.

[11] Chen, J. Y.; Yang, M. X.; Zhang, Q. A.; Cho, E. C.; Cobley,

C. M.; Kim, C.; Glaus, C.; Wang, L. H. V.; Welch, M. J.;

Xia, Y. N. Gold nanocages: A novel class of

multifunctional nanomaterials for theranostic applications.

Adv. Funct. Mater. 2010, 20, 3684-3694.

[12] Kennedy, L. C.; Bickford, L. R.; Lewinski, N. A.; Coughlin,

A. J.; Hu, Y.; Day, E. S.; West, J. L.; Drezek, R. A. A new

era for cancer treatment: Gold-nanoparticle-mediated

thermal therapies. Small 2011, 7, 169-183.

[13] Dreaden, E. C.; Alkilany, A. M.; Huang, X. H.; Murphy, C.

J.; El-Sayed, M. A. The golden age: Gold nanoparticles for

biomedicine. Chem. Soc. Rev. 2012, 41, 2740-2779.

[14] Wang, Y. C.; Black, K. C. L.; Luehmann, H.; Li, W. Y.;

Zhang, Y.; Cai, X.; Wan, D. H.; Liu, S. Y.; Li, M.; Kim, P.;

Li, Z. Y.; Wang, L. H. V.; Liu, Y. J.; Xia, Y. A.

Comparison study of gold nanohexapods, nanorods, and

nanocages for photothermal cancer treatment. ACS Nano

2013, 7, 2068-2077.

[15] Khlebtsov, N.; Dykman, L. Biodistribution and toxicity of

engineered gold nanoparticles: A review of in vitro and in

vivo studies. Chem. Soc. Rev. 2011, 40, 1647-1671.

[16] Nie, Z.; Petukhova, A.; Kumacheva, E. Properties and

emerging applications of self-assembled structures made

from inorganic nanoparticles. Nat. Nanotechnol. 2010, 5,

15-25.

[17] Jones, M. R.; Osberg, K. D.; Macfarlane, R. J.; Langille, M.

R.; Mirkin, C. A. Templated techniques for the synthesis

and assembly of plasmonic nanostructures. Chem. Rev.

2011, 111, 3736-3827.

[18] Glotzer, S. C.; Solomon, M. J. Anisotropy of building

blocks and their assembly into complex structures. Nat.

| www.editorialmanager.com/nare/default.asp

Nano Res.

Mater. 2007, 6, 557-562.

[19] Bai, F.; Wang, D.; Huo, Z.; Chen, W.; Liu, L.; Liang, X.;

Chen, C.; Wang, X.; Peng, Q.; Li, Y. A versatile bottom-up

assembly approach to colloidal spheres from nanocrystals.

Angew. Chem. Int. Ed. 2007, 46, 6650-6653.

[20] Zhuang, J.; Wu, H.; Yang, Y.; Cao, Y. C. Supercrystalline

colloidal particles from artificial atoms. J. Am. Chem. Soc.

2007, 129, 14166-14167.

[21] Wang, L. B.; Xu, L. G.; Kuang, H.; Xu, C. L.; Kotov, N. A.

Dynamic Nanoparticle Assemblies. Acc. Chem. Res. 2012,

45, 1916-1926.

[22] Gong, J.; Li, G.; Tang, Z. Self-assembly of noble metal

nanocrystals: fabrication, optical property, and application.

Nano Today 2012, 7, 564-585.

[23] Hao, X.; Shang, X.; Wu, J.; Shan, Y.; Cai, M.; Jiang, J.;

Huang, Z.; Tang, Z.; Wang, H. Single-particle tracking of

hepatitis B virus-like vesicle entry into cells. Small 2011, 7,

1212-1218.

[24] Lin, J.; Wang, S.; Huang, P.; Wang, Z.; Chen, S.; Niu, G.;

Li, W.; He, J.; Cui, D.; Lu, G.; Chen, X.; Nie, Z.

Photosensitizer-loaded gold vesicles with strong plasmonic

coupling effect for imaging-guided

photothermal/photodynamic therapy. ACS Nano 2013, 7,

5320-5329.

[25] He, J.; Huang, X.; Li, Y.-C.; Liu, Y.; Babu, T.; Aronova, M.

A.; Wang, S.; Lu, Z.; Chen, X.; Nie, Z. Self-assembly of

amphiphilic plasmonic micelle-like nanoparticles in

selective solvents. J. Am. Chem. Soc. 2013, 135,

7974-7984.

[26] He, J.; Liu, Y.; Babu, T.; Wei, Z.; Nie, Z. Self-assembly of

inorganic nanoparticle vesicles and tubules driven by

tethered linear block copolymers. J. Am. Chem. Soc. 2012,

134, 11342-11345.

