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Article
Self-Assembled Peptide-Lanthanide Nanoclusters for Safe Tumor Therapy:
Overcoming and Utilizing Biological Barriers to Peptide Drug Delivery
Jin Yan, Wangxiao He, Siqi Yan, Fan Niu, Tianya Liu, Bohan Ma, Yongping
Shao, Yuwei Yan, Guang Yang, Wuyuan Lu, Yaping Du, Bo Lei, and Peter X Ma
ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b00081 • Publication Date (Web): 27 Jan 2018
Downloaded from http://pubs.acs.org on January 28, 2018

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5 Self-Assembled Peptide-Lanthanide Nanoclusters for Safe Tumor Therapy:
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7 Overcoming and Utilizing Biological Barriers to Peptide Drug Delivery
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12 Jin Yan 1†, Wangxiao He 2,5,* †, Siqi Yan 3†, Fan Niu 2, Tianya Liu 2, Bohan Ma 2, Yongping Shao 2, Yuwei
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15 Yan 2, Guang Yang 4, Wuyuan Lu 2, 5, , Yaping Du 1, Bo Lei 1,*, Peter X Ma 1, 6
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Frontier Institute of Science and Technology, State Key Laboratory for Mechanical Behavior of Materials, Xi’an
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22 Jiaotong University, Xi’an 710049, China.
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Center for Translational Medicine, Key Laboratory of Biomedical Information Engineering of Ministry of Education,
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27 School of Life Science and Technology and Frontier Institute of Science and Technology, Xi’an Jiaotong University,
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29 Xi’an 710049, China.
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32 Department of Ophthalmology, the First Affiliated Hospital, Health Science Center of Xi’an Jiaotong University,
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34 Xi’an 710049, China. 4 Department of oncology, BenQ Medical Center, Nanjing Medical University, Nanjing 210029,
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China.
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Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School
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of Medicine, Baltimore, MD 21201, USA.
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44 Department of Biologic and Materials Sciences, Department of Biomedical Engineering, Macromolecular Science and
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46 Engineering Center, Department of Materials Science and Engineering, University of Michigan, Ann Arbor 48109,
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49 USA
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10 ABSTRACT: Developing sophisticated nanomedicine platform to deliver therapeutics effectively and safely
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12 into tumor/cancer cells remains challenging in nanomedicine field. In particular, reliable peptide drug delivery
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15 systems capable of overcoming biological barriers are still lacking. Here, we developed a simple, rapid and robust
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17 strategy to manufacture nanoclusters of ~90 nm in diameter that are self-assembled from lanthanide-doped
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nanoparticles (5nm), two anti-cancer peptides with different targets (BIM and PMI) and one cyclicpeptide iNGR
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22 targeted to cancer cells. The peptide-lanthanide nanoclusters (LDC-PMI-BIM-iNGR) enhanced the resistance of
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24 peptide drugs to proteolysis, disassembled in response to reductive conditions that are present in the tumor
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27 microenvironment and inhibited cancer cell growth in vitro and in vivo. Notably, LDC-PMI-BIM-iNGR exhibited
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29 extremely low systemic toxicity and side effect in vivo. Thus, the peptide-lanthanide nanocluster may serve as an
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32 ideal multifunctional platform for safe, targeted and efficient peptide drug delivery in cancer therapy.
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34 KEYWORDS: multifunctional nanocluster, stimuli-responsive, peptides, targeted delivery, p53 activator, safe
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37 cancer therapy
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10 Over the past few decades, nanotechnology has emerged as a promising means to deliver anticancer
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12 therapeutics for its preferential and selective accumulation at tumor sites.1 However, the clinical applications of
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15 most nanomedicines are hampered by the lack of specific localization of the therapeutic agents at sites of interest.2
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17 Chemotherapeutic drugs escaped from target site often distribute freely and kill healthy cells, resulting in
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undesirable side effects and limited therapeutic efficacies. The lack of efficacy and clinical safety for
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22 nanotherapeutic systems remain the principal causes of failure in clinical application.
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24 An ideal nanotherapeutic system should not only deliver therapeutic agents to desired sites effectively, but
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27 also manage to obliterate the off-target side effect. Such requirements have promoted the recent development of
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29 stimuli-responsive nanoparticles, which can release the drugs at the target site under the trigger of tumor
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32 microenvironment.3-5 Although conceptually impressive, those systems are still at an early stage of development
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34 due to their narrow therapeutic windows attributed to the toxic side effects of the small molecules. Peptide drugs,
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37 with higher specificity and less side effects, offer ideal alternatives to small molecules as therapeutics in
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39 nanotherapeutic system.6 However, peptide drugs encounter a series of biological obstacles including rapid
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degradation by enzymolysis, lack of accumulation at the tumor sites, and weak internalization by cancer cells.7-9
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44 Although various strategies have been proposed to overcome these obstacles to peptide application, to our
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46 knowledge, none of them was able to solve all these problems at the same time.
