Dual-Color Emissive Upconversion Nanocapsules for Differential

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Dual-Color Emissive Upconversion Nanocapsules for Differential Cancer Bioimaging in vivo Oh Seok Kwon, Hyun Seok Song, João Conde, Hyoung-il Kim, Natalie Artzi, and Jae-Hong Kim ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b07075 • Publication Date (Web): 04 Jan 2016 Downloaded from http://pubs.acs.org on January 5, 2016

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Dual-Color Emissive Upconversion Nanocapsules for Differential Cancer Bioimaging in vivo Oh Seok Kwona,b,1, Hyun Seok Songc,d,1, João Condec,e, Hyoung-il Kima, Natalie Artzic,f,* and Jae-Hong Kima,* a

Department of Chemical and Environmental Engineering, School of Engineering and Applied Science,

Yale University, New Haven, CT 06511, U.S.A. b

BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology

(KRIBB), Yuseong, Daejeon 305-600, Republic of Korea c

Harvard–MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, 77

Massachusetts Ave., Cambridge, MA 02139, U.S.A. d

Division of Bioconvergence Analysis, Korea Basic Science Institute (KBSI), Yuseong, Daejeon 169-

148, Republic of Korea. e

School of Engineering and Materials Science, Queen Mary University of London, London, UK.

f

Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA

02115, U.S.A.

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O.S.K. and H.S.S contributed equally to this work

*To whom correspondence should be addressed: Prof. N. Artzi: Tel: +1-617-715-2061; Fax: +1-617-253-2514; E-mail: [email protected] Prof. J. -H. Kim: Tel: +1-203-432-4386; Fax: +1-203-432-4387; E-mail: [email protected]

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ABSTRACT: Early diagnosis of tumor malignancy is crucial for timely cancer treatment aimed at imparting desired clinical outcomes. The traditional fluorescence-based imaging is unfortunately faced with challenges such as low tissue penetration and background autofluorescence. Upconversion (UC)based bioimaging can overcome these limitations as their excitation occurs at lower frequencies and the emission at higher frequencies. In this study, multifunctional silica-based nanocapsules were synthesized to encapsulate two distinct triplet-triplet annihilation UC chromophore pairs. Each nanocapsule emits different colors, blue or green, following a red light excitation. These nanocapsules were further conjugated with either antibodies or peptides to selectively target breast or colon cancer cells, respectively. Both in vitro and in vivo experimental results herein demonstrate cancer-specific and differential-color imaging from single wavelength excitation as well as far greater accumulation at targeted tumor sites than that due to the enhanced permeability and retention effect. This approach can be used to host a variety of chromophore pairs for various tumor-specific, color-coding scenarios and can be employed for diagnosis of a wide range of cancer types within the heterogeneous tumor microenvironment.

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KEYWORDS

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upconversion; triplet-triplet annihilation; dual-color; nanocapsule; cancer; imaging; diagnosis.

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Nanomedicine gained growing interest in diagnosing and treating cancers. Various organic and

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inorganic photoluminescence nanoparticles made of dye-doped or loaded polymers, quantum dots,

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magnetic nanoparticles, and upconverting nanophosphors have been developed in the past decades for in

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vivo detection and imaging.1 More recently, core-shell structured nanoparticles have been explored for

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simultaneous tumor targeting, imaging, and in situ treatment.2-4 The core hosts not only imaging agents

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but also therapeutic drugs that can be released at the target site for treatment, while the nanometric

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particles enable enhanced permeability and retention (EPR), leading to effective accumulation at the

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tumor site.1,

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accumulation at the tumor site and provide selectivity towards specific cancer cell populations.

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However, imparting targeting capabilities to the nanocarriers may improve the

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Most of the conventional imaging approaches rely on fluorescence that exhibits ineffective tissue

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penetration at the excitation range of ultraviolet or low wavelength visible light.1, 10-13 This inevitably

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dictates the use of high power irradiation, which further results in optical noise from background

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autofluorescence and potential cell damage. With the goal of achieving photoluminescence imaging

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using a light source with wavelength that lies within the phototherapeutic window of 600-1000 nm,

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photon frequency upconversion (UC) has been pursued as an alternative strategy.14


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The UC is an anti-Stokes process through which two or more photons of low frequency are

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converted into a single photon with higher frequency.15 Lanthanide ion-doped inorganic crystals have

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been benchmark materials for decades, wherein UC occurs through sequential absorption of two photons

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by long-lived excited-state lanthanide activators or by sensitized energy transfer between excited states.

