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Development of Y-shaped peptide for constructing nanoparticle systems targeting tumorassociated macrophages in vitro and in vivo
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Mater. Res. Express 1 025007 (http://iopscience.iop.org/2053-1591/1/2/025007) View the table of contents for this issue, or go to the journal homepage for more
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Development of Y-shaped peptide for constructing nanoparticle systems targeting tumor-associated macrophages in vitro and in vivo Lu Yan1,2,3,6, Yunxiang Gao3,6, Ryan Pierce3, Liming Dai3,8, Julian Kim2,4,5,8 and Mei Zhang2,4,7 1
Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, People’s Republic of China 2 Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44016, USA 3 Department of Macromolecular Science and Engineering, School of Engineering, Case Western Reserve University, Cleveland, OH 44106, USA 4 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA 5 Seidman Cancer Centre, University Hospitals, Cleveland, OH 44106, USA E-mail:
[email protected] (Mei Zhang) Received 30 November 2013, revised 24 February 2014 Accepted for publication 7 March 2014 Published 10 April 2014 Materials Research Express 1 (2014) 025007 doi:10.1088/2053-1591/1/2/025007
Abstract
Tumor-associated macrophage (TAM) is increasingly being viewed as a target of great interest in tumor microenvironment due to its important role in the progression and metastasis of cancers. It has been shown that TAM indeed overexpresses unique surface marker legumain. In this study, we designed and synthesized a Y-shaped legumain-targeting peptide (Y-Leg) with functional groups allowing for further conjugation with imaging and therapeutic moieties (vide infra). The in vitro cell experiments using FITC-conjugated Y-Leg revealed its specific and selective interaction with M2-polarized macrophages (i.e., TAMs) with preference to M1 macrophages, and that the interaction was not interfered with by conjugating FITC to its functional group. Further, we constructed a nanotube system by grafting Y-Leg onto oxidized carbon nanotubes (OCNTs) loaded with paramagnetic Fe3O4 nanoparticles. The intravenous injection of the resultant Y-Leg-OCNT/Fe3O4 nanotubes to 4T1 mammary tumor-bearing mouse led to the magnetic resonance imaging (MRI) of TAM6 7 8
Authors made an equal contribution to this work. Corresponding author to whom any correspondence should be addressed. Co-corresponding authors.
Materials Research Express 1 (2014) 025007 2053-1591/14/025007+14$33.00
© 2014 IOP Publishing Ltd
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infiltrated tumor microenvironment, revealing the targeting specificity of Y-Legconjugated nanotubes in vivo. The Y shape of peptide and its functional groups containing amines and imidazole can protonate at different pHs, contributing to the in vitro and in vivo targeting specificity. This study represents the first development of novel peptide and peptide-grafted nanotube system targeting M2-polarized TAMs in vivo. The methodology developed in this study is applicable to the construction of various multifunctional nanoparticle systems for selectively targeting, imaging and manipulating of TAMs for the diagnosis and treatment of cancers and inflammatory diseases identified with macrophageinfiltrated disease tissue. Keywords: carbon nanotubes, tumor-associated macrophage, legumain, targeted imaging, biomaterials
Introduction
Breast cancer cells are highly heterogeneous, making them notoriously difficult to target [1]. Recent studies have suggested that therapy directly towards tumor microenvironment could circumvent the challenge of targeting highly heterogeneous breast cancer cells and impact the survival of patients regardless of tumor types and stages [2, 3]. Increasing evidence has shown that macrophage represents one of the most important stromal cells in tumor microenvironment and plays a significant role in the progression and metastasis of many types of cancers such as breast cancer, melanoma, prostate cancer, and lung cancer [4, 5]. Specifically, the TAM has been identified in breast cancer patients, and the degree of TAM accumulation has been found to associate with angiogenesis, the development of immune suppression and poor patient outcome [6, 7]. Moreover, TAM-targeted therapeutics have been demonstrated to effectively delay tumor metastasis and improve survival in animal models of mammary cancer [8, 9]. Therefore, intervention that specifically assesses and manipulates TAM is anticipated to lead to advancement towards diagnosing and treating cancers