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49 With the enhanced permeability and retention effect (EPR) in tumor tissues as well as effective cellular
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51 internalization ability,10,11 nanoparticles provide an ideal solution to targeted delivery and stimuli-responsive
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54 release of peptide drugs. Besides, in the delivery process, nanoplatform can protect peptides from enzymolysis.
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56 Importantly, while low proteolytic stability is one of the biggest obstacles for peptide drug application, this
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2 property can be exploited for rapid clearance of peptide drugs after their release from nanoplatform to prevent
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5 extended off-target toxicity. Thus, nanoplatform-based peptide delivery system holds the promise of overcoming
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7 the aforementioned biological barriers all at once.
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10 The p53 tumor suppressor pathway is perturbed in the majority of human cancers.12,13 Although p53 gene is
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12 frequently mutated or deleted, many cancer cells have WT p53 gene except that its expression is inhibited by
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15 overexpression of the negative regulator MDM2.14,15 MDM2 is an E3 ubiquitin ligase that targets p53 for
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17 poly-ubiquitination which leads to proteasomal degradation of p53.15,16 Therefore, disrupting the interaction
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between MDM2 and p53 to restore p53 expression and its tumor suppressor activity is a promising strategy to
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22 treat cancers.17 PMI is a potent inhibitor of MDM2-p53 interaction discovered by phage display screening and
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24 subsequent mutational optimization process.18 PMI can activate p53 efficiently, and activate the downstream
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27 BH3-only family protein (such as Bim, PUMA and NOXA) to promote apoptosis by sequestering anti-apoptotic
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29 Bcl-2 family proteins from BAX and BAK.18-20 However, the tumor-inhibiting activity of PMI is greatly limited
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32 by its low proteolytic stability and poor cell-member penetration.18 Therefore, it is meaningful to overcome these
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34 biological obstacles of PMI by nanoplatform-based peptide delivery system. What’ more, BIM peptide is the BH3
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37 domain of the pro-apoptotic protein, Bim.21-23 Hence, it is rationale to combine BIM with PMI to get a better
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39 anti-tumor efficacy.
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Herein, we report the development of a peptide-lanthanide clustered nanosystem, named
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44 LDC-PMI-BIM-iNGR, which effectively overcomes barriers to peptide application in cancer therapy, and
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46 achieves safe and reliable antitumor efficacy in vitro and in vivo (Scheme 1). LDC-PMI-BIM-iNGR was
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49 constructed through a thiol-poly(ethyleneglycol)-thiol (SH-PEG-SH) polymer-induced molecular assembly of
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51 three peptides (PMI, BIM, and a cyclicpeptide iNGR targeted to cancer cells) with biocompatible
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54 lanthanide-doped fluorescent nanoparticles (LDN, GdOF:45%Ce,15%Tb) (Scheme 1).17, 24, 25 For peptide delivery,
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56 LDC-PMI-BIM-iNGR was designed to protect the peptide cargos (PMI and BIM) from enzymolysis, accumulate
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2 in tumor tissues with the guidance of iNGR peptide, get internalized by the tumor cells (Scheme 1), and then
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5 stimuli-responsively release potent peptide drugs for robust antitumor efficacy (Scheme 1).
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28 Scheme 1. Schematic diagram of LDC-PMI-BIM-iNGR serving as a smart and safe peptide delivery platform for targeted
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cancer therapy and labeling. The lanthanide-doped nanoclusters (LDC) were constructed through a
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33 thiol-poly(ethyleneglycol)-thiol (SH-PEG-SH) (II) induced molecular assembly of anti-cancer peptides (PMI&BIM (I)) and
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35 targeting peptide (iNGR (III)) with ligand-free lanthanide-doped fluorescent nanoparticles (LDN). These nanoclusters can
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38 protect the peptides from enzymolysis, target tumor cells specifically and get internalized by tumor cells. In reductive cytosol,
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40 LDC can be reduced to release potent peptide drugs to kill cancer cells.
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43 RESULTS AND DISCUSSION
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45 Preparation and characterization of stimuli-responsive LDC-PMI-BIM-iNGR nanoclusters. LDN
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48 capable of conjugating free sulfhydryl group was synthesized as previously described,17 and reacted with
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50 hydrochloric acid (pH 4.0) to expose the surface.18 The three peptides, cysteine modified PMI (PMI-Cys) and
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BIM (BIM-Cys) as well as sulfhydryl PEG modified iNGR (iNGR-PEG-SH) were synthesized by total chemical
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55 synthesis and site-directed modification (Scheme 1). The molecular weights of peptides and pharmacological
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57 features were characterized by electrospray ionization-mass spectrometry (ESI-MS), high-performance liquid
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2 chromatography (HPLC) and isothermal titration calorimetry (ITC) (Fig. S1).