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Bioimaging application based on trivalent lanthanide-doped inorganic nanoparticles has been attempted

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mostly with infrared excitation, but it suffers from requirement for extremely high power excitation (101

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~ 104 W cm-2) and inherently poor quantum yield owing to low absorption and emission cross sections.16

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Such limitations sparked the recent surge in search for an alternative UC strategy, particularly an organic

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chromophore-based system that achieves efficient triplet-triplet annihilation (TTA)-based UC under low

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power density excitation (< 10-2 W cm-2). In TTA-UC, the photon energy absorbed by a sensitizer

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chromophore is transferred to an acceptor chromophore through triplet−triplet energy transfer, and two

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excited acceptor molecules undergo TTA process, producing singlet fluorescence of higher frequency.

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However, there are a couple of major challenges involved in applying TTA-UC for bioimaging, where

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the matrix is oxygen-rich aqueous phase. First, presence of ground-state triplet oxygen is detrimental,

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since it quenches chromophores’ triplet-states in a non-radiative manner.17, 18 Second, chromophores

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known to date are soluble only in organic solvents, significantly limiting the host media options.19, 20

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To overcome these limitations, several studies have attempted to embed TTA-UC chromophores

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within polymeric18 or inorganic21 nanoparticles. These approaches partly resolved the oxygen quenching

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problem, but limited the chromophore mobility in rigid matrices, resulting in retardation of

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intermolecular collision and energy transfer, which were found to be detrimental with respect to

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quantum yield. Therefore, approaches to encapsulate chromophore-containing fluidic media in micelles,

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dendrimers, and polymer shell structures22-24 have been recently explored. Albeit this architecture has

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proven to achieve more efficient TTA-UC, their clinical utility is limited due to lack of structural

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rigidity,22 poor stability against pH, temperature variation,18 difficulty of shell surface modification for

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multifunctional applications24,

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device).

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and large size (e.g., polymer shells fabricated using a microfluidic

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We herein report the successful fabrication of TTA-UC nanocapsules tailored for in vitro and in

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vivo cancer cell imaging. These nanocapsules, previously demonstrated for aqueous photocatalysis by

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surface loading of semiconductors,26 are made of biocompatible, water-stable (i.e., water dispersible)

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silica shells that enable not only robust encapsulation of chromophore-containing organic medium but

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also facile surface functionalization with cancer specific antibodies and peptides for selective tumor


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targeting. We synthesized two different nanocapsules for in vitro and in vivo validation. Each core

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contains a distinct pair of TTA-UC chromophores such that two different emissions, 470 nm (blue) and

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515 nm (green), are achieved under a single wavelength excitation (635 nm; red) (see Scheme 1). Each

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shell is functionalized with distinct bioprobes that target different cancer cells. This enables, for the first

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time in literature, the discrimination between different cancer cells through imaging with more than one

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upconverted photoluminescence color emitted from low energy photon irradiation, which represents a

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significant improvement over conventional fluorescence-based imaging approaches.27 As a proof of

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concept, we show that this platform has the ability to distinguish different cancer cell types, breast and

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colon cancer, using the appropriate targeting moieties. We envision using this platform to distinguish

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between different cells residing in the heterogeneous tumor microenvironment28 (we have shown that

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already discriminate various cancer types,29, 30 which are critical to the development of cost-effective

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differential cancer cell detection and treatment methods.31, 32

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Scheme 1. A schematic of TTA-UC process leading to the delayed fluorescence from acceptors,

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perylene (blue) and BPEA (green). For emission of a single higher-energy photon from one 1ACC*, two

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TTET processes are required to form two 3ACC* which then undergo TTA.

