Neuroendocrine Tumor-Targeted Upconversion Nanoparticle-
Based Micelles for Simultaneous NIR-Controlled Combination
Chemotherapy and Photodynamic Therapy, and Fluorescence
Imaging
Guojun Chen,
Department of Materials Science and Engineering, University of Wisconsin–Madison, Madison,
WI 53715, USA. Wisconsin Institute for Discovery, University of Wisconsin–Madison, Madison, WI
53715, USA
Prof. Renata Jaskula-Sztul,
Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35233, USA
Corinne R. Esquibel,
Laboratory for Optical and Computational Instrumentation, University of Wisconsin–Madison,
Madison, WI 53706, USA
Irene Lou,
Department of Surgery, University of Wisconsin–Madison, Madison, WI 53705, USA
Qifeng Zheng,
Department of Materials Science and Engineering, University of Wisconsin–Madison, Madison,
WI 53715, USA. Wisconsin Institute for Discovery, University of Wisconsin–Madison, Madison, WI
53715, USA
Ajitha Dammalapati,
Department of Surgery, University of Wisconsin–Madison, Madison, WI 53705, USA
April Harrison,
Department of Surgery, University of Wisconsin–Madison, Madison, WI 53705, USA
Prof. Kevin W. Eliceiri,
Wisconsin Institute for Discovery, University of Wisconsin–Madison, Madison, WI 53715, USA.
Department of Biomedical Engineering University of Wisconsin–Madison, Madison, WI 53706,
USA. Laboratory for Optical and Computational Instrumentation, University of Wisconsin–
Madison, Madison, WI 53706, USA
Prof. Weiping Tang,
School of Pharmacy, University of Wisconsin–Madison, WI 53705, USA
Prof. Herbert Chen, and
Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35233, USA
Correspondence to: Herbert Chen; Shaoqin Gong.
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.
HHS Public Access
Author manuscript
Adv Funct Mater
. Author manuscript; available in PMC 2017 October 06.
Published in final edited form as:
Adv Funct Mater
. 2017 February 23; 27(8): . doi:10.1002/adfm.201604671.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Prof. Shaoqin Gong
Department of Materials Science and Engineering, University of Wisconsin–Madison, Madison,
WI 53715, USA. Wisconsin Institute for Discovery, University of Wisconsin–Madison, Madison, WI
53715, USA. Department of Biomedical Engineering University of Wisconsin–Madison Madison,
WI 53706, USA
Abstract
Although neuroendocrine tumors (NETs) are slow growing, they are frequently metastatic at the
time of discovery and no longer amenable to curative surgery, emphasizing the need for the
development of other treatments. In this study, multifunctional upconversion nanoparticle
(UCNP)-based theranostic micelles are developed for NET-targeted and near-infrared (NIR)-
controlled combination chemotherapy and photodynamic therapy (PDT), and bioimaging. The
theranostic micelle is formed by individual UCNP functionalized with light-sensitive amphiphilic
block copolymers poly(4,5-dimethoxy-2-nitrobenzyl methacrylate)-polyethylene glycol (PNBMA-
PEG) and Rose Bengal (RB) photosensitizers. A hydrophobic anticancer drug, AB3, is loaded into
the micelles. The NIR-activated UCNPs emit multiple luminescence bands, including UV, 540 nm,
and 650 nm. The UV peaks overlap with the absorption peak of photocleavable hydrophobic
PNBMA segments, triggering a rapid drug release due to the NIR-induced hydrophobic-to-
hydrophilic transition of the micelle core and thus enabling NIR-controlled chemotherapy. RB
molecules are activated via luminescence resonance energy transfer to generate
1
O
2
for NIR-
induced PDT. Meanwhile, the 650 nm emission allows for efficient fluorescence imaging. KE108,
a true pansomatostatin nonapeptide, as an NET-targeting ligand, drastically increases the tumoral
uptake of the micelles. Intravenously injected AB3-loaded UCNP-based micelles conjugated with
RB and KE108—enabling NET-targeted combination chemotherapy and PDT—induce the best
antitumor efficacy.