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5 For the synthesis of LDC-PMI-BIM-iNGR, the anti-cancer peptides, PMI-Cys and BIM-Cys, were firstly
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7 conjugated with LDN to obtain LDN-PMI-BIM (Scheme 1). Briefly, 5 mg of LDN was dissolved in 10 ml of
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10 reaction buffer (20% acetonitrile and 80% standard PBS) containing 1 mM PMI-Cys and 1 mM BIM-Cys. After a
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12 20-min incubation with stirring，1 mg of HS-PEG-SH was added to crosslink LDN-PMI-BIM (about 4 nm) into
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15 nanoclusters with a size of around 90 nm in diameter. Finally, 1 mg of sulfhydryl-PEGylated targeting peptide
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17 iNGR-PEG-SH was added to the mixture to saturate the free sulfhydryl groups at the surface of the
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LDC-PMI-BIM (Scheme 1). After 40-min incubation at 35ºC with stirring，~7mg LDC-PMI-BIM-iNGR can be
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22 collected by centrifugation (the yield of ~60%).
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24 The successful conjugations of peptides with LDN were confirmed by Fourier transform infrared spectra
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27 (FTIS, Fig. 2B) and ultraviolet–visible spectra (UVS, Fig.S2A). In FTIS, a new peak at 1650 cm-1 were detected
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29 in LDN-PMI-BIM and LDN-PMI-BIM-iNGR samples, which were attributed to the stretching vibration C=O
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32 groups from peptides (Fig.2B). Meanwhile, more sharp-angled peak at 3400cm-1 (characteristic peak of N-H
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34 stretching vibration) can be founded in LDN-PMI-BIM and LDN-PMI-BIM-iNGR samples, which was further
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37 evidence of the successful conjugations of peptides with LDN. In UVS, after peptide conjugation, two typical
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39 UV-vis absorption peaks of aromatic structures and peptides at 325 nm and 280 nm can be observed (Fig. S2A).
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To further determine the efficiency of peptide loading in the nanoclusters，LDC-PMI-BIM-iNGR was dissolved in
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44 PBS buffer containing 6 M guanidine hydrochloride and 1 M dithiothreitol to break the conjunction between
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46 peptide and LDN, and then the amount of released peptides were quantified by high-performance liquid
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49 chromatography (HPLC, Fig. S2B). Our results showed that the loading quantities of the PMI-Cys and BIM-Cys
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51 were 25.2 mmol/mg and 12 mmol/mg, respectively. (mmol/mg=peptide molarity/LDN mass).
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54 High-resolution TEM (HRTEM, Fig.1C) revealed that the LDN cores in LDC-PMI-BIM-iNGR remained as
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56 uniform nanocrystals with a mean diameter of 5.6 ± 0.4 nm (Fig. 1C1). The clear-cut lattice fringes shown in the
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2 Fig.1C2 HRTEM images and the symmetric faculaes shown in the Fig.1 C3 fast Fourier transform (FFT) patterns,
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5 verified that the LDN core kept a high nature of crystallization with single crystalline structure after peptide and
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7 PEG modification. Additionally, LDC-PMI-BIM-iNGR is well dispersed in water, and emits strong green light
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10 and weak red light under excitation at 365 nm (Fig.1D, Fig.S3A), a feature that can be utilized for nuclear and
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15 The molecular assembly and stimuli-responsive properties (Fig.1A) of smart LDC-PMI-BIM-iNGR
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17 nanoclusters were characterized by monitoring changes in their morphologies and in the hydrodynamic diameter
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distribution, using transmission electron microscopy (TEM) and dynamic light scattering (DLS), respectively
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22 (Fig.1E&F). LDN showed a spherical nanostructure with a hydrodynamic diameter of about 4.1 nm (Fig.1E&F).
