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Biological and Medical Applications of Materials and Interfaces

Glutathione-Priming Nanoreactors Enable Fluorophore Core/Shell Transition for Precision Cancer Imaging Zhiqiang Lin, Changrong Wang, Yang Li, Ridong Li, Lidong Gong, Yue Su, Zheng Zhai, Xinyu Bai, Shiming Di, Zhao Li, Anjie Dong, Qiang Zhang, and Yuxin Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b11063 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 17, 2019

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ACS Applied Materials & Interfaces

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Glutathione-Priming Nanoreactors Enable Fluorophore Core/Shell

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Transition for Precision Cancer Imaging

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§ # † † † *,† Changrong Wang, Yang Li, Ridong Li, Lidong Gong, Yue Su, Zheng Zhiqiang Lin,

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† & § *,‡ *,† † † Yuxin Yin Zhai, Xinyu Bai, Shiming Di, Zhao Li, Anjie Dong, Qiang Zhang,

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†

Institute of Systems Biomedicine, Beijing Key Laboratory of Tumor Systems Biology, School

of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China ‡

Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing

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100191, China.

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§ Department of Polymer Science and Technology, School of Chemical Engineering and

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Technology, Tianjin University, Tianjin 300072, China # Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA

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& Department of Hepatobiliary Surgery, Peking University People’s Hospital, Beijing 100044,

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China

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KEYWORDS: nanoreactors, core/shell transition, precision imaging, glutathione,

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bioresponsive

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ABSTRACT: In an attempt to develop an imaging probe with ultra-high sensitivity for a

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broad range of tumors in vivo and inspired by the concept of chemical synthetic nanoreactors, we

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designed a type of glutathione-priming fluorescent nanoreactors (GPNs) with albumin-coating

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shell and hydrophobic polymer core containing disulfide bonds, protonatable blocks and

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indocyanine green (ICG), a near-infrared fluorophore. The albumin played multiple roles

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including biocompatible carriers, hydrophilic stabilizer, “receptor” of the fluorophores, and even

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targeting molecules. The protonation of hydrophobic core triggered the outside-to-core transport

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of acidic glutathione (GSH), as well as the core-to-shell transference of ICGs after disulfide bond

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cleavage by GSH, which induced strong binding of fluorophores with albumins on the GPN shell,

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initiating intensive fluorescence signals. As a result, the GPNs demonstrated extremely high

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response sensitivity and imaging contrast, proper time window and broad cancer specificity. In

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fact, an orthogonal activation pattern were found in vitro with an ON/OFF ratio up to 24.7 folds.

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Furthermore, the nanoprobes specifically amplified the tumor signals in five cancer-bearing

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mouse models and actualized tumor margin delineation with a contrast up to 20 folds,

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demonstrating much better imaging efficacy than the other four commercially-available probes.

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Therefore, the GPNs provide a new paradigm in developing high-performance bioresponsive

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nanoprobes.

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1. INTRODUCTION

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During surgery, real-time and precision cancer imaging holds great potential to significantly

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improve cancer therapy outcomes, empowering surgeons to perform better and faster.1, 2 Over the

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past several years, near-infrared (NIR) fluorescence image-guided surgery has shown great

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clinical applicability in cancer imaging, lymph-node identification and vital structure

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visualization in the surgical theatre.3 A common strategy of fluorescent imaging usually focused

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on cancer molecular imaging probes targeting the specific receptors on cell surface as epidermal

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growth factor receptor, folate receptor-, integrin αvβ3 and so on.4, 5 Despite success of such

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probes in visualizing some subset of cancers in clinical trials, they are in lack of broad tumor

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applicability and produce high tissue background because of their incapability of achieving

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fluorescence quenching in non-cancerous tissues.6 Alternative fluorescent probes on basic

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research stage were mainly the bioresponsive small-molecule tracers which could be specifically

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activated in cancer nodules. Unfortunately, they are often short of high cancer to normal tissue

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ratio (CNR) in fluorescent signals and response sensitivity in vivo.7 Thus, it is an urgent need to

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develop a fluorescent probe to illuminate tumors more universally and specifically.3

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Currently, stimuli-responsive nanoprobes are increasingly attracting our attention in terms

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of precision cancer imaging because of their broad specificity as well as high sensitivity. 8, 9 Such

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“smart” nanoprobes are capable of responding to specific physiological or pathological triggers

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such as pH, enzymes, glucose, and redox for being activated at disease site. For example, redox-

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sensitive systems have commonly been developed by disulfide-containing materials because of


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the abundant glutathione/glutathione disulfide couple in animal cells and the reaction of thiol–

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disulfide exchange. So far, a great number of novel bioresponsive systems have been designed

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and featured with strong novelty. However, under the mild biological stimuli conditions with

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complex environments in vivo, many of them have been impeded by inadequate response

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efficacy (e.g., selectivity, sensitivity and response time) and output performance like CNR,

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which were regarded as two important basic criteria for their further translation and possible

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commercialization.10, 11

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Nanoreactors are an emerging and promising technology in a large variety of applications such as organic synthesis, polymerization, nanoparticle synthesis and so on.12,

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In typical

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nanoreactors, there are hollow nanostructures for chemical reactions and permeable shells for

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sheltering and controlling the catalytic processes.12, 13 Nanoreactors offer the feasibility for high-

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efficiency transformation of reactants into resultants, and significantly accelerate the reaction

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rate. For one of the surface-catalyzed core-shell nanoreactors, one reactant diffuses from a bulk

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solution to the core of nanoreactors and reacts with the other reactant inside the nanoparticles,

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hence generating a product. (Figure. S1). Since the reaction occurs at the internal surface of

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nanoreactors, the reaction rate depends mainly on the reactant diffusion rate and the permeability

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of the shell, which can be modulated by material design and stimulus in the environment.