with dramatic TAM infiltration. Targeting TAMs has been hindered by the lack of targeting molecules. It has been recently shown that legumain can be used as a specific ligand for tumor-specific drug delivery due to its overexpression by TAMs [10]. Legumain is an asparaginyl endopeptidase, originally isolated in plants before eventual identification in mammals [11]. It is highly homogeneous across species [11]. Legumain is highly specific, cleaving asparagine at the P1 site of the substrate [11], which has been used in several cancer treatment strategies that target primary tumor microenvironment [10]. For instance, Liu et al developed a compound called legubicin that attaches a peptide to doxorubicin for delivering chemotherapeutic drug to the tumor microenvironment [10]. This approach was expanded by Wu et al to create a cell-impermeable molecule that can be cleaved by legumain in the tumor microenvironment and internalized by cells [12]. In addition, Luo developed legumain-based DNA vaccine to target TAM with a concomitant reduction of TAMsecreted factors, and to increase cytotoxic T cell responses in the tumor microenvironment [13]. However, no study has ever been performed on the design and development of functionalized peptide for constructing multifunctional nanoparticle systems to deliver imaging and therapeutic components to TAM-infiltrated tumor microenvironment.
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In this study, we designed and developed Y-shaped peptide with functional groups, called Y-Leg. The Y-Leg was conjugated to fluorescein and nanotube to assess the in vitro and in vivo targeting of TAMs, respectively. Results of this study revealed great promise of Y-Leg for constructing multifunctional nanotube systems to allow for TAM-targeted imaging and manipulation. Since macrophages have been shown to play a significant role in various cancers and many inflammatory diseases [14], the newly-developed Y-Leg-grafted nanotube systems will lead to very broad implications for fundamental investigations and clinical applications.
Experimental methods Synthesis of peptide and FITC-conjugated peptide
The peptide was prepared using solid phase synthesis with Fmoc chemistry according to the published method [15]. After the peptide segment Ala-Ala-Asn-Leu-His-Lys was prepared, the Lys was co-blocked with His-Lys to form a Y-shaped peptide Ala-Ala-Asn-Leu-His-Lys-(HisLys)2. The peptide Ala-Ala-Asp-Leu-His-Lys-(His-Lys)2 was synthesized using the same method and served as Mw isotype control. The Ala-attached Wang resin, as well as Fmocconjugated Ala, Asn, Asp, Leu, His, and Lys were purchased from ANASPEC. In order to prepare FITC-conjugated Y-Leg, the Y-shaped peptide and its isotype control containing amines were reacted with NHS-fluorescein in the presence of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Briefly, the Y-shaped peptide or isotype control conjugated to the resin were added with excess NHS-fluorescein and EDC and incubated at room temperature for 3 h, followed by repeatedly washing to remove unreacted NHS-fluorescein and EDC. The FITC-conjugated Y-Leg and MisTg-Y-Leg were, respectively, cleaved from the resin according to the published method and lyophilized to yield Y-Leg-FITC and MisTg-Y-Leg-FITC, respectively. Preparation and culture of M1 and M2 macrophages
The preparation and culture of M1 and M2 macrophages from bone marrow-derived cells were conducted according to the published method [16]. Briefly, the bone marrow from tumor-free Balb/c mouse was harvested and prepared into a single-cell suspension. The single-cell suspension of bone marrow was then stimulated by IFN-γ 20 ng ML−1 and LPS 5 ng mL−1 in the presence of GM-CSF 25 ng mL−1 to produce M1-polarized macrophages, and stimulated by IL4 20 ng mL−1, IL-1β 10 ng mL−1 and IL-10 15 ng mL−1 in the presence of GM-CSF 25 ng mL−1 to produce M2-polarized macrophages. The growth factor GM-CSF was added to cell culture every 3 d. The stimulated cells were cultured for at least 12 d until the majority of cell colonies were formed. The day-14 cells were used for further experiments. For the preparation of co-cultured M1 and M2 macrophages, the day-7 M1 and M2 cells were transferred to a new culture plate at 1:1 ratio and co-cultured in the presence of GM-CSF for another 7 d for further experiments. LPS was purchased from Invitrogen and Sigma. The culture medium as well as recombinant proteins IFN-γ, IL4, IL-1β, IL-10, and GM-CSF were purchased from Invitrogen. The intracellular legumain ELISA kit was purchased from MER&CIEL company (www. meretciel.com). Balb/c mice were purchased from Charles River.