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24 In sharp contrast, as shown in the TEM image of LDC-PMI-BIM-iNGR in Fig. 1E, the self-assembled
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27 nanoclusters had a bigger raspberry-like structure, which presented a monodispersed nanocluster morphology
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29 composed of several LDN cores. The mean hydrodynamic diameter of LDC-PMI-BIM-iNGR was around 89.3 nm
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32 (Fig. 1F), which was consistent with TEM observation and was an advantageous size for improved
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34 pharmacokinetics and vascular extravasation.26, 27 More importantly, LDC-PMI-BIM-iNGR showed a sensitive
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37 redox-responsive behavior in an intracellular-mimic reducing environment28 (10mM dithiothreitol, DTT, in ph7.4
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39 PBS). After reduction for 4 h, the LDC-PMI-BIM-iNGR presented a monodispersed nanocrytal morphology with
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a significantly decreased hydrodynamic diameter (about 5.0 nm), demonstrating the stimuli-responsive
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Figure 1. Synthesis and physicochemical properties of smart LDC-PMI-BIM-iNGR nanoclusters. (A) Schematic illustration
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33 showing the self-assembly of LDN into the redox-sensitive LDC-PMI-BIM-iNGR nanocluster, and the disintegration of the
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35 nanocluster into small particles in the reducing intracellular environment; (B) FTIR spectra of LDN, LDC-PMI-BIM and
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38 LDC-PMI-BIM-iNGR, demonstrating their surface chemical structures before and after self-assembly; (C) HRTEM images
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40 (C1 and C2) and corresponding FFT pattern (C3) of an individual particle of LDC-PMI-BIM-iNGR, exhibiting single crystal
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43 structures; (D) Fluorescence spectra and quantum yield of LDN, LDC-PMI-BIM-iNGR before and after responding to
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45 reducing milieu, significant stimuli-responsive fluorescence-switch property was observed; (E) TEM images and (F)
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hydrodynamic distributions of ligand-free LDN, LDC-PMI-BIM-iNGR showing the self-assembly and disassembly process of
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50 nanocluster system responding to reducing milieu.
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Excellent performance on proteolysis resistance and stimuli-responsive release of peptide cargos. As
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55 our design indicates, LDC-PMI-BIM-iNGR is capable of overcoming enzymolysis during peptides delivery by
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57 providing steric hindrance against the enzyme binding (Fig. 2A). For proteolysis resistance test, all samples were
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2 incubated in an extracellular-milieu mimicking phosphate buffer solution with oxidized glutathione and
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5 chymotrypsin, a protease with dual specificities for both basic and bulky hydrophobic residues (Both PMI and
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7 BIM peptides have many hydrophobic residues). Compared with non-clustered LDN-PMI-BIM-iNGR (half-time,
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10 22.1h), LDC-PMI-BIM-iNGR showed a significantly higher resistance against enzymolysis (half-time>48h),
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12 whereas the free peptides had a half-life less than 6 min (Fig.2B). Another designed function of
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15 LDN-PMI-BIM-iNGR is to release peptide drugs in response to the reductive intracellular environment. To assess
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17 the reduction-induced release of peptides from LDC-PMI-BIM-iNGR, nanoclusters were incubated with PBS
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buffer containing 10 mM DTT (mimicking intracellular reductive condition).28 As shown in Fig. 2C, PMI and
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22 BIM peptides can be released from the nanoclusters in a redox-dependent manner. These results demonstrated the
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2 superior functions of LDC-PMI-BIM-iNGR on proteolysis resistance and peptide drug delivery (B) LDC-PMI-BIM-iNGR protects
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5 peptides from proteolysis by chymotrypsin; (C) Redox-dependent release of peptides from LDC-PMI-BIM-iNGR at pH 7.4.
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7 LDC-PMI-BIM-iNGR inhibits the viability of cancer cells in a p53 pathway-dependent manner. As
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10 expected, LDC-PMI-BIM-iNGR, LDC-PMI-BIM, LDC-PMI-iNGR dose dependently inhibited the growth of the
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12 p53 wild type cancer cell line HCT116 p53+/+ with IC50 values of 81 nM, 166 nM and 125 nM (PMI concentration)
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15 respectively. By contrast, the positive control drug Nutlin-3 showed an IC50 of 5.5 µM while free PMI exhibited
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17 no inhibitory effect (Fig. 3A). Moreover, according to the western blotting analysis (normalized against Actin
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protein), after treatment with LDC-PMI-BIM-iNGR, LDC-PMI-iNGR or Nutlin-3, the expressions of p53, MDM2,
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22 p21, NOXA and PUMA proteins in HCT116p53+/+ cells were significantly increased, indicating the activation of
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24 p53 pathway (Fig. 3B&Fig. S5A-D). Interestingly, an isogenetic p53 null cell line, HCT116 p53-/- were highly
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27 resistant to the treatment of LDC-PMI-BIM-iNGR (Fig. 3C), suggesting that the tumor-inhibiting activity of our
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29 nanoclusters is p53-dependent. Additionally, Annexin V- PI staining further revealed that LDC-PMI-BIM-iNGR
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32 induced apoptotic rather than necrotic cell death in HCT116 p53+/+ cells (Fig.3D). Together, these findings strongly
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34 support that LDC-PMI-BIM-iNGR can reduce the viability of HCT116p53+/+ cells by reactivating the p53 pathway