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Inspired by the concept of nanoreactors, we proposed to design a type of core-shell

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bioresponsive polymer-based nanoreactors to improve the rate of reaction between stimuli-

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responsive materials and pathological triggers. Herein, we reported a fluorescent Glutathione-

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Priming Nanoreactor (GPN) in response to reductive microenvironment of tumor tissue to

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address the challenges of redox-sensitive probes lack of response sensitivity and fluorescence

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activation (Figure. 1). Glutathione (GSH) is recognized as a ubiquitous target of cancers with

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over 4 folds higher expression than normal tissues. Moreover, the intracellular GSH levels are

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significantly higher than extracellular concentrations by several hundred folds.14, 15

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2. MATERIALS AND METHODS

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2.1 Materials. Amicon ultra-15 centrifugal filter tubes (MWCO = 100 KD) were from

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Millipore. ICG-OSU was purchased from AAT Company. Monomers 2-(diisopropyl amino)

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ethylmethacrylate (DPA-MA), 2-aminoethyl methacrylate (AMA) were from Polyscience


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Company. GSH was obtained from Beijing Lablead Biotech CO., LTD. Albumins were provided

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by SBJ Bioscience Company. Other organic solvents were analytical grade from Sigma-Aldrich

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or Fisher Scientific Inc. All the cell lines were obtained from National Infrastructure of Cell Line

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Resource (Beijing, China). Cells were cultured in DMEM medium supplemented with 10% fetal

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bovine serum (FBS, Gibco) and 1% penicillin–streptomycin solution at 37 ℃ under the

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atmosphere of 5% carbon dioxide.

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2.2 Synthesis of dye-free block copolymers. The dye-free copolymers were synthesized

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by the method of Reversible Addition-Fragmentation Chain Transfer Polymerization (RAFT).

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Firstly, the mPEG5k-CTAm, DPA-MA and azodiisobutyronitrile (AIBN) were solved in N, N-

10

Dimethylformamide (DMF) with certain ratio. After three cycles of freeze pump-thaw to remove

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oxygen and reaction over 24 hours, the products were dialyzed by de-ionization (DI) water for 2

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days and then lyophilized for 2 more days. Secondly, a certain amount of the above lyophilized

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powder together with BMA, AIBN and PFPMA were solved in DMF and then performed three

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cycles of freeze pump-thaw in Ar atmosphere. After 24-hour reaction, the products were

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dialyzed by DI water for 2 days and then lyophilized for 2 more days. Finally, the lyophilized

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powders together with cystamine were solved in DMF and reacted for 24 h. The resultants were

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further dialyzed and lyophilized to obtain the final products.

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2.3 Synthesis of dye-conjugated block copolymers (Polymer-ICG). The conjugation of

19

polymers with indocyanine green (ICG) was performed via N-hydroxysuccinimide-ester

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chemistry. In briefly, the certain amount of polymers and ICG-OSU were solved in methanol /

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Tetrahydrofuran (THF) mixture solvents. After 24-hour reaction, the products were passed

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through an acidic Al2O3 column to remove free dyes. Then, the purified product powders were

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further ultrafiltrated to remove salt and solvents and lyophilized to obtain Polymer-ICG

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conjugates.

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2.4 Synthesis of albumin-conjugated block copolymers (Polymer-albumin). The

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hydrophobic blocks were synthesized using a similar RAFT polymerization method to polymer

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synthesis described above. Then the hydrophobic blocks were conjugated into PEG containing

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double bonds by a esterification reaction to obtain double bonds-containing polymers. For

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synthesis of albumin-conjugated polymers, a certain amount of polymers were solved in

30

tetrahydrofuran (THF) and added to DI water dropwise. After rotary evaporation to remove the


4

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organic solvents, the nanoparticles with double bond facing outside were obtained. Finally, the

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albumins were added to react with the polymers under ultraviolet radiation. After 24 hours, the

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final products were lyophilized to obtain the Polymer-albumin conjugates. To verify successful

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coupling of polymer with albumin, we performed SDS-PAGE analysis. Briefly, various samples

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including EPB-albumin, albumin and physical mixture of EPB with albumin were boiled in

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loading buffer and then run in a 10% SDS-PAGE gel. The resulting gel was subsequently stained by

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Coomassie Blue for visualization of the protein bands. The band location as an indicator of molecular

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weight was used for verifying sucessful coupling of EPB wth albumin.

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2.5 Preparation and characterization of the GPNs. The GPNs were prepared following

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two steps. At the first step, the lyophilized powders of Polymer-ICG was first dissolved in 0.2 mL

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THF solution. Then, 0.6 mL distilled water was added to the polymer solution dropwise under

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sonication to obtain nanoparticle solution, which was purified subsequently through micro

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ultrafiltration system (100 KD) for 4-5 times to remove THF. The obtained solution is Polymer-

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ICG nanoparticles. At the second step, a certain amount of Polymer-albumin was added into the

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solution of Polymer-ICG nanoparticles to obtain the GPNs with a certain density of albumin

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modification. For in vitro characterization, the final polymer concentrations were adjusted to 1

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mg/mL by distilled water for further fluorescence characterization. The morphology of the GPNs

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was characterized by transmission electron microscopy (JEOL 1200EX). The particle size and

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zeta-potential were determined by dynamic light scattering (DLS) methods using Malvern

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Zetasizer Nano-ZS instrument.