1. Introduction
Neuroendocrine tumors (NETs), such as medullary thyroid cancers, carcinoids, islet cell
tumors, and small cell lung cancers, frequently metastasize to the liver.
[1]
Unfortunately,
patients with isolated NE liver metastases have poor survival outcomes.
[1c,2]
Furthermore,
patients with NETs often have debilitating symptoms, such as uncontrollable diarrhea, skin
rashes, flushing, and heart failure due to excessive hormone secretions,
[3]
thus leading to a
poor quality of life. While surgical resection can be potentially curative, many patients are
not candidates for operative intervention due to widespread metastases. Moreover, other
forms of therapy, including chemoembolization, radioembolization, radiofrequency ablation,
and cryoablation, have had limited efficacies.
[3a,4]
Therefore, besides surgery, there are no
curative treatments for NETs and their hepatic metastases. However, even surgical resection
is often followed by disease recurrence, thereby emphasizing the need for the development
of other forms of therapy.
Nanotheranostics, the integration of diagnostic and therapeutic capabilities into one
nanoplatform, may enable simultaneous imaging and therapy, thereby making personalized
medicine possible. Nanotheranostics are of great interest for targeted cancer theranostics for
the following reasons. (1) Nanoparticles (NPs) with high surface-to-volume ratios can offer
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high loading capacities for multiple payloads, including anticancer agents (e.g., drugs,
peptides, genes, etc.), imaging probes (e.g., dyes, radioisotopes, etc.), and tumor-targeting
ligands (e.g., small molecules, peptides, antibodies, aptamers, etc.).
[5]
(2) NPs can
effectively deliver these payloads to tumor lesions due to their passive (via the enhanced
permeability and retention (EPR) effect) and active (via cell-specific ligand conjugation)
tumor-targeting abilities.
[6]
(3) NPs can deliver multiple agents simultaneously, thus
enabling combination therapies, such as chemotherapy and photodynamic therapy (PDT),
which can significantly enhance their therapeutic indexes.
[7]
Photodynamic therapy (PDT) is clinically approved and known as a minimally invasive
medical technology for neo-plastic disease treatment.
[8]
PDT was the first drug–device
combination approved by the U.S. Food and Drug Administration (FDA) two decades ago.
[8]
Typically, it involves three key components: photosensitizer, light, and tissue oxygen.
[8,9]
Upon excitation of the photosensitizer under lights with proper wavelengths, the
photosensitizer is able to transfer the absorbed photon energy to the oxygen molecules in the
surroundings, thereby generating cytotoxic singlet oxygen (
1
O
2
) to kill cancer cells. PDT
can provide high specificity for the treatment of particular lesions through the control of
light exposure to the photosensitizer, thereby minimizing any potential detrimental side
effects on normal tissues.
[9,10]
However, one major limitation with current PDT is its
relatively low tissue penetration depth since most photosensitizers are excited by visible or
even UV light, thus limiting its applications.
[11]
Near-infrared (NIR) light in the range of
700–1100 nm, known as the optical tissue penetration window, can penetrate deeper into
biological tissues than UV or visible light and thus is ideal for phototherapies including PDT
and optical imaging.
[11]
Lanthanide ion (Ln
3+
, such as Er
3+
, Tm
3+
, Yb
3+
)-doped upconversion nanoparticles
(UCNPs) have attracted much attention in recent years for biomedical applications due to
their unique ability of converting NIR light to higher-energy photons (e.g., UV and visible
light).
[11b,c,12]
Therefore, photosensitizers attached to lanthanide-doped UCNPs can be
activated by NIR light via resonance energy transfer to effectively generate
cytotoxic
1
O
2
.
[13]
Photosensitizers can be loaded onto the UCNPs either via physical
adsorption or chemical conjugation. Physical adsorption is less desirable due to the high
possibility of desorption and/or leakage of the photosensitizers from the UNCPs, resulting in
low/limited efficacies.
[11c,12a,b,14]
Conjugating photosensitizers onto UCNPs via covalent
bonds can effectively overcome this limitation and thus is more desirable for UCNP-based
PDT.