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37 and addition of the BIM peptide can potentiate the anti-tumor efficacy of PMI.22, 23
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39 Real-time Monitoring of morphological changes of nucleus during LDC-PMI-BIM-iNGR induced
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apoptosis. We have previously shown that LDN had a distinctive capability to emit two different fluorescences,
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44 and it can be used as a probe to simultaneously label the nucleolus yellow and the cytoplasm green.29 Therefore,
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46 LDC-PMI-BIM-iNGR is endowed with a capability to monitor the pharmacological effects of peptide drugs on
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49 the morphology of subcellular structures in real time (Fig. 3E&Fig. S6). After 24 h treatment of
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51 LDC-PMI-BIM-iNGR, a small population of cancer cells (~12.8%) underwent early apoptosis (Fig.3F&S7),
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54 which was characterized by the collapse of the chromatin against the nuclear periphery and fusion into a few large
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56 clumps (circle 1 in Fig.3E1).30, 31 After 48 h, some nucleuses were condensed into a single dense sphere (circle 2
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2 in Fig.3E2), and other chromatins budded outward into smaller sphere surrounded by nuclear envelope (circle 3 in
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5 Fig.3E2), both of which were consistent with the increased apoptosis level (41.4%, Fig.3F&S7).30, 31 After 72 h,
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7 96.2% of cells were dead or in apoptosis (Fig.S7) and the cell nucleus showed important features of the late
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10 apoptosis: diffused and granular apoptotic bodies (circle 4 in Fig.3E3) or ring-like structures (circle 5 in
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12 Fig.3E3).32, 33 Of note, these morphology changes of nucleus were also confirmed by a Hoechst staining in situ
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15 (Fig. S6). This imaging capability of LDC-PMI-BIM-iNGR may have promising applications for cell morphology
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17 test and drug efficacy evaluation in the development of anti-cancer drugs as well as in the clinical settings.
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55 Figure 3. In vitro anti-cancer efficacy and therapy monitoring of LDC-PMI-BIM-iNGR. (A) Dose-dependent growth
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57 inhibition of HCT116p53+/+ cells upon various treatments as determined by the Amar blue cell viability assay; (B) Protein
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2 expression analysis of p53, p21, MDM2, NOXA and PUMA in HCT116p53+/+ cells after 48 h treatment with 100 nM
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5 LDC-PMI-BIM-iNGR and LDC-PMI-iNGR by western blot, normalized against Actin; (C) Growth inhibition of
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7 HCT116p53+/+ and HCT116p53-/- cells 72 h after treatment with 100 nM LDC-PMI-BIM-iNGR and LDC-PMI-iNGR; (D)
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10 Effects of Nutlin3a and LDC-PMI-BIM-iNGR on HCT116 p53+/+ cells as determined by Annexin V-PI staining and flow
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12 cytometric analysis; (E) CLSM images of HCT116p53+/+ cells at 24 h (E1), 48 h (E2) and 72 h (E3) after 100 nM
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LDC-iNGR-PMI-BIM treatment, scale bars: 60 µm; (F) Apoptosis levels measured by FACS in HCT116 p53+/+ cells treated
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17 with 100 nM LDC-iNGR-PMI-BIM or 6µM Nutlin-3a for 24-72 hours.
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Targeted antitumor activity of LDC-PMI-BIM-iNGR in vivo. The excellent anti-cancer and
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22 stimuli-responsive performance of LDC-PMI-BIM-iNGR in vitro further compelled us to study its in vivo activity.
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24 Mice bearing HCT116p53+/+ tumors were used to investigate the tumor targeting ability and therapeutic efficacy of
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27 our nanoclusters. The targeting ability was determined by tracking the LDN fluorescent signals, which can be
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29 used to gauge the quantity and distribution of LDC-PMI-BIM-iNGR in tumor and normal tissues (Figs. 4A-B).
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32 Facilitated by the tumor-targeting iNGR peptide and the EPR effect of nanoclusters, LDC-PMI-BIM-iNGR is
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34 expected to be primarily taken by tumor tissues but much less by the normal tissues (Fig.4A). 10, 11, 25, 26 Indeed, a
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37 strong fluorescent signal was detected at the tumor site after 4 h post-injection, while only a low (kidney and liver)
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39 or background (brain, lung, spleen and heart) level of fluorescence was observed in normal tissues, confirming the
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tumor-targeting ability of LDC-PMI-BIM-iNGR nanoclusters (Fig. 4B).
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44 To test the therapeutic efficacy of LDC-PMI-BIM-iNGR, xenograft tumors were established and randomly
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46 divided into 7 groups (n = 5/group) to respectively receive a 2-week treatment regimen as follows: PBS,
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49 doxorubicin, PMI peptide, PMI&BIM, LDC-PMI-iNGR, LDN-PMI-BIM-iNGR, or LDC-PMI-BIM-iNGR (The
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51 dosage of all groups were 2.5 mg/kg). The volumes of tumors were measured every day. As shown in Fig. 4C, the
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54 free PMI peptide or the combination of free PMI and BIM peptides displayed hardly any therapeutic effects when
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56 compared to the PBS control, whereas LDC-PMI-iNGR and LDN-PMI-BIM-iNGR inhibited tumor growth by
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2 76.8% and 75.3% respectively on day 12, close to the inhibition level of the clinical chemotherapeutics
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5 Doxorubicin (DOX), 76.8%. LDC-PMI-BIM-iNGR exhibited the strongest suppression of tumor growth (90.1%).