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2.6 Determination of fluorescence activation ratio of the GPNs. Fluorescence intensity

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of the GPNs in different conditions was measured on a fluorospectrophotometer (Lengguang,

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Shanghai, China). For each determination, a stock solution of the GPNs in DI water at the

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concentration of 1.0 mg/mL was prepared. Also, a series of GSH stock solutions with different

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concentrations were prepared. Then the above solutions were diluted to appropriate levels and

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mixed together as needed. Each solution with a fixed concentration of the GPNs (100 µg/mL)

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was excited at 780 nm for emission spectra scanning from 800 nm to 850 nm. The maximum

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emission intensity at 808 nm was recorded to quantify the fluorescence intensities of the GPNs.

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To calculate the SNR of the GPNs under different conditions, we determined fluorescence

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intensities of the GPNs with or without reductive GSH, respectively. The excitation and emission


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wavelength were set up at 780 nm and 808 nm respectively. Fluorescence intensities of the GPNs

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with GSH (“ON” state), without GSH (“OFF” state) and blank GSH solution were recorded as

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FIon, FIoff and FI0. The SNR of the GPNs was calculated by the following formula (Equation. 1):

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SNR（on/off ratio） = （FIon – FI0） / (FIoff – FI0)

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Equation 1. Calculation of signal-to-noise ratio (SNR) by the fluorescence intensities of the

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GPNs at “ON” state and “OFF” state.

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2.7 Response sensitivity of the GPNs. The sensitivity of the GPNs in response to GSH was

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assayed by fluorospectrophotometry. In briefly, the fluorescent intensities of the GPNs in a series

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of various concentrations of GSH were determined by a fluorospectrophotometer. The excitation

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and emission wavelength were set up to 780 nm and 808 nm respectively. Then, the normalized

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fluorescent intensities of the GPNs as a function of GSH concentrations were plotted. For

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investigations of time-response sensitivity, the fluorescent intensities of the GPNs without GSH

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were firstly determined by time sweep mode. After 6 min scanning, the sample was withdrawn

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and added with GSH stock solutions as soon as possible. The final concentration of the GPNs

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and GSH were fixed at 100 µg/ml and 10 mM respectively. The curve of fluorescent intensities

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as a function of scanning time was plotted to explore the time sensitivity of the GPNs response to

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GSH.

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2.8 SPR study of the GPNs. SPR technology (OpenSPR Starter Pack, Nicoya, Canada)

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was applied in this study to investigate the interaction of the albumin with ICG. In briefly, sensor

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chips

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dimethylaminopropyl] carbodiimide hydrochloride (EDC). Then the albumins were covalently

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conjugated to the surface of the chips with acetamide. After SPR response signals were stable,

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the polymer-ICG nanoparticles without albumin coating in different concentrations of GSH

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solutions were injected into the channel at 5 mL/min flow rate. Phosphate buffer was used to

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wash the unconjugated ICG and residual nanoparticles off the sensor chip prior to each injection.

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2.9 Determination of the ICG core/shell distribution in the GPNs. For quantitative

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determination of the ICG core/shell distribution, the GPNs at different concentrations of GSH

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solutions were firstly boiled at 100 ℃ for 10 min to denature the albumin for release of free ICG.

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The released ICG molecules and polymer-conjugated ICG were subsequently separated via ultra-

were

firstly

activated

by

N-hydroxysuccinimide


(NHS)

and

1-ethyl-3-[3-

6

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centrifugation (10 KD) and lyophilized respectively. Finally, the lyophilized powers were further

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solved in the THF/methanol mixed solutions (3:1) or methanol to determine the ICG levels using

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ultraviolet spectrophotometry.

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2.10 Imaging of the GPNs at cell level. The different tumor cell lines were cultured in the

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wells of a 96-well plate. When the cells were grown to 80%, the medium was changed by serum-

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free medium. Then, the GPN solutions were added to each well. After 1 h incubation of the

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GPNs with tumor cells, the medium was removed and the cells were slightly washed with PBS.

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Finally, the plate was placed to the imaging platform of Lumina XR System (PerkinElmer). The

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excitation and emission wavelengths were set up to 780 nm and 845 nm, respectively.

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2.11 Animal models. Animal protocols related to this study were reviewed and approved

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by the Institutional Animal Care and Use Committee of Peking University Health Science Center.

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Female NOD-SCID mice (6-8 weeks) were chosen to develop cancer models.

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For subcutaneous tumor xenograft models, various tumor cells including 4T1, MCF-7,

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HT29, 293T and Karpas (106/mouse) were injected into the left armpit of mice respectively. For

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development of tumor bearing mouse models for margin validation, we established cancer cells

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invading into the surrounding muscles to mimic the real tumor boundary. In briefly, 2×106

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cancer cells in 15 μL serum-free medium were injected into the submental triangle area of

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NOD/SCID mice to allow the cancer cell to invade into the muscle tissue underneath. The

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cancers were grown for 7 days prior to imaging experiments.

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2.12 Imaging of the GPNs in vivo and ex vivo. For investigations of the GPN imaging at

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subcutaneous tumor models, 2.5 mg/kg of the GPNs was administrated intravenously into the

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subcutaneous tumor models (n = 3 for each group). Time-course fluorescent images were

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captured on a Lumina XR in vivo imaging system. The excitation/emission wavelengths were set

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up at 780 / 845 nm.

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After imaging at the time point of 24 hour, the mice were euthanized and sacrificed by

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cervical dislocation. Tumors and organs including hearts, livers, spleens, lungs and spleens were

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collected and then imaged by the Lumina XR system. Fluorescence intensity values of ex vivo

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tumors were quantified and normalized to that of the heart (normal tissue).