[12b,14b,15]
In contrast to “free” photosensitizers employed by traditional PDT that are
subject to fast clearance and lack tumor-targeting abilities, conjugating photosensitizers onto
UCNPs can also effectively increase the accumulation of photosensitizers in the target tumor
tissues/cells due to the unique tumor-targeting abilities of the nanoparticles.
It has been demonstrated recently that UCNP-based combination chemotherapy and PDT
can lead to much better therapeutic outcomes than chemotherapy or PDT alone.
[16]
However, these previous studies either did not carry out any in vivo studies, only
investigated the anticancer efficacy of intratumorally injected nanoparticles, or used a
relatively high laser power density (e.g., 2.5 W cm
–2
).
[16]
In this study, we developed a
unique NET-targeting UCNP-based micelle capable of NIR-controlled combination
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chemotherapy and PDT, as well as fluorescence imaging (Scheme 1A) and studied their in
vivo tumor targeting behavior and anticancer efficacy in NET-bearing mice through
intravenous injection. The laser power density used for this study was 500 mW cm
−2
, which
is well below the conservative limit of 980 nm laser intensity (726 mW cm
−2
) for biological
studies and clinical applications.
[17]
The following four factors were taken into consideration
for the design of this multifunctional UCNP-based nanoplatform. (1) UCNPs can offer high-
quality imaging due to their low background autofluorescence.
[12b,18]
(2) Covalent
conjugation of photosensitizers onto the UCNPs can enable a more efficient NIR-activated
PDT. (3) NIR-controlled fast drug release at tumor sites can potentially enhance the
therapeutic efficacy of chemotherapy. (4) KE108 peptide, a true pansomatostatin synthetic
nonapeptide, can potentially serve as an effective tumor targeting ligand for medullary
thyroid cancers, a common type of NETs.
More specifically, the NaYF
4
:Yb/Tm/Er UCNPs emitted light in the UV, visible, and far-red
regions. The far-red emission (650 nm) of the UCNPs was employed for the UCNP-based
theranostic micelle imaging in vitro and in vivo. Rose Bengal (RB) photosensitizer
molecules were covalently conjugated onto the UCNP core. Since the UCNP’s luminescence
band around 540 nm overlapped with the absorption peak of RB, RB molecules were
activated via resonance energy transfer by the NIR-activated UCNPs to effectively
generate
1
O
2
for PDT. As shown in Scheme 1B, the hydrophobic core of the UCNP-based
theranostic micelle was formed by a photosensitive poly(4,5-dimethoxy-2-nitrobenzyl
methacrylate) (PNBMA) polymer that can undergo a hydrophobic-to-hydrophilic transition
under the UV light emitted by NIR-activated UCNPs due to photo induced polymer side-
group cleavage. The NIR-triggered hydrophobic-to-hydrophilic transition of the micelle core
subsequently caused a rapid release of the encapsulated hydrophobic drug (e.g., AB3, a
histone deacetylase (HDAC) inhibitor whose chemical structure is shown in Figure S1,
Supporting Information), thus leading to superior anticancer efficacy.
[19]
Finally, the UCNP-
based theranostic micelles were also conjugated with KE108 peptide, an NET-targeting
ligand, which can specifically and efficiently target all five subtypes of somatostatin
receptors (SSTRs) overexpressed by NET cells. We have recently demonstrated that KE108
possesses superior tumor targeting abilities in carcinoid xenograft animal models over other
commonly used somatostatin analogs, such as octreotide.
[20]
Our studies have demonstrated
that NIR-controlled combination chemotherapy and PDT enabled by these unique UCNP-
based theranostic micelles administered intravenously were very effective in suppressing the
tumor growth of medullary thyroid cancer. We have also shown that these UCNP-based
theranostic micelles can effectively serve as imaging probes specifically targeting the
medullary thyroid tumors (Scheme 1C).
2. Results and Discussion
2.1. Synthesis and Characterization of UCNP-Based Theranostic Micelles
The NaYF
4
:Yb
3+
/Er
3+
/Tm
3+
UCNP core was first prepared using a thermal decomposition
method in oleylamine.