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Fluorescence-assisted cell sorting (FACS)
Cell expression of surface protein F4/80 and legumain was quantified by fluorescence staining followed by FACS analysis. Cells were treated with or without Y-Leg-FITC for 20 min, and then washed by PBS thrice. MisTg-Y-Leg-FITC served as control. Cell pellets were incubated with rabbit IgG (isotype IgG) in stain buffer, and then stained with rabbit-anti-mouse alexa fluor 647-conjugated F4/80 antibody (F4/80-AF647) and PE-conjugated legumain (legumain-PE) for 30 min in dark. The stained cells were washed by PBS twice and fixed by fixative buffer, followed by FACS analysis (BD Biosciences, LSR II) to quantify the AF647-, PE- and FITCpositive cells. All the fluorescence-conjugated antibodies (polyclonal), stain buffer, and fixative buffer were purchased from BD Biosciences. All experiments were repeated twice and all data were expressed as the mean values ± SE. Confocal fluorescence microscopy
Cell interaction with Y-Leg-FITC and MisTg-Y-Leg was visualized by confocal fluorescence microscopic imaging (Zeiss, LSM 510). The M2-polarized cells were cultured on glass slide placed in a cell culture plate. At the end of culture, cells were treated with Y-Leg-FITC and MisTg-Y-Leg peptide for 20 min, respectively, and then washed by PBS. For the uptake experiments, the cells on cover slide were incubated with rabbit IgG (isotype IgG), and then stained with rabbit-anti-mouse F4/80-AF647 and CD206-R-PE for 30 min at room temperature in the dark; for the lysosome co-localization experiment, the cells on cover slide were incubated with lysotracker (Invitrogen) for 20 min in the dark. After staining, cells on cover slides were repeatedly washed by PBS, fixed by formalin and mounted to glass slide using mounting buffer (Invitrogen). The antibodies (polyclonal) were purchased from BD Biosciences. The mounting reagent was purchased from Invitrogen. Construction and characterization of peptide-grafted oxidized carbon nanotube (OCNT) loaded with Fe3O4 nanoparticles
The carbon nanotubes (CNTs) of diameter ∼10 nm and length ∼100 nm were obtained from our previous study [17] and oxidized to introduce controlled percentage (∼19%) of defect-sites to the outer wall [18]. In order to prepare amine reactive OCNT, we suspended our freshly prepared OCNTs in solution in the presence of 0.1 M of N-hydroxysuccinimide (NHS). The solution was kept at 80 °C overnight under constant stirring. Thereafter, the suspension was filtered through a 0.22-μm PVDF membrane and washed with an excessive amount of water to get rid of free NHS. NHS activated OCNT thus obtained was mixed with the Y-Leg or control peptide at mass ratio 1:2 in an aqueous solution at room temperature, followed by the addition of 0.2 M of 1-ethyl-3-(3-dimethylpropyl)-carboiimide (EDC). The mixture was sonicated in a water bath for 1 h and filtrated through 0.22 μm PVDF membrane. After repeatedly washing with water to remove the unconjugated peptide, the Y-Leg-conjugated OCNT and control were obtained. The peptide-conjugated OCNT in water was added with 500 mg magnetic Fe3O4 nanoparticles (5 nm diameter, NN-Labs, LLC), and then dispended in 50 mL water followed by sonication for 1 h. The resulting suspension was kept at room temperature for 2 d to allow the precipitation of large particles. The supernatant was collected and dried to produce samples labeled as Y-Leg-OCNT/Fe3O4 and MisTg-Y-Leg-OCNT/Fe3O4, respectively. The degree of oxidation as well as the concentration of peptide and Fe3O4 on the OCNT were characterized by 4