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2.13 Activation of the GPNs at microscopic level. Tumors as well as surrounding tissues

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were collected 24 h after probe injection. The collected samples were frozen in O.C.T.

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compound for preparation of 8 μm frozen section slides. Each section was imaged at 800 nm

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excitation using a fluorescence flatbed scanner (Odyssey, LI-COR Biosciences) and then stained

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by H&E. Validations of tumor cells and normal cells were determined according to H&E

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staining. Fluorescence pictures of the sections were exported from a software of Image Studio.

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Colocalization of H&E staining results with fluorescence were performed to observe the regions

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of the GPN activation.

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2.14 Safety studies. Safety investigations were performed in NOD/SCID mice via i.v.

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injections. The mice were randomly divided into 2 groups (n=5 for each group). All the mice

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were injected with 2.5 mg/kg of the GPNs or PBS as controls. At day 1 and 7, the blood samples

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(~800 µL /mouse) from each group were collected and centrifuged at 5,000 g for 10 min to

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separate serum. The serum levels of alanine transaminase (ALT) and glutamic oxaloacetic

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transaminase (GOT) were determined for evaluation of liver functions. Blood urea nitrogen

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(BUN) and creatinine (CRE) were assayed for evaluation of kidney functions. For

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histopathological evaluation on safety, the mice were sacrificed at day 1 post administration for

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collection of main organs including heart, liver, spleen, lung and kidney. Sections from different

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organs were made and stained by H&E.

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2.15 Statistical analysis. Quantitative data were indicated as means ± standard deviation

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(SD). Either a Student’s t test or a one-way analysis of variance (ANOVA) was performed to

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assess the experimental results. A p-value of less than 0.05 was regarded to be statistically

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significant difference.

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3. RESULTS AND DISCUSSION

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3.1 Design and synthesis of the GPNs. As shown in Figure. 1, the GPNs are designed as

3

polymeric core/shell nanostructures. The core is comprised of hydrophobic polymer blocks with

4

plenty of redox-sensitive disulfide bonds and indocyanine greens (ICG) fluorophores, a type of

5

near infrared (NIR) fluorescent dye approved by the Food and Drug Administration (FDA). The

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shell consists of hydrophilic polyethylene glycol (PEG) conjugated with albumins which have

7

strong binding affinities with ICG molecules. In non-reductive environment, the GPNs keep

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silent due to the homoFRET-induced fluorescence quenching of ICG in the hydrophobic core,

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which is crucial for inhibiting background signals of the GPNs in normal tissues. In reductive

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environment such as tumor tissues, the disulfide bonds in the cores are cleaved by GSH to

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release free ICG molecules. Driven by the strong binding affinities with albumins, the released

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ICGs transit from core to shell, resulting in OFF/ON transition of fluorescence and achieving

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nonlinear amplification of signals in tumor. Meanwhile, the separation of ICG molecules as

14

resultants promotes the oxidation-reduction reaction in core, and thus enhancing the response

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sensitivity of the GPNs to reductive agents.

16

For proof of concept, we firstly synthesized a series of nanoprobes consisting of

17

poly(ethyleneglycol)-b-poly(2-(diisopropylamino)ethyl

methacrylate-butyl

methacrylatex)

18

polymers (PEG-b-P(DPA-r-BMA)) conjugated with indocyanine green (ICG), abbreviated as

19

EPB-ICG (Figure. S2). Considering the conjugation of ICG and albumin on the same unimer

20

chain would cause difficulty in polymer purification, we synthesized EPB-ICG and EPB-albumin

21

respectively, as shown in Figure.S2 and S3. Structures of EPB, EB90 and EPB-ICG were verified

22

by 1H NMR (Figure. S4-S8) while EPB-albumin was confirmed by SDS-PAGE electrophoresis

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(Figure. S9). Higher molecular weight of EPB-albumin than either physical mixture or albumin

24

in the gel suggested successful

25

purification of different polymers, we engineered the GPNs following two steps. Firstly, EPB-

26

ICG nanoparticles were prepared by a solvent ultracentrifugation method. Then, various ratios of

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EPB-albumins were inserted into nanoparticles to obtain albumin-coating GPNs (Figure. S10).

coupling of EPB with albumin. After construction and


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Figure 1. Schematic design of GSH-Priming Nanoreactors (GPNs). (a) The GPNs for precision cancer imaging. The GPNs keep “OFF” state due to homoFRET-induced fluorescence quenching of ICGs in the hydrophobic core under non-redox environment during circulation and at normal tissues. Once reaching cancers, the nanoprobes turned “ON” state via fluorophore core/shell transition in response to the stimuli of GSH, highly expressed in the vast majority of tumors. (b) The molecular basis of GPN activation. GSH primes the cleavage of the disulfide bonds in the hydrophobic blocks of polymers, leading to ICG release, subsequent core/shell transition and binding with surface albumins. The core/shell transition of ICGs enables the fluorescence activation of the GPNs as well as accelerates the chemical reaction process in the core owing to separation of resultants. 3.2

In vitro optimization of the GPNs. According to the concept of core/shell

15

nanoreactors and design of the GPNs, the core offers a place for chemical reaction between

16

disulfide bonds and GSH, closely related to their response sensitivity to GSH. Meanwhile, shell

17

structures affect the amount of transited ICG molecules, which is responsible for fluorescence


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activation effect. Both are crucial for performance of the GPNs. Therefore, optimization studies

2

of the GPNs were conducted from the perspective of core structures and shell components,