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RESULTS AND DISCUSSION

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Nanocapsule Fabrication and Characterization. Two different types of silica nanocapsules (SNCs)

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that achieve different photon frequency shifting schemes were synthesized. The first SNC (referred to

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herein as SNC-B) encapsulates palladium (II) tetraphenyltetrabenzoporphyrin (PdTPBP) as a sensitizer

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and perylene as an acceptor for red-to-blue UC. The second SNC (referred to as SNC-G) encapsulates


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the same sensitizer and 9,10-bis-(phenylethynyl)anthracene (BPEA) acceptor for red-to-green UC. SNCs were formed by first fabricating silica-shell, oleic acid (OA)-core nanocapsule structures following the self-assembly procedure we previously established.26 Herein, OA serves as a solvent to dissolve chromophore, as a surfactant to form fundamental micelle structure, and as an oxygen quencher. Briefly, the OA phase containing a chromophore pair was emulsified in water by sonication under vigorous mixing (Figure 1A). The micelles self-assembled with the addition of (3-aminopropyl)triethoxysilane (ATES), which were further reacted with tetraethyl orthosilicate (TEOS) to form rigid silica shells (Figures 1B and S1). The surface of SNC was then functionalized with the amino group (-NH2) by adding (3-aminopropyl)trimethoxysilane (APMS) (Figure 1C) to covalently attach cancer specific bioprobe using 4-(4,6-dimethoxy-1,3,5-triazin-2-ly)-4-methylmorpholinium chloride (DMT-MM) as a coupling reagent. As a proof of concept, we designed the particles to selectively accumulate in breast or colon cancer tumors using specific targeting moieties. MUC1 is a heterodimeric membrane protein that functions as an oncoprotein interacting with receptor tyrosine kinases, aberrantly overexpressed in breast cancer.33-35 Hence, anti-MUC1 antibody, which specifically binds to breast cancer cell, was attached to the SNC-B (Figures 1D and 1F). TCP1 is a vasculature-targeting peptide for colorectal cancer, which exhibits the homing ability to deliver therapeutic agents to colon tumor neovasculature.36, 37 Therefore, the TCP1 peptide was used for targeting colorectal cancer cells and was conjugated to the SNC-G (Figures 1E and 1G).


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Figure 1. A procedure to fabricate bioprobe-attached SNC-B and SNC-G (A through E) and the schematic illustration of the cancer-specific, dual color imaging of (F) breast and (G) colorectal-cancer cells.


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Scanning electron microscopy (SEM; JSM-6700F) and transmission electron microscopy (TEM; JEOL JEM-3010 (JEOL) (inset) images of the SNCs prior to bioprobe attachment is depicted in Figure 2A. The SNCs were highly spherical (Figure 2A inset TEM image; the most representative SNC shown) and about 21 nm in average shell thickness (Figure 2A inset histogram). A Dynamic light scattering (DSL) analysis revealed that particles present in the aqueous phase as polydispersed agglomerates with an average diameter of 216.8 ± 36.2 nm. After MUC1-antibody was attached, the surface of the SNC became slightly rougher (Figures 2B and S3) compared to its precursor’s smooth surface, confirming a successful surface coating. The binding of bioprobes on the surface of SNCs was confirmed via an immunocytochemistry analysis. We introduced Alexa-Fluor® 488-conjugated antibody to SNC-Bs coated with anti-MUC1 antibody and fluorescein isothiocyanate (FITC)-conjugated TCP1 peptides to SNC-Gs. In both cases, we observed an intense green fluorescence emission under 488 nm excitation using an epi-fluorescence microscope (Figures 2C and 2D), confirming the successful binding of bioprobes onto the surfaces of SNCs (Figure S2).

Figure 2. SEM and TEM images (insets) of SNCs (A) before and (B) after the introduction of the bioprobes on their surfaces. The inset in (A) shows the size distribution of the SNCs. (C) Fluorescence image of SNC-B coated with anti-MUC1 antibody. For the visualization, Alexa Fluor 488-conjugated antibody that specifically binds to anti-MUC1 antibody was treated. (D) Fluorescence image of SNC-G coated with fluorescein isothiocyanate (FITC)-conjugated TCP1 peptide.