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quantifying the heating-induced weight loss using TGA analysis (TA Instrument, Q50). The nanotube diameter and length were determined by atomic force microscopic (AFM) imaging (Agilent 5500 atomic force microscope) of spin-coated samples on mica substrates. Magnetic resonance imaging
A 9.4 tesla Bruker Biospec MR scanner with a 35 mm diameter transmit/receive volume coil was used to image mice pre- and post-injection of the peptide-conjugated nanotubes. For initial localizer scans, multi-echo spin echo MR acquisition was performed to obtain the high resolution T2 relaxation time maps (20 echoes, echo spacing = 5 ms). This multi-slice acquisition provides quantitative T2 maps over the entire tumor before and after the contrast agent injection. A long repetition time over 5 s and prospective respiratory gating was used to limit the effects of T1 relaxation and respiratory motion on the T2 assessment, respectively. Imaging results were analyzed by OsiriX software. The monoexponential and biexponential relaxation models were established to calculate the T2/T2* relaxation times for each image pixel and each image slice to identify tumor regions with accumulation of nanotube and Fe3O4. Animal model
A million 4T1 cells were inoculated to the mammary fat pad of Balb/c mice. Starting with a day-10 tumor, we injected the nanotube to mice via tail vein at 125 μg kg−1. MisTg-Y-Leg conjugated nanotube served as control. Six mice per group were used for Y-leg and MisTg-YLeg groups. Mice were MRI scanned pre-injection and 8 h post intravenous (i.v.) injection. After the scans, mice were euthanized and the tumors were harvested, formalin-fixed, paraffinembedded and sliced according to the standard method. The tumor tissue slices were deparaffinized and incubated with rabbit IgG (isotype IgG) before staining with rabbit-antimouse F4/80-AF647 and legumain-FITC antibodies. The stained tumor tissue slices were imaged by confocal fluorescence microscopy (Zeiss, LSM 510). The iron stain was conducted using HT-20 iron stain kit from Sigma. Rabbit IgG and all antibodies (polyclonal) were purchased from BD Biosciences. 4T1 tumor cell line was from ATCC. Balb/c mice were purchased from Charles River. Statistic analysis
Power analysis determined 6 mice/group allowed establishing a significant correlation (R > 0.8). An analysis of covariance (ANCOVA) was used to test for statistically significant differences among groups. Results and discussion Development of Y-shaped functionalized peptide Y-leg
It has been shown that legumain is overexpressed by TAMs and M2-polarized macrophages but hardly seen on circulating monocytes and M1 macrophages [10–13]. Circulating monocytes can infiltrate to specific tissue/organ/tumor and are polarized by environmental cytokines to differentiate into antitumoral (M1) and tumor-promoting (M2) macrophages (M1 and M2 paradigm) [19, 20]. This has been observed in various types of cancers, such as breast cancer, 5
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Figure 1. Development of Y-shaped peptide with functional groups. A. The sequence of
Y-shaped legumain-targeting peptide (Y-Leg) and the legumian-miss-targeting Mw isotype control MisTg-Y-Leg. Y-Leg and MisTg-Y-Leg contain one segment targeting or miss-targeting legumain, respectively; they both contain segments composed of histine (His) and lysine (Lys) that provide functional groups for further conjugation. B. The GC-MS characterization of the Y-Leg and MisTg-Y-Leg.