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7 The average weights of the tumors excised at the end of the experiment also demonstrated the same trend (Fig. 4D
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10 & Fig. S8). The geometric mean of the tumor weights in the LDC-PMI-BIM-iNGR group (19.4mg) were just 21%
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12 of that in the LDN-PMI-BIM-iNGR group (90.5 mg, P <0.01)，suggesting the cluster structure significantly
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15 improve the therapeutic efficacy. Meanwhile, as we expected, the mean tumor weights of the LDC-PMI-iNGR
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17 group (82.2 mg) were 4.2-fold higher than that of the LDC-PMI-BIM-iNGR group, indicating that BIM peptide
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can enhance the therapeutic efficacy of PMI. To further characterize the anti-tumor efficacy in vivo at pathological
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22 level, histological analyses, including hematoxylin and eosin (H&E, Fig. 4E) staining, and terminal
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24 deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end (TUNEL, Fig4F) staining were carried
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27 out. As shown in Fig. 4E, the dense texture and morphologic integrity of tumor tissues can be observed in control,
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29 PMI and PMI/BIM-combo groups. By contrast, necrotic tumor tissues were observed in DOX,
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32 LDN-PMI-BIM-iNGR and LDC-PMI-BIM groups (Fig.4E). As expected, LDC-PMI-BIM-iNGR treated groups
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34 showed large areas of necrotic tumor with numerous apoptotic cancer cells (Fig.4E). Furthermore, TUNEL
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37 staining (brown) was performed to determine the level of apoptosis in tumor tissues (Fig.4F).34 Compared with the
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39 other groups, the LDC-PMI-BIM-iNGR group exhibited apparently stronger staining in the tumor regions,
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demonstrating that much more cancer cells entered the apoptotic process. These results indicated that
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44 LDC-PMI-BIM-iNGR markedly impedes the growth of the tumors by promoting apoptosis in vivo.
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38 Figure 4. Targeted in vivo antitumor activity of LDC-PMI-BIM-iNGR in human colon tumor model. (A) Schematic diagram
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40 of targeted accumulation process of LDC-PMI-BIM-iNGR in tumor region; (B) Ex vivo fluorescent images of tumors and
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43 major organs from LDC-PMI-BIM-iNGR treated mice; (C) Tumor growth curves of indicated treatment groups; (D) The
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45 average weights of the tumors excised at the end of the experiment, ** stand for P<0.01; (E) H & E ( ×200) and (F) TUNEL
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(×200) staining of HCT116p53+/+ solid tumors tissues after various treatments for 12 days
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50 Therapeutic safety evaluation on LDC-PMI-BIM-iNGR in vitro. Systemic toxicity from the freely
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distributed off-target drugs poses a major challenge to cancer chemotherapy. To minimize the off-target effects, a
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55 dual-targeting strategy was adopted in our design of LDC-PMI-BIM-iNGR nanocluster (Fig. 5A). Firstly, the
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57 Asn–Gly–Arg (NGR) sequence in iNGR peptide can specifically bind to CD13, which are highly expressed in
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2 diverse cancer cell lines.24 Then, the internalization process of iNGR can be triggered when the C-end rule motif
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5 sequence tNGR (CRNGR) is exposed through a proteolytic process that specifically occurs in cancer cells.16 This
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7 strategy allows enrichment of LDC-PMI-BIM-iNGR specifically in CD13-over-expressed cancer cells. Indeed,
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10 we observed that LDC-PMI-BIM-iNGR preferentially labeled the cancer cells (HCT116) while not the normal
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12 cells (BMSCs and HUVECs) with bright fluorescence (Fig.5B). The second level of specificity of our
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15 nanoclusters results from the PMI peptide, whose target, MDM2 is only overexpressed in cancer cells but not in
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17 the normal cells. In accordance with this, LDC-PMI-BIM-iNGR had no inhibitory activity for the normal cells
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BMSC and HUVEC, as well as the untargeted p53 null cell line, SW480, whereas DOX induced growth inhibition
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22 for all cell lines (Figs. 5C-D).
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24 The low proteolytic stability of peptides advantageously offers another level of protection against off-target
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27 effects. Peptide drugs can be rapidly degraded before they diffuse and bind to unwanted targets. For instance, 100
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29 nM LDC-PMI-BIM-iNGR effectively induced a time-dependent growth inhibition in various cancer cells, such as
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32 HCT116p53+/+, SJSA (a cell line from primitive multipotential sarcoma of femur) and MCF-7 (a breast cancer cell
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34 line), whereas free PMI exhibited no inhibitory activity even at 6 µM (Fig.5E). Meanwhile, according to previous
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37 reports,21 the free BIM peptide has no inhibitory activity for cancer cells at a concentration within the nM range.
