Tumor Cell-Specific Nuclear Targeting of Functionalized Graphene

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Tumor Cell-Specific Nuclear Targeting of Functionalized Graphene Quantum Dots In Vivo Chenjie Yao, Yusong Tu, Lin Ding, Chenchen Li, Jiao Wang, Haiping Fang, Yanan Huang, Kangkang Zhang, Quan Lu, Minghong Wu, and Yanli Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00466 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 14, 2017

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Bioconjugate Chemistry


Tumor Cell-Specific Nuclear Targeting of Functionalized Graphene Quantum Dots In Vivo Chenjie Yao,†,+ Yusong Tu,§,+ Lin Ding,† Chenchen Li,† Jiao Wang, Haiping Fang, Yanan ∥

⊥

Huang,† Kangkang Zhang,† Quan Lu,*,‡ Minghong Wu,*,† Yanli Wang*,†,‡

†

Institute of Nano-chemistry and Nano-biology, Shanghai University, Shanghai 200444, P.R.

China ‡

Program in Molecular and Integrative Physiological Sciences, Harvard T.H. Chan School of

Public Health, Boston, Massachusetts, 02115, USA §

College of Physics Science and Technology, Yangzhou University, Jiangsu 225009, P.R.

China ∥

School of Life Science, Shanghai University, Shanghai 200444, P.R. China

⊥

Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, P.

R. China

+

These authors contributed equally to this work.

KEYWORDS：：graphene quantum dots, targeted nanoparticle, nucleus, cancer

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ABSTRACT Specific targeting of tumor tissues is essential for tumor imaging and therapeutics but remains challenging. Here, we report an unprecedented method using synthetic sulfonic-graphene quantum dots (sulfonic-GQDs) to exactly target the cancer cell nuclei in vivo without any bioligand modification, with no intervention in cells of normal tissues. The key factor for such selectivity is the high interstitial fluid pressure (IFP) in tumor tissues, which allows the penetration of sulfonic-GQDs into the plasma membrane of tumor cells. In vitro, the sulfonicGQDs are repelled out of the cell membrane because the repellsive force between negatively charged sulfonic-GQDs and the cell membranes which contribute to the low distribuation in normal tissues in vivo. However, the plasma membrane-crossing process can be activated by incubating cells in ultrathin-film culture medium because the attachment of sulfonic-GQDs on cell memebranes. Molecular dynamics simulations demonstrated that, once transported across the plasma membrane, the negatively charged functional groups of these GQDs will leave the membrane with a self-cleaning function retaining a small enough size to achieve penetration through the nuclear membrane into the nucleus. Our study showed that IFP is a previously unrecognized mechanism for specific targeting of tumor cell nuclei and suggested that sulfonic-GQDs may be developed into novel tools for tumor-specific imaging and therapeutics.

INTRODUCTION Efficiently targeting nanomaterials to regions of disease in vivo has enormous potential to improve the precision of cancer therapy. However, our ability to do so remains very limited.1 So far, two general strategies have been utilized to enhance the specificity of nanoparticle delivery to tumors: active and passive targeting strategies.2, 3 The active targeting strategies rely on coating nanoparticles with antibodies to selectively direct them to sites expressing the target antigen. Many targeting design methods have been developed by modifying the ligand 2 ACS Paragon Plus Environment

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type or ligand density to target specific or overexpressed receptors in diseased cells in vivo.4-12 However, the translation of such active targeting strategies to the clinic suffers from a myriad of obstacles,4, 5 including the expression of biomarkers in non-target cells13 and inefficient targeting in vivo due to multiple biological barriers.14-16 So far, the two most developed are doxorubicin-containing immunoliposomes and transferrin-targeted oxaliplatin liposomes2; however, to date, none have been approved clinically due to their poor targeting performance in vivo.2 Passive targeting strategies take advantage of the leaky nature of tumor micro-vessels which facilitates greater accumulation of nanoparticles due to the enhanced permeability and retention (EPR). Couple nanoparticles such as the doxorubicin-liposome carriers Myocet, Doxil, polymeric micelles, nanoparticles, and polymer-drug conjugates are currently in human clinical trials.3, 17 However, targeting performance of nanoparticles designed based on passive targeting strategies is still very poor. First, nanoparticles must escape the reticuloendothelial system (RES) during circulation before extravasation in the tumor tissue, yet after circulation, most of the nanoparticles are distributed in normal main organs such as the liver, lung, spleen and heart. Second, the high interstitial fluid pressure (IFP) in tumor tissues is a large barrier for extravasation of the nanoparticles.2 Normalizing the abnormal tumor vasculature with antiangiogenic therapy can improve the delivery and efficacy of nanomedicines.3,