melanoma, prostate cancer, and lung cancer [4, 5]. Increasing evidence has shown that M1 macrophages aggressively engulf bacteria/transformed cells and mediate adaptive immunity, thus exerting tumoricidal activity [19, 20], while M2 macrophages promote angiogenesis and mediate immune suppression, thus exerting tumor-promoting activity [19, 20]. The fact that leguamin is overexpressed by TAM and M2, but not monocytes and M1, provides an opportunity to use legumain as a specific ligand for targeting TAMs. In addition, it has been shown that liver, kidney, and lungs express no surface legumain [10–13]. Collectively, these provide strong rationale for legumain as a highly specific way of systemically targeting ‘bad’ macrophages (TAM or M2) dramatically infiltrated to mammary tumor microenvironment. Legumain is a member of the endopeptidase family [10, 11]. It specifically digests substrate-containing asparagine (Asn) at the P1-site [10, 11]. Based upon this, we designed a Yshape legumain-targeting peptide, called Y-Leg, which contained two segments (figure 1(A), upper). One segment contains Asn at the P1 site so that can be specifically recognized by legumain. This segment is called Legumain-targeting segment. The other segment contains histidine and lysine and is called non-targeting segment. Lysine is the cationic amino acid containing primary amines so that it provides flexibility for further functionalization. Histidine contains imidazole that absorbs proton at endothemal-lysosomal pH (5.4–6.5) so that it is
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Figure 2. Y-Leg shows specificity and selectivity for targeting legumain-expressing cells (M2-polarized macrophages, i.e. TAMs). A. Synthesis of FITC-conjugated Y-Leg and MisTg-Y-Leg. The two Lys(s) of the Y-Leg were reacted with NHS-activated FITC. MisTg-Y-Leg peptide was conjugated with FITC in the same manner and served as control. B. FACS analyses of the legumain-non-expressing M1 and legumainexpressing M2 macrophages incubated with Leg-FITC and MisTg-Y-Leg-FITC. Unstained cells served as control to gate positive and negative cells. The MisTg-YLeg-FITC-treated cells served as negative control to identify the cell events, showing specific interaction of the Y-Leg-FITC with legumain-expressing cells. P1- and P1’gated cell events were F4/80+Legumian− M1 macrophages, while P2- and P2’-gated cell events were F4/80+Legumian+ M2 macrophages. C. FACS analyses of the cocultured M1 and M2 macrophages incubated with Leg-FITC and MisTg-Y-Leg-FITC, respectively. Unstained cells served as control to gate positive and negative cells. The MisTg-Y-Leg-FITC-treated cells served as negative control to identify the cell events having selective interaction with Y-Leg-FITC. P3- and P3’-gated cell events were F4/ 80+Legumian− M1 macrophages, while P4- and P4’-gated cell events were F4/ 80+Legumian+ M2 macrophages. D. Confocal fluorescence microscopic images of M2 macrophages incubated with the Y-Leg-FITC and MisTg-Y-leg-FITC, respectively, providing visualized evidence for the interaction of Y-Leg-FITC with M2 macrophages. The emission wavelength for AF647, PE, and FITC are 632 nm, 525 nm, and 488 nm, respectively.
known to facilitate endosome-lysosome escape within cells [21]. In addition to Y-Leg, we synthesized another Y-shaped peptide with Asp, instead of Asn, at the P1 site (figure 1(A), lower). This peptide served as a miss-targeting peptide (MisTg-Y-Leg) and used as Mw isotype control for Y-Leg. Both Y-Leg and MisTg-Y-Leg were prepared using solid phase synthesis with Fmoc chemistry and characterized by GC-MS (figure 1(B)). The GC-MS results revealed the successful synthesis of targeting and miss-targeting peptides. The Y-Leg shows specificity and selectivity of targeting M2-polarized macrophages (TAMs)