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Photoluminescence Characteristics. Chemical structures and photoluminescence spectra of the UC chromophores dissolved in OA are shown in Figure 3. PdTPBP shows characteristic absorption at 446 nm (Soret band) along with two Q-band features at 575 nm and 635 nm (red) where neither BPEA nor perylene absorbs (Figure 3B). The UC emission intensity from PdTPBP/BPEA and PdTPBP/perylene pairs in OA increased with increasing 635 nm excitation power; displaying precipitous increase under low-power density excitation (< 50 mW cm-2), followed by a gentle gradient beyond 1,000 mW cm-2, and ultimately reaching a plateau (ca. 4.3 % and ca. 3.3 % for PdTPBP/BPEA and PdTPBP/perylene, respectively) at the highest power (Figures 4 and S4), when acceptor concentrations were the highest (Figure S5). A double logarithm plot of normalized integrated photoluminescence intensity versus incident power exhibited a characteristic transition from quadratic (slope = 2) to linear dependency (slope = 1) for both chromophore pairs,38 while singlet fluorescence from a standard dye, methyelene blue, exhibited linear excitation power dependence.

Figure 3. (A) Molecular structures of the sensitizer (PdTPBP) and acceptors (perylene and BPEA). (B) Normalized absorption and emission spectra of PdTPBP, perylene, and BPEA in OA at room


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temperature. The emission spectra were obtained with excitation at 635 nm for PdTPBP, 407 nm for perylene and 438 nm for BPEA.

Figure 4. (A) The dependence of UC emission intensities from PdTPBP/BPEA and PdTPBP/perylene pairs on the incident laser power density. Dark-field photographs on the right show UC emission from chromophore pairs in OA samples, taken through a 600 nm short-pass filter under excitation at 635 nm.


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(B) and (C) Log-log plots of the normalized integrated UC emission intensities of PdTPBP/BPEA (B) and PdTPBP/perylene (C) in OA as a function of the incident laser power density. The dashed lines are linear fits with slopes of 1.0 (gray lines) and 2.0 (green line in (B) and blue line in (C)). The bright-field photographs in (B) and (C) showed the UC emission from chromophore pairs and control samples without a 600 nm short-pass filter. All measurements were carried out in ambient atmosphere. The concentrations of sensitizer (PdTPBP) and acceptors (BPEA and perylene) employed for these inset photos are 11.96 µM, 1.1 mM, and 1.45 mM, respectively.

Bioprobe-loaded SNCs that encapsulate these chromophores, when suspended in water without deoxygenation, exhibited very similar photoluminescence behaviors as the above-discussed bulk solutions. Under irradiation with 635 nm laser, bright blue UC emission by SNC-B and green UC emission by SNC-G were visible to the naked eye through a 600 nm shortpass filter (Figure 5A). At high SNC concentration (10 wt%), intense light scattering by SNCs that settled at the bottom of the vial under the quiescent condition was observed, whereas the emission was detected only along the path of incident laser beam at low SNC concentration (0.03 wt%) under gentle mixing (Figure 5A). The PLS spectra upon laser excitation at 635 nm show phosphorescence emission at λmax = 800 nm (i.e., PdTPBP phosphorescence) as well as upconverted anti-stokes emissions at λmax = 470 nm (blue) for SNC-B and λmax = 515 nm (green) for SNC-G in the aqueous suspension (Figures 5B and 5C). Figure 5D shows that normalized UC emission intensity from SNCs increases with increasing incident laser power density, following a quadratic dependency, which is typical of TTA-UC in weak annihilation regime. The incident laser power was increased only up to 538 mW cm-2 due to interference from light scattering above this value. Moreover, the prepared SNCs showed long term stability in oxygen-rich water (Table S1). The integrated UC intensity only slightly decreased (2~3%) at 158 mW cm-2 after more than a month of storage in the dark.


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Figure 5. (A) Blue and green emission from SNC-B and SNC-G, respectively, suspended in water without deoxygenation under 635 laser beam irradiation, taken using a 600 nm shortpass filter. UC emission spectra of (B) SNC-B and (C) SNC-G as a function of incident laser power density (mW cm-2) under excitation at 635 nm. (D) Normalized integrated UC emission intensity plotted as a function of normalized incident light power density. The highest incident power density was 342 mW cm-2.