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39 These results suggested that PMI or BIM peptides extracellulary leaked from the nanoclusters would not have any
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effects to cells.
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46 Figure 5. Therapeutic safety evaluation of LDC-PMI-BIM-iNGR in vitro. (A) Schematic diagram of tumor therapeutic safety
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49 benefitted from LDC-PMI-BIM-iNGR; (B) Confocal laser scanning microscope (CLSM) images of HCT116, BMSC and
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51 HUVEC cells after incubation with LDC-PMI-BIM-iNGR (20 µg/mL), all images were taken under the same exciting light
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54 and detector gain (scale bars 60 µm); (C) Selective cancer cell cytotoxicity of LDC-PMI-BIM-iNGR determined by
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56 LIVE/DEAD staining (×600); (D) Selective cytotoxicity of LDC-PMI-BIM-iNGR against HCT116p53+/+, SW480, HMSC and
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2 HUVEC, using DOX as a positive control; (E) Time-dependent cytotoxic activity of 6 µM nutlin-3, 6 µM free PMI and 100
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5 nM LDC-PMI-BIM-iNGR against HCT116p53+/+ (E1), SJSA (E2) and MCF-7 (E3) cell lines.
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7 Therapeutic safety evaluation on LDC-PMI-BIM-iNGR in vivo. To further verify the biosafety of
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10 LDC-PMI-BIM-iNGR in vivo, drug toxicity tests were carried out on three randomly divided mice groups (n =
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12 6/group) by treating each individual group with PBS, doxorubicin or LDC-PMI-BIM-iNGR (5 mg/kg for all
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15 groups) respectively for one week. Physical appearance and body weight were monitored every two days (Figs.
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17 6A-C), and major organs were excised at the end of the experiment. After 1 W administration, the body weight of
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mice in the LDC-PMI-BIM-iNGR group showed little difference from the control group (PBS), whereas the
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22 DOX-treated group lost weight significantly (Fig.6B). Meanwhile, mice in the DOX group showed symptoms of
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24 adynamia, dehydration and fading of eye color, but the LDC-PMI-BIM-iNGR group of mice remained as healthy
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27 as the control group (Fig. 6C). Histologically, no morphological or pathological changes were observed in livers,
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29 kidneys and lungs of the mice from the control or LDC-PMI-BIM-iNGR group (Fig.6D), whereas some
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32 significant pathological changes, such as necrosis and fatty degeneration in liver, necrosis and inflammation in
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34 kidney as well as thickening of alveolar walls in lung, were discovered in the DOX group (Fig.6A&D). These
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37 results further demonstrated the biosafety of our LDC-PMI-BIM-iNGR nanocluster as a cancer therapeutic
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32 Figure 6. Biosafety evaluation of LDC-PMI-BIM-iNGR in vivo. (A) Schematic diagram of tumor therapeutic safety benefitted
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34 from LDC-PMI-BIM-iNGR; (B) Changes in body weight of nude mice with tumors upon various treatments; (C) Photos of
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mice with tumors after 8 days of various treatments; (D) the representative histological H&E staining images of liver (×200),
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39 kidney (×200) and lung (×200) tissue sections, demonstrating significantly lower tissue necrosis levels in
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LDC-PMI-BIM-iNGR treated group.
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44 CONCLUSIONS
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46 In summary, a kind of peptide-lanthanide nanocluster (LDC-PMI-BIM-iNGR) with effective anti-cancer efficacy
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49 in vitro and in vivo was fabricated facilely through self-assembly of LDN-Peptides ultra-small nanocrystals. These
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51 nanoclusters demonstrated excellent capacity for targeted and safe cancer therapy while overcoming all biological
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54 barriers to peptide drug delivery. LDC-PMI-BIM-iNGR platform could efficiently protect peptide drugs from
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56 enzymolysis, selectively deliver them into the tumor cells and gradually release peptide drugs in response to
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2 reductive intracellular environment. With the high tumor-targeting ability and short half-life of peptide drugs,
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5 LDC-PMI-BIM-iNGR greatly mitigated systemic toxicity and side effects, thereby inhibiting tumor growth with
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7 therapeutic safety. In brief, LDC-PMI-BIM-iNGR could serve as an ideal multifunctional platform for targeted
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10 peptide drugs delivery with real-time tracking ability and superior therapeutic efficacy and safety.