It has been shown that TAMs infiltrating to 4T1 mammary tumor microenvironment are M2polarized macrophages [22]. TAMs and M2 macrophages are legumain-expressing cells while M1 macrophages are legumain-non-expressing cells. In this study, therefore, we prepared bone marrow-derived M1- and M2-polarized macrophages to examine the targeting specificity and selection of Y-Leg. In order to quantify and visualize the specific interaction of Y-Leg with legumainexpressing cells, we labeled Y-Leg with fluorescence marker FITC by conjugating aminereactive FITC to the non-targeted segment of Y-Leg, as shown in figure 2(A). The MisTg-YLeg was also conjugated with FITC and served as control. After incubating Y-Leg-FITC and MisTg-Y-Leg-FITC with legumain-expressing M2 and legumain-non-expressing M1 macrophages, respectively, the cells were stained with F4/80-AF647 and legumain-PE, followed by FACS analysis (figure 2(B)). Compared to unstained cells and MisTg-Y-Leg-FITC-treated cells, the F4/80+legumain− M1 macrophages (P1 gated cells) are FITC−, indicating no interaction of Y-Leg-FITC with M1 macrophages. By contrast, the F4/80+legumain+ M2 macrophages (P2 gated cells) are FITC+ by 90.6%, indicating positive interaction of Y-LegFITC with M2 macrophages. This revealed the specific interaction of Y-Leg-FITC with legumain-expressing cells. 8
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In order to examine the targeting selectivity of Y-Leg-FITC with legumain-expressing cells, the Y-Leg-FITC and MisTg-Y-Leg-FITC were, respectively, incubated with the cocultured M1 and M2 macrophages. After staining with F4/80-AF647 and legumain-PE, cells were fixed and analyzed by FACS (figure 2(C)). Compared to the unstained cells and MisTg-YLeg-FITC-treated cells, the co-cultured M1 and M2 cells were F4/80+ by over 95% (figure 2(C), II), indicating they are mature macrophages; the co-cultured cells were legumain+ by 53.6% (figure 2(C), II), indicating percentage of M2 macrophages. The P3-gated legumainnon-expressing cells (M1) were legumain− (figure 2(C), III), while the P4-gated legumainexpressing cells (M2) were legumain+ by 89.6% (figure 2(C), IV). The legumain+ cells were not observed in MisTg-Y-Leg-treated co-cultured cells. Collectively, these results revealed a good targeting selectivity of Y-Leg. In addition, the interaction of Y-Leg with M2 macrophages was visualized by confocal fluorescence microscopic imaging, using MisTg-Y-Leg as control (figure 2(D)). After incubating Y-Leg-FITC with M2 macrophages, the FITC signal (green) was observed in F4/80+CD206+-defined M2 cells, providing additional visualized evidence for the good targeting and selectivity of Y-Leg. The results of figures 2(A)–(D) not only demonstrated the targeting specificity and selectivity of Y-Leg using leguamin-expressing (M2) and leguamin–non-expressing cells (M1), but also revealed that the targeting specificity and selectivity are not interfered with by the modification (i.e. FITC conjugation) of the non-targeted segment within Y-Leg. The grafting of Y-Leg onto carbon nanotube systems leads to TAM-targeted delivery of imaging and therapeutic agents in vivo
In order to explore the potential of Y-Leg for TAM-targeted delivery of imaging and therapeutic agents in vivo, we grafted Y-Leg to nanotubes, as shown in figure 3(A). To start with, we prepared carbon nanotubes (CNTs) of defined structure through our previously-reported procedure [17], and oxidized the resulting CNTs according to our published method [18] into oxidized CNTs (OCNTs) with a controlled degree of oxidation (19.5%) for the outer wall. The resultant OCNT showed an excellent biocompatibility and dispersion stability [23]. We then grafted Y-Leg to the oxidized-sites of OCNT via the chemical reaction between amines of YLeg and NHS-activated defect-sites of the OCNT (figure 3(A)). Further, the non-oxidized-sites of the OCNT were attached with paramagnetic Fe3O4 nanoparticles (∼5 nm diameter, NN-Labs, LLC) for T2-weighted magnetic resonance imaging (MRI) of cells and tissues. The TGA analysis on the heating-induced weight loss of Y-Leg-OCNT/Fe3O4 revealed a 20.3% peptide grafting and 19.3% Fe3O4 mass loading (figure 3(B)). The diameter and length of the Y-LegOCNT/Fe3O4 was visualized and analyzed by atomic force microscopic imaging (figure 3(B)) and the associated height profile, revealing a ∼5 nm diameter and ∼100 nm length. In this study, we chose 4T1 model in Balb/c mouse to examine whether the Y-Leg grafted nanotube can systemically target TAMs in tumor microenvironment. The 4T1 model is a highly aggressive synergistic xenograft mammary cancer model [24]. The spread of 4T1 cells to liver and/or lungs in Balb/c closely mimics human breast cancer growth and metastasis [24]. Growth of 4T1 tumor in mammary fat-pad is associated with rapid infiltration of TAM [24]. Therefore, this model is ideal for accessing diagnostic and therapeutic modality targeting TAMs in tumor microenvironment. After i.v. administration of Y-Leg-OCNT/Fe3O4 nanotubes and the mis-targeted counterparts, respectively, to 4T1-bearing mouse at 125 ug kg−1, the mice were anesthetized 9