In vitro Dual-Color Differential Cancer Cell Imaging. The specific affinity of the bioprobes to the targeted cancer cells was first confirmed via in vitro immunocytochemistry using conventional cell lines. Human breast cancer cell lines (MCF7 and MDA-MB-231), colorectal cancer cells (LoVo), and a control cell line (3T3 human-fibroblasts) were used. Nanoparticles conjugated with MUC1 antibody for targeting MUC1 were treated with Alexa-Fluor® 488-conjugated secondary antibody to allow intracellular trafficking. Fluorescence emission was observed only with MCF7 and MDA-MB-231 cell lines (Figure S6A). Western blot analysis of these cells (Figure S6B) suggests that only breast cancer cells exhibit unique bands that are associated with MUC1. These results collectively suggest that MUC1


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is expressed only in breast cancer cells and MUC1 antibody binds merely to breast cancer cells. TCP1 peptide conjugated with FITC was also added to LoVo colorectal cancer cells and 3T3 fibroblast control cells. Fluorescence was detected exclusively in the colorectal cancer cells when compared to 3T3 fibroblasts (Figure S6C), confirming that TCP1 peptide binds only to colorectal cancer cells while sparing healthy cells. Parallel in vitro imaging was performed using SNC-B and SNC-G along with the same cancer cell lines. All cells were incubated with SNCs for 5 min and washed with 0.1 mM NaCl. With the addition of SNC-B decorated with anti-MUC1, MDA-MB-231 breast cancer cells showed bright blue UC emission (Figures 6A and S7A) when viewed through a 475 nm bandpass filter under a fluorescence microscope (see Figure S8 for filter configuration). Likewise, intense green emission was also observed (Figure 6B and S7B) through a 515 nm bandpass filter only with LoVo colorectal cancer cells, when treated with SNC-G loaded with TCP1 peptide. Locations of fluorescence emission matched with the locations of cells, as confirmed by superimposing UC emission images onto fluorescence images obtained with staining cells with DRAQ5 (Figures 6A and 6B). Minimal UC emissions were detected from 3T3 fibroblast control cells, as expected, due to non-specific ionic binding of SNCs to cell surfaces (MCF7; 15.983±0.357, MDA-MB-231; 14.422±1.843, 3T3; 4.111±0.178, fluorescence intensity (arbitrary unit, ×104) per cell, Figure 6C) (LoVo; 19.174±0.709, 3T3; 1.517±0.670 fluorescence intensity (arbitrary unit, ×104) per cell, Figure 6D). To verify the selective uptake of the nanocapsules, we performed UC imaging analysis using LoVo colorectal cancer cells following treatment with MUC1 antibody-conjugated SNC-B and MDA-MB-231 breast cancer cells following treatment with TCP1-conjugated SNC-G, and found that minimal UC emissions were obtained (Figure S7C and D). This background fluorescence can be set as a threshold above which positive signal was considered cell specific. However, when we co-cultured cancer cells with control cells, for example LoVo with 3T3 fibroblast, we observed UC emission only from LoVo cells (stained with DRAQ5) and not from 3T3 fibroblasts (stained with DiI, indicated by arrows) (Figure S7E). Finally, MCF7 cells with their nuclei stained with Hoechst 33258 and further treated with SNC-B showed characteristic blue emission from Hoechst 33258 (through blue channel using a confocal laser scanning microscope) as well as green and red fluorescence original from the UC chromophores (through green and red channels, respectively), pointing at their co-localization (Figure S7F). These in vitro results collectively suggest that the SNC-B and SNC-G selectively bind to breast and colorectal cancer cells, respectively, and emit sufficiently intense upconverted emission of which the color is cancer specific.


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The final in vitro experiment involved the testing of cytotoxicity of SNCs based on 3-(4,5dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The viability of 3T3 fibroblasts showed negligible decrease at 24-h exposure with 0.2 µg/mL of bare SNC-Bs and retained greater than 80% of their original viability even after 24-h exposure with 10 µg/mL (Figure S7G).