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12 EXPERIMENTAL SECTION
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15 Fabrication of LDC-PMI-BIM-iNGR. The as-prepared LDN were re-dispersed in PBS (pH 7.4) buffered
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17 saline (containing 20% acetonitrile). PMI, BIM or iNGR-PEG (Cys-Peptide or -SH) was conjugated with LDN by
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the the sulfhydryl groups of cysteine of peptide using ligand chemistry. In detail, 5 mg of LDN was dissolved in
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22 10 ml of reaction buffer (20% acetonitrile and 80% standard PBS) containing 1 mM PMI-Cys and 1 mM
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24 BIM-Cys. After a 20-min incubation with stirring at 30℃，1 ml HS-PEG-SH solution (1 mg/ml in deionized water)
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27 were added for further functionalized reaction at 30℃ for 1h. Finally, 1 mg of sulfhydryl-PEGylated targeting
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29 peptide iNGR-PEG-SH was added to the mixture to saturate the free sulfhydryl groups at the surface of the
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32 LDC-PMI-BIM. After 40-min incubation at 35ºC with stirring，~7mg LDC-PMI-BIM-iNGR can be collected by
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34 10000g centrifugation (the yield of ~60%).
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37 Physicochemical properties of the LDN and its conjugates. The nanocrystals morphology and lattice
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39 structure were observed on a high-resolution transmission electron microscope (HRTEM, F20, FEI) operated at
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200 kV. The hydrodynamic size distribution was obtained from the dynamic light scattering (DLS) measurement
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44 (Malvern Zetasizer Nano ZS system). The surface chemical structure of modified nanocrystals was evaluated by
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46 Fourier transform infrared (FT-IR) spectroscopy (Nicolet 6700) and UV−vis absorption spectra (Shimadzu 3000
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49 spectrophotometer).
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51 Proteolytic stability. Proteolytic resistance of free PMI, LDC-PMI-BIM-iNGR and LDN-PMI-BIM-iNGR
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54 were incubated at 100 µM each in PBS with 10 µg/ml chymotrypsin – an intracellular protease with dual
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56 specificities for both basic and bulky hydrophobic residues. RP-HPLC was used to monitor and quantify
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2 time-dependent peptide hydrolysis.
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5 Animal handling and tumor xenografts. Animal procedures were in agreement with the guidelines of the
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7 Institutional Animal Care and Use Committee. Tumor cells were harvested when they reached near confluence by
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10 incubation with 0.05% trypsin-EDTA. Cells were pelleted by centrifugation and resuspended in sterile PBS.
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12 HCT116 cells (4 × 106cells/site) were implanted subcutaneously into bilateral hip of four- to five-weeks-old male
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15 athymic nude mice. Treatment of three groups of mice (six mice per group) was initiated after the tumor had been
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17 established in 2 weeks as a palpable mass (a volume of ~ 50mm3). Tumor length and width were measured with
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calipers, and the tumor volume was calculated using the following equation: tumor volume(V)=length × width
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22 × width / 2. For histological examination, the tumor, liver, kidney and lung tissues were fixed with
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24 paraformaldehyde, dehydrated, sliced into 5.0µm sections and subjected to H&E or immune-histochemical
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27 staining assay.
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29 In vivo imaging and biodistribution analysis. Tumor bearing mice were injected with 200µl
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32 LDC-PMI-BIM-iNGR (1mg/ml). IVIS Spectrum In Vivo Imaging System was used to take the ex vivo images of
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34 mice. Light with a wavelength at 500 nm was used as the excitation source. Furthermore, mice were humanely
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37 killed at 4 h post-injection, and then the heart, liver, spleen, lung, kidney and tumor were collected from each
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39 mouse without delay. The fluorescence intensity in all organs was further analyzed by the IVIS Spectrum In Vivo
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Imaging System.
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44 ASSOCIATED CONTENT
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46 Supporting Information
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49 General information, synthetic procedures of LDN, synthesis and modification of the peptide, protocols of the
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51 necessary cell experiments and 8 supplementary figures were included in Supporting Information . This material is
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54 available free of charge via the Internet at http://pubs.acs.org
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56 AUTHOR INFORMATION
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2 Corresponding authors
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5 * Email (B. Lei) rayboo@xjtu.edu.cn
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7 * Email (W. He): WHe@ihv.umaryland.edu
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10 Author Contributions
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12 † J. Yan, W. He and S. Yan contributed equally to this work.
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15 ACKNOWLEDGMENTS
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17 The authors acknowledge the valuable comments of potential reviewers. This research was supported by State
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Key Laboratory for Mechanical Behavior of Materials (No. 20161801), the Natural Science Basic Research Plan
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22 in Shaanxi Province of China (No. 2015JQ5165) and National Natural Science Foundation of China (No.
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24 51502237).
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26
27 REFERENCES
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39 Evidence from Using an Improved TUNEL Histochemical Method. Mol. Brain Res. 2001, 93, 64-69.
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60 ACS Paragon Plus Environment