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Figure 3. The Y-Leg grafted nanotube hybrids led to systemic targeting of TAM-
infiltrated tumor microenvironment in the mammary cancer mouse model. A. Schematic of our Y-Leg conjugated OCNT loaded with Fe3O4 nanoparticles. B. Characterization of Y-Leg-OCNT/Fe3O4 by TGA and AFM. C. MR scans of mice pre- and postinjection (via tail vein) of the Y-Leg-OCNT/Fe3O4 and MisTg-Y-Leg-OCNT/Fe3O4, respectively. MisTg-Y-Leg-OCNT/Fe3O4 served as control. D. Confocal fluorescence microscopic images of tumors treated by Y-Leg-OCNT/Fe3O4 and MisTg-Y-LegOCNT/Fe3O4, respectively. Tumor slices were stained with F4/80-AF647, legumianFITC, and iron-staining reagent, revealing the accumulation of imaging agent (Fe3O4 nanoparticles) in the TAM-infiltrated mammary tumor microenvironment.
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Figure 4. Schematic of the Y-Leg in mediating specific interaction with legumain-
expressing TAMs. A. Schematic of cyclic peptide with targeting segment and two functional ‘feet’ for conjugation with nanomaterials. B. Schematic of Y-Leg peptide with the targeting segment and two functional ‘feet’ for further conjugation. The conjugation of the Y-Leg to nanomaterials facilitates the orientation of the targeting segment for an improved targeting specificity. C. Schematic of cellular mechanism of the endosomallysosomal exclusion of nanomaterials for the exclusion of imaging or therapeutic agents carried by nanomaterials. D. Schematic of imidazole-containing Y-Leg for mediating the endosomal-lysosomla escape within cells. Once imidazole-containing materials are internalized by cells and transferred to the endosomal-lysosomal compartment, the pH sensitive imidazole can pump in hydrogen ions, leading to the disruption of endosome and lysosome. Consequently, nanomaterials are released to cytoplasm. E. Confocal fluorescence microscopic imaging of PMJ2R macrophages treated by Y-LegFITC and MisTg-Y-Leg-FITC, respectively, followed by lysotracker stain.
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with isofluorine and scanned by a 9.4 Tesla MR scanner pre- and post-i.v. administration to assess relative accumulation of nanotubes in tumor (figure 3(C)). The post-contrast T2-weighted MR image obtained from Y-Leg-conjugated nanotubes exhibited growing regions of low signal intensity (dark region in yellow circles in figure 3(C)), indicating accumulation of the ironbased contrast agent within the tumor. By comparison, the MisTg-Leg-conjugated nanotubes exhibited virtually no signal change and agent accumulation (figure 3(C)). These MR images clearly demonstrated that the i.v. injected Y-Leg-OCNT/Fe3O4 nanotubes can deliver the T2 contrast agent Fe3O4 nanoparticles to the intratumoral region with high specificity and facilitate the MR-guided imaging of tumor microenvironment. Having shown the intratumoral accumulation of the nanotubes at 8 h post i.v. administration, we harvested the tumors at 8-hour post injection and performed fluorescence immunohistological studies on the tumor slides using confocal fluorescence microscopy (figure 3(D)). Compared to the tumor tissue treated with MisTg-Y-Leg-conjugated nanotube, the TAM-infiltrated microenvironment treated by Y-Leg-conjugated nanotube was found to be positive for iron stain in regions stained with F4/80-AF647 and legumain-FITC. Although the iron stain did not completely overlap with F4/80+legumain+-defined TAMs, yet it appeared at tumor microenvironment where a dramatic accumulation of TAMs occurred. This provides strong evidence that the i.v. administration of Y-Leg-OCNT/Fe3O4 indeed facilitates the delivery of imaging agent (Fe3O4 nanoparticles) to the TAM-infiltrated tumor microenvironment. Clearly, therefore, figure 3 demonstrates that the functionalized Y-Leg can be used to construct nanoparticle system to deliver imaging (Fe3O4) agents to TAM-infiltrated tumor microenvironment with high specificity. The development of functionalized Y-shaped peptide for targeting TAMs will lead to the construction of various multifunctional nanoparticle systems for selective targeting, imaging, and modulating of TAM for cancer diagnosis and treatment