[12b]
The hydrophilic amino-functionalized UCNPs (NH
2
-UCNPs)
were synthesized via a ligand-exchange approach using 2-aminoethyl dihydrogenphosphate
(AEP) as a surface coating agent to replace the original oleylamine ligand. The crystal
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structure of the NaYF
4
:Yb
3+
/Er
3+
/Tm
3+
UCNPs and the presence of elements including Na,
F, Y, Yb, Er, and Tm in these UCNPs was characterized by X-ray diffraction (XRD) (Figure
S2A, Supporting Information) and energy-dispersive X-ray (EDX) spectroscopy (Figure
S2B, Supporting Information), respectively. The Fourier transform infrared (FTIR)
absorption spectra shown in Figure 1A confirmed the successful coating of AEP on the
surface of the UCNPs. The two new absorption bands around 1115 and 1014 cm
−1
were
attributed to the O=P stretching vibration mode and P–O–C vibration mode, respectively.
Meanwhile, the long alkyl chain (−(CH
2
))–,
n
> 4) vibration mode located at 736 cm
−1
attributed to oleylamine disappeared after ligand exchange. The average size of the NH
2
-
UCNPs determined by transmission electron microscopy (TEM) was around 14 nm in
diameter (Figure 1B). The photosensitizer RB and the alkyne functional groups
(dibenzocyclooctyne acid, DBCO) were conjugated onto the UCNPs via an amidation
reaction to form RB/alkyne-UCNPs. The photosensitive amphiphilic block copolymers
polyethylene glycol (PEG)-PNBMA-N
3
and KE108-PEG-PNBMA-N
3
were prepared by
atom transfer radical polymerization (ATRP) (detailed characterization can be found in the
Supporting Information, Figure S3 and Table S1, Supporting Information) and then
conjugated onto the RB/alkyne-UCNPs via catalyst-free click chemistry,
[21]
as shown in
Figure 2.
Figure 3A (black line) shows the luminescence emissions of the NH
2
-UCNP upon 980 nm
excitation, including UV light (340–370 nm), 460, 540, and 650 nm. The red line in Figure
3B represents the UV–vis absorption spectrum of the resulting UCNP-RB/PNBMA-PEG
nanoparticles. The strong absorption peaks at the UV region are attributed to the
hydrophobic PNBMA blocks, which can form a hydrophobic micelle core wherein
hydrophobic drugs can be encapsulated. As reported previously, when excited by UV light,
the hydrophobic PNBMA segments can undergo a hydrophobic-to-hydrophilic transition,
resulting from the cleavage of the
o
-nitrobenzyl groups on the PNBMA chain, as indicated
in Scheme 1B. Hence, upon NIR activation, the UCNP would emit UV light, which would
subsequently trigger a hydrophobic-to-hydrophilic transition of the micelle core formed by
the PNBMA, thereby inducing a rapid drug release as described in detail later. Changes in
the chemical structure of the PNBMA polymer segments after 980 nm laser illumination (10
min; 0.5 W cm
−2
) were confirmed by
1
H NMR as shown in Figure S4 (Supporting
Information). The peaks ascribed to
o
-nitrobenzyl groups were significantly decreased after
980 nm laser irradiation for 10 min. The absorbance of the RB photosensitizers also
overlapped with the 540 nm luminescence emission of the UCNPs under 980 nm irradiation,
thereby enabling NIR-controlled PDT via luminescence resonance energy transfer as
discussed later. RB is a photosensitizer with a proven record for producing singlet oxygen
with high yields.
[22]
In this study, ≈100 RB molecules were conjugated per UCNP in order
to effectively generate singlet oxygen.
[12b]
Furthermore, the far-red luminescence emission
at 650 nm of the NIR-activated UCNPs was conveniently used for fluorescence imaging in
vitro and in vivo.
The individual UCNP functionalized with amphiphilic block copolymer PNBMA-PEG and
RB (i.e., UCNP-RB/PNBMA-PEG) can form a stable micelle in an aqueous solution due to
its globular structure as well as a large number of amphiphilic arms with a proper
hydrophobic-to-hydrophilic ratio.
[23]
The morphologies of the UCNP-based theranostic
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