Figure 6. (A) Photoluminescence images of MDA-MB-231 breast cancer cells and 3T3 fibroblast control cell treated with MUC1-conjugated SNC-B. (B) Photoluminescence images of LoVo colorectal cancer cell and 3T3 fibroblast control cell treated with TCP1-conjugated SNC-G. Cells were stained with DRAQ5 for the visualization of nucleus. Scale bar = 100 µm. Quantitative analysis of upconversion luminescence intensity from (C) breast cancer cell-lines, MCF7 and MDA MB 231, and 3T3 fibroblasts, and (D) colorectal cancer cell-line, LoVo, and 3T3 fibroblasts (***, P < 0.005 *, P < 0.05 using twotailed Student’s t-test).

In vivo Dual-Color Differential Tumor Imaging. To demonstrate in vivo imaging of different cancer cells, a cancer mouse model bearing two types of tumors was treated with two distinct SNCs: i.e., SNCB to target breast cancer cells and SNC-G to target colorectal cancer cells. A schematic illustration of a custom-built UC in vivo cancer cell imaging system is depicted in Figure S9. We successfully observed strong blue emission at the location of breast cancer cells from SNC-B conjugated with MUC1 and strong green emission at the location of colorectal cancer cells from SNC-G conjugated with TCP1 (Figure 7A). In the absence of targeting moiety, SNCs still showed nominal


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accumulation at the tumor site because of the EPR effect at the leaky tumor vasculature (Figure 7B). Due to an inherent tumor defective vasculature together with a poor lymphatic drainage, SNC nanoparticles can exploit these characteristics to achieve a passive accumulation effect.7-9 However, the signal was significantly enhanced when targeting moieties were conjugated to the particles. The conjugation of bioprobes increased the accumulation of SNCs about 3-fold at both tumor sites (green targeting; 62.4±9.1, green non-targeting; 21.9±2.9, blue targeting; 50.2±9.8, blue non-targeting; 18.7±3.4, fluorescence intensity (arbitrary unit, ×104), Figures 7D, E). As expected, no signal was detected at the tumor area in the absence of SNCs (Figure 7C). Our in-vivo results suggest that targeted SNC formulations provided a statistically significant accumulation at the tumor sites at 16 hours postinjection, compared to non-targeted SNCs that exhibited low sensitivity and poor specificity. Results further confirm that SNCs enable simultaneous cancer type differentiation based on high-contrast color under low frequency excitation.

Figure 7. Bright field image (left) and blue and green merged photoluminescence image (right) of a mouse bearing breast and colorectal cancers. (A) With the injection of bioprobes-conjugated SNCs, (B)


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bare-SNCs and (C) with no treatment. Fluorescence intensity from (D) SNC-B and (E) SNC-G (**, P < 0.01; *, P < 0.05 using two-tailed Student’s t-test).

CONCLUSIONS The TTA-UC scheme herein realized using nano-scale encapsulation architecture for in vitro and in vivo imaging of cancer cells presents unique advances over existing bioimaging approaches. First, the use of TTA-UC permits the use of low-frequency light source within the phototherapeutic window that enables deep tissue penetration while avoiding autofluorescence interference compared to fluorescencebased imaging. Second, the encapsulation strategy allows hosting of various UC chromophore pairs for different color imaging, such that simultaneous multicolor imaging for multiple cancer cell types can be pursued. Third, the silica surface of SNC is not only biocompatible as evident by low cell toxicity but also capable of hosting various other functionalities including different bioprobes and potentially treatment drugs, opening up further multifunctional applications. These probes are covalently bound to the surface, presenting significant advantage in terms of stability compared to past approaches that rely on physical absorption of probe molecules,39, 40 as was validated using two cancer models.