3

respectively. Figure. 2a illustrates the effect of albumin density on the fluorescence activation

4

ratio of the GPNs. A higher albumin conjugation density resulted in significantly higher

5

fluorescence activation intensity and a marginal increase of “OFF” signals (Table. S1). However,

6

95% modification of albumin also reduced the fluorescence intensity of the GPNs. Considering

7

the factors of the fluorescence activation intensity at “ON”state and signal-to-noise ratio (SNR),

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the albumin density of 88% was regarded as the optimal one for the GPNs (Table. S1). To verify

9

the importance of the DPA blocks in hydrophobic core of nanoparticles, we synthesized EB,

10

polymers without DPA as a control. Figure. 2b demonstrates the fluorescence activation effect of

11

the GPNs with different core structures at the same albumin-coating density. Interestingly, the

12

GPNs containing DPA blocks presented a 24.7-fold ON/OFF ratio, significantly higher than that

13

without DPA, suggesting that DPA block is indispensable for the GPNs (Table. S2).

14

Although many studies reported the importance of reduction and pH condition on the

15

cleavage of disulfide bonds, few focused on the impact of core microenvironment in

16

nanoparticles on the response action of bioresponsive nanoparticles.16, 17, 18 To further explore the

17

role of DPA blocks, we compared the protonation capacity of these polymers by titration. 19 In

18

the titration curves, the EPB polymers manifested a smooth pH platform at around pH 6.3 owing

19

to the protonation of DPA blocks containing tertiary amines (Figure. S11). Owing to electrostatic

20

repulsion, the protonation of DPA blocks might cause core swelling, thus promoting the

21

transport of hydrophilic GSH, an acidic peptide, into the hydrophobic cores of nanoparticles. In

22

other words, the DPA segments enhanced the GSH distribution proportion in the core of the

23

GPNs. It is noteworthy that DPA protonation did not cause dissociation of the GPNs, evident

24

from the changeless particle size of the GPNs in solutions with different pH levels (Figure. S12).

25

Besides the DPA block, EP30B60 polymer consisted of another segment, BMA block, with strong

26

hydrophobicity. Thus, both segments would codetermine the behaviour of the GPN nanoparticles

27

in response to environment change. Specifically, electrostatic repulsion caused by protonation of

28

DPA under acidic environments was prone to result in swelling or dissociation of the GPNs

29

while the hydrophobicility of BMA tended to maintain the association structure of nanoparticle.

30

As shown in Fig. S12, the GPNs containing 2-folds more BMA than DPA (60 vs 30 per polymer

31

chain) kept intergrate micelle structure with constant particle size at various pH levels.


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Figure 2. Optimization and in vitro characterization of the GPNs. (a) The effect of albumin density on the fluorescence activation of the GPNs. (b) The effect of core structures on the fluorescence activation of the GPNs. FIon and FIoff indicated the fluorescence intensity of the GPNs under the condition of 10 mM GSH and PBS. FI0 is background fluorescent signal of the solution. (c) The change of GPN particle size at solutions with various GSH concentrations. (d) Transmission electron micrographs of the optimized GPNs (EPB60-ICG1) at PBS and reductive solutions (10 mM GSH). Scale bar: 50 nm. (e) Fluorescence activation of the optimized GPNs under the conditions of different GSH levels. (f) Normalized fluorescence intensity as a function of GSH levels for the optimized GPNs. (g) Time sensitivity of the GPNs in response to GSH.


12

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1


Besides

the

polymer

structure,

we

also

investigated

the

influence

of

ICG

2

(excitation/emission wavelengths= 780/810 nm) conjugate number on the fluorescence activation

3

of the GPNs. As listed in Table. S3, the GPNs with one ICG conjugation number per polymer

4

chain possessed the highest SNR (24.7 folds). However, if ICG conjugation density is too high, it

5

might induced the increased formation of ICG dimers.5 So, EP30B60-ICG1 modified with 88% of

6

albumin density was chosen as the optimal polymer composition for the GPNs. These optimized

7

GPNs were exploited for subsequent studies. Under the condition of 10 mM GSH, the optimized

8

GPNs showed high fluorescent ON/OFF ratio up to 24.7 folds (Table. S1-S3).

9

3.3 In vitro characterization of the GPNs. For the in vitro characterization of above

10

GPNs, transmission electron microscope (TEM) clearly demonstrated the spherical morphology

11

of the nanoparticles around 50 nm under phosphate buffered solution (pH 7.4) (Figure. 2d). The

12

shape and particle size of the GPNs did not change with the addition of 10 mM GSH. It

13

suggested that the cleavage of disulfide bonds did not break the integrity of these nanostructures

14

maintained by the strong hydrophobic force of BMA blocks (Figure. 2d). Moreover, the GPNs

15

did not show remarkable change in particle size under the conditions of different pH, suggesting

16

that acidosis was not sufficient to cause dissociation of these nanoparticles (Figure. S12). So, the

17

GPNs presented a novel activation mechanism totally different from the bioresponsive

18

dissociation of nanoparticle as previously reported (Figure. S12).5, 10 Statistically, particle size

19

and polydispersion index (PDI) of the GPNs kept stable in various GSH levels by dynamic light

20

scattering (DLS) determination, consistent with the results of TEM (Figure. 2c). In vitro

21

fluorescent imaging of the GPNs in different GSH concentrations revealed that the GPNs were

22

able to be activated by 3 mM GSH, lower than typical response concentrations of other redox-

23

sensitive polymers (Figure. 2e and 2f).20, 21 A plot of fluorescence intensity as a function of GSH

24

concentrations indicated the orthogonal GSH-modulated fluorescence activation where the ICG