To the best of our knowledge, such a Y-shaped peptide has not been reported previously. It has been shown in literature that the cyclic constrained peptide that has two ‘feet’ for conjugation and one targeting segment in the loop (figure 4(A)) can exhibit an exponential increase in binding affinity with specific ligand [25]. Similarly, our newly-developed Y-Leg can provide two functional sites (‘feet’) containing amines (figure 4(B)) for further modification. The conjugation of the two ‘feet’ to the surface could facilitate the orientation of the targeting segment (figure 4(B)), which anticipate enhancing the spatial interaction of Y-Leg with cells overexpressing targeting ligand. It is also worth noting that the Y-Leg contains histidine in the non-targeted segment (figure 2(A)), and that histidine contains imidazole capable of absorbing proton at pH ∼ 5.5–6.4 to facilitate the endosomal-lysosomal escape within cells (figure 4(C)). Since the inclusion of nanomaterials by endosomal-lysosomal compartments is one of the major mechanisms of cells to exclude particles and establish resistance (figure 4(D)) [26], the capability of molecule to escape the endosomal-lysosomal compartment could lead to the prolonged presence within cells. In this study, we incubated Y-Leg-FITC with PMJ2R macrophage cells and then stained macrophages with lysosome tracker (lysotraker) (figure 4(E)) to co-localize Y-Leg-FITC with specific subcellular compartments. By comparison to MisTg-Y-Leg-FITC the Y-Leg-FITC was observed in both endosome-lysosome (regions indicated by yellow arrows in figure 4(E)) and 12
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intracellular regions (cytosol) (regions indicated by white arrows in figure 4(E)), suggesting the ability of Y-leg-FITC for mediating endosomal-lysosomal escape. This may explain the observation of the accumulation of Y-Leg-conjugated nanotubes in TAM-infiltrated tumor microenvironment at 8 h post injection. Such a long-lasting imaging window is favorable for applying therapeutic modalities in clinic. Indeed, the newly-developed Y-Leg conjugated nanotubes have led to the systemic targeting and MRI-visualized imaging of TAMs in tumor microenvironment. In addition, the delivery of OCNT and iron oxide nanoparticle hybrids to the tumor microenvironment provides great potential to targeted modulate (i.e., thermal ablate) TAMs. Although the elimination of macrophage has been demonstrated effective in delaying mammary cancer metastasis and improving survival in mice models, the multifunctional nanoparticle systems for selective targeting, imaging, and manipulating of TAMs have not been accomplished to date. TAM represents up to 40–60% total mass of breast tumor, prostate tumor, and brain tumor [27–29], and its infiltration to tumor microenvironment has been identified in primary, recurrent and metastatic tumor in humans regardless of the tumor types and stages [30, 31]. Therefore, our demonstration of the Y-Leg-conjugated carbon nanotube and iron oxide nanoparticle hybrids for TAM specific targeting and imaging has important implications for many diagnostic and therapeutic modalities for various cancers and inflammatory diseases with dramatic infiltration of TAMs. Acknowledgement
This study is supported by NIH-NCI annual pilot via Case Comprehensive Cancer Center at Case Western Reserve University (Imaging Pilot (M.Z. CON 500659), 2012-2013). We are also grateful for the support from the National Nature Science Foundation of China (81301320), and the Nature Science Foundation of Zhejiang Province, China (LY13H180013).
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