EXPERIMENTAL SECTION Materials. Tetrahydrofuran (THF, >99.9%), OA (99%), ATES (99%), APMS (97%), DMT-MM (96%), TEOS (99.999%), and perylene were purchased from Sigma-Aldrich, PdTPBP from Frontier Scientific, and BPEA from Gelest Inc. All reagents and solvents were used as received without further treatment. Stock solutions of PdTPBP (0.47 mM), perylene (19 mM), and BPEA (13 mM) in THF were prepared and stored in the dark. MCF7, MDA-MB-231 and 3T3 cells were cultured in Dulbecco's modified Eagles medium (DMEM, Invitrogen) with 10% fetal bovine serum (Gibco, Life Technologies), 2 mM Lglutamine (Invitrogen), and 100 units/mL penicillin/streptomycin (Invitrogen). LoVo cells were cultured in Ham's F-12K medium (Gibco, Life Technologies) containing 2 mM L-glutamine with 10% fetal bovine serum (Gibco, Life Technologies) and 100 units/mL penicillin/streptomycin (Invitrogen).

SNC-B and SNC-G Fabrication. Two pairs of chromophores were dissolved in OA and stored overnight at 70oC to evaporate THF to prepare core phases for SNC-B (0.2 µM PdTPBP and 25 µM perylene) and SNC-G (0.2 µM PdTPBP and 17 µM BPEA), respectively. An aliquot (500 µL) of each


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mixture was emulsified into the distilled water (28.8 mL) via sonication (40 KHz) for 1 h followed by vigorous stirring for 1 h. ATES (1.67 mmol) was added dropwise to the emulsified mixture, followed 11 mmol of TEOS. The mixture was left at room temperature for ca. 2 h, then placed in oven at 55oC for 30 h. The final products were obtained after several washing steps with distilled water and ethanol. The surface of as-prepared SNC was modified using 5.6 mmol APMS under gentle mixing at 55oC for 30 h and washed with distilled water. The amino groups on the surface of SNCs further reacted with carboxylic groups of anti-MUC1 antibody and TCP1 peptide using 1 wt% DMT-MM solution (10 µL) as a conjugation reagent under gentle mixing for 24 h. The final products, bioprobe-attached SNC-B and SNC-G, were washed with distilled water and stored in dark.

Photoluminescence Measurements. A UV-visible spectrophotometer (8453, Agilent Technologies) and spectrofluorophotometer (RF-5301, Shimadzu) were used to obtain the static absorption spectra and Stocks emission spectra, respectively. Stokes and Anti-stokes emission spectra of the aqueous SNC-B and SNC-G suspensions were obtained with a diode laser (635 nm, beam of cross-sectional area of beam = 2 × 4.5 cm2) as an excitation source. The irradiation power was adjusted using neutral density filters and measured using a Nova II power meter/photodiode detector head (Ophir). The laser was irradiated to a cylindrical glass vial containing sample at an angle of approximately 45o and emission was collected through two focusing lenses and an optical chopper (160 Hz) into a monochromator (Oriel Cornerstone, Newport Corp.). The signal was detected by an Oriel photomultiplier tube and processed by an Oriel Merlin radiometry detection system (Newport Corp.).

In vitro Fluorescence and UC Imaging. Two human breast cancer cells (MCF7 and MDA-MB-231), one human colorectal cancer cell (LoVo), and one non-cancer cell (3T3 human fibroblasts) were used throughout this study. For imunocytochemistry and fluorescence imaging, MCF7, MDA-MB-231 and 3T3 cells were first fixed with chilled absolute ethanol for 5 min and washed by phosphate buffer saline (PBS) several times. The fixed cells were then incubated with anti-MUC1 antibody (abcam, 1:1000 dilution in PBS) for 2 h at 37oC and washed using PBS several times. The cells were further treated with Alexa-Fluor 488-conjugated anti-mouse antibody (Life Technologies, 1:1500 dilution in PBS) and washed with PBS several times for fluorescence imaging. LoVo and 3T3 cells were incubated with FITC-conjugated TCP1 peptide (CTPSPFSHC) (0.01 mg/mL in culture media) for 30 min at 37 oC and washed with PBS several times. Alternatively, MCF7, MDA-MB-231 and 3T3 cells were treated with