25

signal was eliminated at the lower GSH levels (to simulate normal tissues) but dramatically

26

activated at higher GSH levels (to simulate tumor tissues) (Figure. 2f). Below 3 mM of GSH, the

27

ICG molecules clustered in the core resulted in fluorescence annihilation of the GPNs because of

28

homo-fluorescence resonance energy transfer (homoFRET) effect.22 Above 3 mM of GSH, the

29

GPNs were dramatically activated to emit strong fluorescence. Further, time-dependent

30

sensitivity is equally essential for bioresponsive nanomaterials since the cleavage of stimuli-

31

sensitive chemical bonds usually needs some time.23 As seen in Figure. 2g, the GPNs could


13


Page 14 of 28

1

respond to GSH instantly in less than 5 seconds. This binary (OFF/ON) imaging effect together

2

with high response sensitivity of the GPNs are crucial to amplify the reductive signals of tumor

3

microenvironment as well as suppress the background noise in normal tissues.

4

In vitro stability is an important parameter for translational studies of a nanoparticles. As

5

listed in Table. S4, both lyophilized powders of EP30B60-ICG1 stored at -20 ℃ for 18 months

6

and suspensions of the GPNs stored at -4 ℃ for 3 weeks could preserve stable particle size, PDI

7

and SNR, respectively. Although a standard experimental design about shelf life is necessary to

8

meet the requirements of the regulations in future, these data of the GPNs at current stage have

9

shown superior stability compared to many existing nanoparticles. 24 We believe that the good

10

stability of the GPNs was predominantly attributed to their larger hydrophilic shell comprised of

11

PEG5000 and albumins. 24

12 13 14 15 16 17

Figure 3. Fluorescence activation mechanism of the GPNs. (a) Schematic diagram of a qualitative method of surface plasmon resonance (SPR) for study of the interaction between albumins and ICGs. (b) SPR response signals of the GPNs as function of time at various GSH levels. (c) A flowchart for quantitative determination of the ICG core/shell distribution proporation at various GSH levels. (d) The percentage of ICGs on shell and in core at different GSH levels.


14

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1

3.4 Fluorescence activation mechanism of the GPNs. For verification of the fluorescence

2

activation mechanism of the GPNs, we qualitatively and quantitatively investigated the core/shell

3

transition of ICG molecules, which is the principal tache for fluorescence activation. For

4

qualitative study, surface plasmon resonance (SPR) was exploited here to study the interaction

5

between ICG molecules and albumins.25 As shown in Figure. 3a, albumins were anchored on the

6

sensor surface via covalent conjugations. Under the subsequent flow of phosphate buffer

7

containing different concentrations of GSH and albumin-free nanoparticles, the SPR response

8

signals were recorded. Clearly, there was a GSH-concentration dependent increase of SPR

9

response signals (Figure. 3b), suggesting increased binding of albumins with ICG molecules. For

10

quantitative calculation, we determined the percentage of ICG in core and on shell using

11

ultraviolet spectrophotometry, respectively (Figure. 3c). Prior to the determination, albumin-

12

binding ICGs and polymer-conjugated ICGs in the GPNs were separated by the combination of

13

albumin denaturation and ultracentrifugation. As a result, at the low concentrations of GSH (
1 mM), the percentage of albumin-binding ICG molecules rose dramatically, in accord

16

with the results of in vitro imaging and fluorescence activation (Figure. 2e).

17

To further prove that fluorescence activation of the GPNs was resulted from the albumin-

18

bound ICGs instead of the free ICGs released into solution, we conducted a dialysis experiment

19

to separate the free ICGs released from nanoprobes and nanoparticle-combined forms of ICGs

20

including conjugated, albumin-bound or physically-loaded forms (Figure. 4a). For all the three

21

nanoprobes, over 90% of ICGs existed in nanoparticle-combined form instead of the free one in

22

solution (Figure. 4b). However, only the GPNs were able to be significantly activated in aqueous

23

solution, indicating the necessity of albumin coating (Figure. 4c).

24

It is noteworthy that fluorescence activation of the GPNs was mainly achieved by the

25

core/shell transition of ICGs. Due to a large number of ICGs clustered in core of the GPNs with

26

extremely small intermolecular distance (usually ﹤10 nm), fluorescence of ICGs was quenched

27

to a great extent. When ICG molecules diffused onto surface albumins of nanoparticles, the

28

fluorescence of ICGs recovered with increase of distance among fluorophores. As a matter of

29

fact, such phenomenon has previously been reported for many other fluorescence dyes, namely

30

aggregation-caused quenching (ACQ) effect.26,

27

Regarding the fluorescence activation or


15


Page 16 of 28

1

quenching, another famous effect is aggregation-induced emission (AIE) in which organic

2

luminophores such as tetraphenylene show higher photoluminescence efficiency in the aggregated

3

state than in solution.28,

4

potentially serve as a platform to integrate both fluorophores with opposite readout in signal

5

changes for specific applications.

29

Hence, the GPNs or similar nanoreactor-like nanosystems can

6 7 8 9 10 11 12 13 14 15 16 17

Figure 4. Quantitative determination of ICG release amount from different nanoparticles under the conditions of 10 mM GSH. (a) The protocol for determination of ICG release experiment. Free ICGs were able to diffuse to outside solution while the conjugated/ albumin-binding/ loaded ICGs stayed inside of the dialysis bag. (b) The percentage calculation of different forms of ICGs by determining the fluorescence intensities of different samples in organic solvents. Over 90% of ICGs existed in nanoparticle-combined form instead of the free form in solution for all the three nanoprobes. (c) The fluorescence intensities of different ICG samples in aqueous systems. For the control nanoprobes, neither conjugated nor loaded ICGs were able to be activated in aqueous system. Only the GPNs were able to be activated in aqueous solution, indicating the necessity of albumin coating.