[27] He, J.; Wei, Z.; Wang, L.; Tomova, Z.; Babu, T.; Wang, C.;

Han, X.; Fourkas, J. T.; Nie, Z. Hydrodynamically driven

self-assembly of giant vesicles of metal nanoparticles for

remote-controlled release. Angew. Chem. Int. Ed. 2013, 52,

2463-2468.

[28] Chou, L. Y. T.; Zagorovsky, K.; Chan, W. C. W. DNA

assembly of nanoparticle superstructures for controlled

biological delivery and elimination. Nat. Nanotech. 2014, 9,

148-155.

[29] Huang, P.; Lin, J.; Li, W.; Rong, P.; Wang, Z.; Wang, S.;

Wang, X.; Sun, X.; Aronova, M.; Niu, G.; Leapman, R. D.;

Nie, Z.; Chen, X. Biodegradable gold nanovesicles with an

ultrastrong plasmonic coupling effect for photoacoustic

imaging and photothermal therapy. Angew. Chem. Int. Ed.

2013, 52, 13958-13964.

[30] Guo, X.; Szoka, F. C. Chemical approaches to triggerable

lipid vesicles for drug and gene delivery. Acc. Chem. Res.

2003, 36, 335-341.

[31] Sawant, R. R.; Torchilin, V. P. Liposomes as ‘smart’

pharmaceutical nanocarriers. Soft Matter 2010, 6,

4026-4044.

[32] Percec, V.; et al. Self-assembly of janus dendrimers into

uniform dendrimersomes and other complex architectures.

Science 2010, 328, 1009-1014.

[33] Zhang, X.; Rehm, S.; Safont-Sempere, M. M.; Würthner, F.

Vesicular perylene dye nanocapsules as supramolecular

fluorescent pH sensor systems. Nat. Chem. 2009, 1,

623-629.

[34] Discher, B. M.; Won, Y. Y.; Ege, D. S.; Lee, J. C. M.;

Bates, F. S.; Discher, D. E.; Hammer, D. A. Polymersomes:

Tough vesicles made from diblock copolymers. Science

1999, 284, 1143-1146.

[35] Huang, H. Y.; Remsen, E. E.; Kowalewski, T.; Wooley, K.

L. Nanocages derived from shell cross-linked micelle

templates. J. Am. Chem. Soc. 1999, 121, 3805-3806.

[36] Nie, Z.; Fave, D.; Kumacheva E.; Zou, S.; Walker, G. C.;

Rubinstein, M. Self-assembly of metal-polymer analogues

of amphiphilic triblock copolymers. Nat. Mater. 2007, 6,

609-614.

[37] Nikolic, M. S.; Olsson, C.; Salcher, A.; Kornowski, A.;

Rank, A.; Schubert, R.; Frömsdorf, A.; Weller, H.; Förster,

S. Micelle and vesicle formation of amphiphilic

nanoparticles. Angew. Chem. Int. Ed. 2009, 48, 2752-2754.

[38] Zubarev, E. R.; Xu, J.; Sayyad, A.; Gibson, J. D.

Amphiphilicity-driven organization of nanoparticles into

discrete assemblies. J. Am. Chem. Soc. 2006, 128,

15098-15099.

[39] Hu, J.; Wu, T.; Zhang, G.; and Liu, S. Efficient synthesis of

single gold nanoparticle hybrid amphiphilic triblock

copolymers and their controlled self-assembly. J. Am.

Chem. Soc. 2012, 134, 7624-7627.

[40] Song, J.; Cheng, L.; Liu, A.; Yin, J.; Kuang, M.; Duan, H.;

Plasmonic vesicles of amphiphilic gold nanocrystals

self-assembly and external-stimuli-triggered destruction. J.

Am. Chem. Soc. 2011, 133, 10760-10763.

[41] Guo, Y.; Harirchian-Saei, S.; Izumi, C. M. S.; Moffitt, M.

G. Block copolymer mimetic self-assembly of inorganic

nanoparticles. ACS Nano 2011, 5, 3309-3318.

[42] Wang, B.; Li, B.; Dong, B.; Zhao, B.; Li, C. Y. Homo- and

hetero-particle clusters formed by janus nanoparticles with

bicompartment polymer brushes. Macromolecules 2010, 43,

9234-9238.

[43] Andala, D. M.; Shin, S. H. R.; Lee, H. -Y. Bishop, K. J. M.

Templated synthesis of amphiphilic nanoparticles at the

liquid-liquid interface. ACS Nano 2012, 6, 1044-1050.

[44] Wei, K.; Li, J.; Liu, J.; Chen, G.; Jiang, M. Reversible

vesicles of supramolecular hybrid nanoparticles. Soft

Matter 2012, 8, 3300-3303.