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100 µg/mL SNC-B and LoVo and 3T3 cells with the same concentration of SNC-G for 5 min at 37 oC and washed with 0.1 mM NaCl. Cells were then cultured with culture media for 24 h and stained with DRAQ5 (abcam). The fixation was performed by using 3.7% paraformaldehyde in PBS. Cells were examined using a fluorescence microscope (Nikon Eclipse Ti). All images were obtained after 0.5 s of exposure. For UC luminescence analysis, 635 nm excitation filter and either 475 nm or 515 nm emission filter (20-nm bandwidth) for blue and green emission, respectively, were used. ImageJ software was used to quantitatively analyze UC emission intensities after correcting background noise and normalizing based on the number of cells in the image. For confocal laser scanning microscopy, MCF7 cells treated with SNC-B were fixed with 3.7% paraformaldehyde in PBS for 15 min at 37 ºC and then permeabilized by the incubation with 0.1% Triton X-100 in PBS for 10 min. Cells were then mounted in ProLong® Gold Antifade Reagent (Invitrogen). Images of cells were taken with a Nikon A1R Ultra-Fast Spectral Scanning Confocal Microscope using blue (λex = 405 nm/λem = 450 nm), green (λex = 488 nm/λem = 525 nm), and red (λex = 561 nm/λem = 600 nm) channels. All images were obtained using the same exposure time.

Cytotoxicity test. To test the cytotoxicity of the SNCs, 3T3 fibroblasts were seeded on a 48-wells plates at 70% confluency, and different concentrations of SNCs were added. MCF7, MDA-MB-231, and LoVo cells were treated by bioprobe-conjugated SNCs under the same condition as the above fluorescence imaging experiments. Cells were then incubated for 24 h at 37 °C in a 5% CO2 incubator, treated with 1 mM of (3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyl-tetrazolium (MTT, Invitrogen), and further incubated for 2 h. The formazan crystals were solubilized by dimethyl sulfoxide (Sigma), and the absorbance was measured at 570 nm using a microplate reader (Varioskan Flash Multimode Reader, Thermo Scientific).

In vivo UC Imaging. Tumors were induced in female SCID hairless congenic mice by the subcutaneous injection of MDA-MB-231 breast cancer cells on the right side of the mice and LoVo colorectal cancer cells on the left side (5×106 cells suspended in 100 µL of Hank’s balanced salt solution from Lonza). For determination of tumor growth, individual tumors were measured using a caliper and tumor volume was calculated as width × (length2) / 2. When the tumor volume reached about 1 cm3, the mice were anesthetized with isoflurane and SNC-B and SNC-G were injected through a tail vein. Sixteen hours after the administration, the mice were anesthetized by 200 mg/kg of ketamine and placed on a custombuilt in vivo imaging system equipped with a dark chamber with an adjustable stage to place the mice


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and tumors were irradiated by a laser source. The power and size of irradiation was adjusted by a beam expander (300 mW on 0.5 cm2). The images were obtained using a high resolution CCD camera after bandpass filters (475 nm for blue images and 515 nm for green images) and analyzed using a gel documentation system (ChemiDoc XRS, BioRad). UC signals were quantified by analyzing images using ImageJ software after background noise correction and normalization as discussed above. All experimental protocols were approved by the MIT Animal Care and Use Committee and were in compliance with NIH guidelines for animal use.

Statistical Approach. Differences between fluorescence measurements were analyzed using a twotailed Student’s t-test through SPSS statistical package (version 23, SPSS Inc.). All error bars used in this report represent standard deviations of measurements from at least 3 independent experiments. Observations with P-values lower than 0.05 were considered statistically significant. All in vivo experiments involved three mice per treatment.

ACKNOWLEDGMENTS This work was supported by the National Science Foundation (CBET-1335934) and the KRIBB Initiative Research Program (KRIBB, Korea). This work was supported by the Korea Basic Science Institute (T35402). This work was also supported by the Korea Basic Science Institute (T35402) and Global Frontier Project (H-GUARD_2014M3A6B2060489) through the Center for BioNano HealthGuard funded by the MSIP. Supporting Information Available: The Supporting Information is available free of charge on the ACS Publications website at DOI:


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TABLE OF CONTENTS


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