18

imaging efficiency of the GPNs at cell level. Five types of cell lines including 4T1, MCF-7, HT-

19

29, 293T and Karpas-299 were cultured in 96-well plates. As shown in Figure. 5a, all the cells

20

were found to activate the GPNs 2-hour post incubation, resulting in strong fluorescent signals.

3.5 In vivo imaging efficiency of the GPNs. After in vitro investigations, we studied the


16

Page 17 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Although none of targeting moieties like receptors or antibodies were conjugated on the surface

2

of the GPNs, the albumin actually acted as the role of targeting molecules, likely owing to the

3

avid uptake of tumor cells for albumin, an important nutritional ingredient for cell culture.30

4

Albumin, as one of the most famous biocompatible carriers for drug delivery, has been widely

5

and extensively investigated by many researchers.31 This experiment together with the in vitro

6

tests were indicative of multiple roles of albumins in the GPNs.

7 8 9 10 11 12

Figure 5. Near-infrared images of the GPNs in various cancer cells and cancer-bearing NOD/SCID mouse models. (a) Images of five types of cells 2 h after incubation with the GPNs. (b) Images of different cancer-bearing mouse models 24 h post injection of the GPNs (2.5 mg/kg). (c) Time-course images of a 4T1-bearing mouse within 24 hours. (n=4).

13

For in vivo imaging, all the above cancer cells were innoculated subcutaneously into the

14

NOD/SCID mice to establish cancer-bearing mouse models and test imaging efficiency of the


17


Page 18 of 28

1

GPNs (2.5 mg/kg). It was displayed that the GPNs were activated in all these cancers with high

2

imaging contrast (Figure. 5b). Specifically, as shown in Figure. 5c, time-course imaging

3

illustrated gradual decline of liver signals but significant increase of tumor fluorescence within

4

24 hours. From 8 h to 24 h, the signals in liver decreased dramatically, suggesting a short half-

5

time of the GPNs in circulation system probably because of the preferrential liver uptake of

6

albumin-coating nanoparticles (Figure. 5c).30, 31 Although the fluorescence of the GPNs could be

7

triggered instantly in reductive environment in vitro, apparent tumor-specific signal activation

8

was not accomplished until 8 h after intravenous injections of nanoprobes possibly owing to the

9

circulation time and the complex physiological environments in vivo. After 24 h, the mice were

10

anaesthetized and the excised organs were imaged. As shown in Figure. S13-S16, the GPNs

11

showed an extremely high imaging contrast (> 10 folds for all the five tumor models).

12

Interestingly, we did not observe significant differences in fluorescence intensities among

13

different cancer models. According to in vitro characterization data in Fig. 2f, a plot of

14

fluorescence intensity as a function of GSH concentrations have indicated an orthogonal

15

correlation with a threshold of 3 mM GSH, instead of the linear correlation typically illustrated

16

by a molecular targeting probe in fluorescence intensity and target level. 32 In other words, the

17

GPNs will emit maximum and saturated signals as long as the GSH level reaches 3 mM.

18

Therefore, the comparable fluorescence intensities of the GPNs in various cancers could be

19

explained by the high GSH levels beyond the threshold in all these tumor models. It is

20

noteworthy that the same NOD/SCID mice for development of various cancer models were

21

exploited to exclude the differences from mice in this study. Further investigations of the GPNs

22

performance in various mouse strains bearing the same tumor will be needed in future.

23

Based on the above findings, high CNR, clear activation time window (8-24 h) and the

24

broad cancer specificity enable the GPNs to be especially applicable for image-guided surgery of

25

cancers.3, 33, 34 Firstly, the high tumor imaging contrast in vivo may overcome the disadvantages

26

of optical imaging on background signals, while the binary imaging performance could help

27

surgeons to discriminate tumor focis from surrounding normal tissues in the surgical theater.

28

Secondly, the GPNs with broad imaging specificity provides a very convenient tool for surgeons.

29

The applicability of conventional targeting probes, by contrast, must be judged by surgeons prior

30

to the surgery according to cancer classification case by case. Thirdly, stable fluorescence signals


18

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1

of the GPNs at a relatively long period of time (8-24 h) are equally important for clinical practice

2

to offer a time window for surgery.

3

3.6 Comparison of the GPNs with other NIR fluorescent probes. To exclude that all

4

nanoprobes could produce the same high CNR regardless of their response sensitivity in vivo, we

5

made a comparison among the optimized GPNs, control GPNs (redox-sensitive nanoprobes) and

6

non-sensitive nanoprobes (PLGA-PEG@ICG) at an equivalent dose of dye (Figure. 6a). The

7

optimized GPNs manifested the strongest fluorescent imaging contrast among all the nanoprobes.

8

While, the PEG-PLGA@ICG nanoprobes showed extremely low tumor fluorescent signals as

9

well as CNR (< 1.5-fold), revealing that the enhanced permeability and retention

10

(EPR) effect alone was not sufficient to yield high tumor imaging efficacy.35 As described

11

previously, a distinct advantage of the GPNs over reported cancer-targeting probes is their

12

capability in identifying a broad range of tumors. Moreover, comparison of the optimized GPNs

13

with 800CW RGD, a commercially-available targeting probes, illustrated the superior imaging

14

efficacy of the GPNs (Figure. 6a).36 Besides, the mice receiving free ICG did not cause

15

observable tumor contrasts but high fluorescent signals in abdomen, probably due to the fast

16

metabolic rate and metabolic pathway of free ICG in vivo. In a word, the GPNs may represent an

17

emerging strategy in achieving higher specificity and broader detection spectrum than traditional

18

ones.