[45] Liu, Y.; Li, Y.; He, J.; Duelge, K. J.; Lu, Z.; Nie, Z.

Entropy-driven pattern formation of hybrid vesicular

assemblies made from molecular and nanoparticle

amphiphiles. J. Am. Chem. Soc. 2014, 136, 2602-2610.

[46] Song, J.; Zhou, J.; Duan, H. Self-assembled plasmonic

vesicles of SERS-encoded amphiphilic gold nanoparticles

for cancer cell targeting and traceable intracellular drug

delivery. J. Am. Chem. Soc. 2012, 134, 13458-13469.

[47] Niikura, K.; Iyo, N.; Higuchi, T.; Nishio, T.; Jinnai, H.;

Fujitani, N.; Ijiro, K. Gold nanoparticles coated with

semi-fluorinated oligo(ethylene glycol) produce sub-100

nm nanoparticle vesicles without templates. J. Am. Chem.

Soc. 2012, 134, 7632-7635.

[48] Song, J.; Pu, L.; Zhou, J.; Duan, B.; Duan, H.

Biodegradable theranostic plasmonic vesicles of

amphiphilic gold nanorods. ACS Nano 2013, 7, 9947-9960.

[49] Llevot, A.; Astruc, D. Applications of vectorized gold

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

Nano Res.

nanoparticles to the diagnosis and therapy of cancer. Chem.

Soc. Rev. 2012, 41, 242-257.

[50] Erathodiyil, N.; Ying, J. Y. Functionalization of inorganic

nanoparticles for bioimaging applications. Acc. Chem. Res.

2011, 44, 925-935.

[51] Cheng, Y.; Samia, A. C.; Li, J.; Kenney, M. E.; Resnick, A.;

Burda, C. Delivery and efficacy of a cancer drug as a

function of the bond to the gold nanoparticle surface.

Langmuir 2010, 26, 2248-2255.

[52] Ghosh, P.; Han, G.; De, M.; Kim, C. K.; Rotello, V. M.

Gold nanoparticles in delivery applications. Adv. Drug

Delivery Rev. 2008, 60, 1307-1315.

[53] He, J.; Zhang, P.; Babu, T.; Liu, Y.; Gong, J.; Nie, Z.

Near-infrared light-responsive vesicles of Au nanoflowers.

Chem. Commun. 2013, 49, 576-578.

[54] Niikura, K.; Iyo, N.; Matsuo, Y.; Mitomo, H.; Ijiro, K.

Sub-100 nm gold nanoparticle vesicles as a drug delivery

carrier enabling rapid drug release upon light irradiation.

ACS Appl. Mater. Interfaces 2013, 5, 3900-3907.

[55] Song, J.; Fang, Z.; Wang, C.; Zhou, J.; Duan, B.; Pu, Lu.;

Duan, H. Photolabile plasmonic vesicles assembled from

amphiphilic gold nanoparticles for remote-controlled

traceable drug delivery. Nanoscale 2013, 5, 5816-5824.

[56] Amstad, Esther.; Kohlbrecher, J.; Müller, E.; Schweizer, T.;

Textor, M.; Reimhult, E. Triggered release from liposomes

through magnetic actuation of iron oxide nanoparticle

containing membranes. Nano Lett. 2011, 11, 1664-1670.

[57] Gopalakrishnan, G.; Danelon, C.; Izewska, P.; Prummer, M.;

Bolinger, P.; Geissbühler, I.; Demurtas, D.; Dubochet, J.;

Vogel, H. Multifunctional lipid/quantum dot hybrid

nanocontainers for controlled targeting of live cells. Angew.

Chem. Int. Ed. 2006, 45, 5478-5483.

[58] Al-Jamal, W. T.; Al-Jamal, K. T.; Tian, B.; Lacerda, L.;

Bomans, P. H.; Frederik, P. M.; Kostarelos, K.

Lipid-quantum dot bilayer vesicles enhance tumor cell

uptake and retention in vitro and in vivo. ACS Nano 2008,

2, 408-418.

[59] Niikura, K.; Iyo, N.; Higuchi, T.; Nishio, T.; Jinnai, H.;

Fujitani, N.; Ijiro, K. Gold nanoparticles coated with

semi-fluorinated oligo(ethylene glycol) produce sub-100

nm nanoparticle vesicles without templates. J. Am. Chem.

Soc. 2012, 134, 7632-7635.

[60] Bian, T.; Shang, L.; Yu, H.; Perez, M. T.; Wu, L. -Z.; Tung,

C. -H.; Nie, Z.; Tang, Z.; Zhang, T. Spontaneous

organization of inorganic nanoparticles into nanovesicles

triggered by UV light. Adv. Mater. 2014, DOI:

10.1002/adma.201401182.