19


Page 20 of 28

1 2 3 4 5 6 7

Figure 6. Comparison of the GPNs with other NIR fluorescent probes in vivo. (a) Images of tumorbearing NOD/SCID mouse models 24 h after injection of different imaging probes. The doses of fluorophores were the same for all groups. (b) Tumor margin identification capacity of different probes. At 24 h post administration of NIR probes, the mice were sacrificed. Tumor tissues together with surrounding normal tissues were collected and prepared to 8-µm sections. Each same section was firstly imaged by NIR fluorescent scanner and then stained by H&E.

8 9


20

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1

3.7 Tumor margin delineation by the GPNs at the histological levels. Finally, to

2

explore the capacity of the GPNs in tumor margin delineation, we collected tumors together with

3

surrounding muscle tissues for histological study 24 h after intravenous injections of various NIR

4

probes. The same section was scanned by a scanner at the excitation wavelength of 780 nm and

5

further stained by haematoxylin and eosin (H&E) staining, which is regarded as the gold

6

standard for tumor validation in clinic.37 As illustrated in Figure. 6b, the excellent colocalization

7

of fluorescent signals with H&E illustrated that the GPNs were able to identify the tumor margin

8

clearly by the binary signals between tumor tissues and surrounding normal tissues with

9

extremely high contrast up to 20 folds at histology level. Although both integrin receptors and

10

GSH are highly expressed in cancer tissues and viewed as a target for cancer therapy or imaging,

11

they represent distinct pathological features of the tumor tissues with high hetergenicities. 38

12

According to these data, GSH might be a better target for tumor margin delineation.

13

3.8 Preliminary toxicity of the GPNs in vivo. Generally, liver and spleen functions might

14

be the most essential indicators for nanosafety due to the preferential distributions of almost all

15

types of nanoparticles in the reticuloendothelial system (Figure. S13-S16).39 Firstly, the HepG2

16

cell, a hepatoblastoma-derived cell line, was chosen to test cytotoxicity, and the GPNs at a

17

concentration range of 0.1-100 µg/ml did not cause any apparent cell death over 72 hours (Figure.

18

S17). With regard to in vivo evaluations, assays on the blood of NOD/SCID mice receiving

19

intravenous GPNs exhibited no obvious abnormity, in terms of alanine transaminase (ALT),

20

aspartate aminotransferase (GOT), blood urea nitrogen (BUN), and creatinine (CRE) (Figure.

21

S18). While at histology levels, the GPNs did not trigger any evident toxicity compared to saline

22

(Figure. S19).

23 24

4. CONCLUSIONS

25

In conclusion, we capitalized on the concept of nanoreactors to enhance the response

26

efficacy and output performance of bioresponsive nanoparticles, resulting in a reduction-

27

responsive nanoreactor-like fluorescent probe, the GPNs in this study. Firstly, the GPNs

28

demonstrate a novel mechanism of fluorescence activation mediated by the core/shell transiting

29

of the fluorophores. Secondly, the GPNs achieve high response sensitivity and imaging contrast


21


Page 22 of 28

1

in vitro and in vivo. The high SNR (24.7 folds) of the GPNs offers a robust tool for precision

2

detection of cancer and clear identification of tumor margin in vivo. Finally, the GPNs are

3

primed by GSH, a universal target in a vast range of cancers, allowing us to image diverse

4

cancers with broad specificity. Therefore, design of the GPNs provides a new paradigm in

5

developing high-performance bioresponsive nanomaterials and holds promise for precision

6

image-guided surgery.

7 8

ASSOCIATED CONTENT

9

Supporting Information.

10

The Supporting Information is available free of charge on the ACS Publications Site at DOI: .

11

In vitro characterization, optimization and stability of various polymers; Schematic

12

diagram of a typical core/shell nanoreactor; Synthetic routes of polymers; 1H NMR

13

spectra and SDS-PAGE analysis of polymers; In vitro preparation and

14

characterization of the GPNs; Ex vivo imaging of and in vivo safety evaluation.

15 16

AUTHOR INFORMATION

17

Corresponding Author

18

*(Z. L.) E-mail: [email protected].

19

*(Q. Z.) E-mail: [email protected].

20

*(Y. Y.) E-mail: [email protected].

21

ORCID

22

Zhiqiang Lin: 0000-0003-1834-2060

23

Qiang Zhang: 0000-0002-8862-3098

24

Yuxin Yin: 0000-0002-8966-772X

25

Anjie Dong: 0000-0002-7348-3221


22

Page 23 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Author Contributions

2

The manuscript was written through contributions of all authors. All authors have given approval

3

to the final version of the manuscript.

4

Notes

5

The authors declare no competing financial interest.

6 7

ACKNOWLEDGMENT

8

This work was supported by grants including the Fundamental Research Funds for the Central

9

Universities (Grant BMU2017YJ001 to Z. Lin), the National Natural Science Foundation of

10

China (81821004 and 81690264 to Q. Zhang, 31420103905 and 81621063 to Y. Yin).

11 12

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The GSH-Priming Nanoreactor as a fluorescent probe can respond to high GSH levels in cancer tissue and instantly turn on by core/shell transition of ICGs to light up the tumor. 82x44mm (300 x 300 DPI)


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Glutathione-Priming Nanoreactors Enable ... - ACS Publications

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