18, 19

Scientists have also tried to utilize other aspects of tumor tissue such as the acidic tumor microenvironment.1, 20 However, this design method failed to accomplish in vivo targeting because many parts of the human body, such as the gastrointestinal tract and vagina, also have an acidic microenvironment.20 So far, the statistic results from more than 100 nanomedicine papers published in the past 10 years showed that only less than 0.7% of any nanoparticle dose gets into tumors.21 Therefore, it remains a major task to find a new approach for tumorspecific targeting that attunes to the tumor micro-environment.21



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Graphene quantum dots (GQDs) have received increasing attention owing to their excellent physicochemical properties, including good chemical stability, photoluminescence, and electronic properties.22-26 Their high biocompatibility and tunable optical properties make GQDs suitable for a wide range of potential applications in biomedical research, such as bioimaging,23, 24, 27 biosensors,23, 27, 28 disease diagnosis

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and therapy.32, 33 Numerous studies

have examined surface functionalization of GQDs for the application in diagnosis, and gene and drug delivery.29,

31, 34-36

However, their large-scale applications are limited by current

synthetic methods that commonly produce GQDs in small amounts and with poor optical properties. In our previous work, we developed a simple bottom-up approach for large-scale synthesis of OH- and amine- functionalized single-crystalline GQDs via a green and low-cost alkali-mediated hydrothermal molecular fusion using an active PAH molecule as a precursor. Our amine- modified GQDs featured excellent optical properties, strong excitonic absorption bands extending to the visible region, large molar extinction coefficients, and long-term photo-stability.37 High interstitial fluid pressure (IFP) is a distinctive characteristic of cancerous tissue but has never been utilized in selective targeting strategies because it posed a physiological barrier to anticancer therapies that use strategies designed on the EPR effect.38, 39 Herein, we synthesized negatively charged sulfonic-graphene quantum dots (sulfonic-GQDs) via a modified alkali-mediated hydrothermal molecular fusion method which show specific tumor cell nuclear targeting perfomance both in vitro and in vivo. IFP is a new cancer-associated targeting trigger and that high IFP allows synthetic sulfonic-GQDs to target a variety of cancer cells in vivo with minimal uptake by cells in normal tissues. This IFP-dependent mechanism provides a new theoretical foundation to design targeted nanomaterials and nanomedicines and represents a promising future for the integration of tumor diagnosis and therapy. 4 ACS Paragon Plus Environment

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RESULTS AND DISCUSSION Synthesis and characterization of sulfonic graphene quantum dots (GODs). GQDs have garnered increasing attention because of their various attractive physicochemical properties and wide range of potential applications, including imaging and therapeutics.40-42 We previously established a new mass-production synthesis method for single-crystalline GQDs via an environmentally friendly and low-cost alkali-mediated hydrothermal molecular fusion method using an active PAH molecule as a precursor.37 However, when we attempted in vivo imaging by tail vein injection, positively charged amine-GQDs showed acute toxicity even at a very low dose. It has been reported that the surface charge of GQDs plays an important role in their in vivo toxicity and behavior.43, 44 To reduce the in vivo toxicity, we synthesized negatively charged sulfonic functionalized GQDs by introducing hydrophilic groups exclusively localized at the periphery without perturbing the hydrophobic graphene core. We realized bottom-up fabrication and in situ functionalization of nearly monodisperse, single-crystalline sulfonic-GQDs to design targeted nanoparticles on a mass-scale (Figure 1a). Sulfonic-GQDs were in situ functionalized at edge sites by sulfonic and hydroxyl groups whose characteristic signals are shown by Fourier transform infrared spectroscopy (FT-IR) (Figure 1e), SO3- at 900 cm-1, S-C at 520 cm-1, O-H at 3, 400 cm-1, and C-OH at 1,270 cm-1), and by X-ray Photoelectron Spectroscopy (XPS; Figure S1e). The content of C1s is 34.8 at%, O1s is 38.74 at%, S2p is 10.53 at% and N1s is 2.12 at%. Its XRD pattern (Figure S2f) is characteristic of graphite with a (002) layer spacing identical to that of bulk graphite (3.45 Å).They were nearly mono-dispersed (Figure 1b, c) with lateral sizes of 1.8 to 3.6 nm (2.5±0.5 nm on average) and thicknesses of 0.5 to 4 nm. Sulfonic-GQDs in aqueous solution emit bright green fluorescence when irradiated by a UV light with a high QY of 55% (using quinine sulfatein sulfuric acidas the reference as the reference; Figure 1a and Figure S2a). In contrast to highly defective GQDs or carbon dots produced by chemical cutting methods, the 5 ACS Paragon Plus Environment


molecularly fused sulfonic-GQDs are single-crystalline, as shown by high-resolution TEM (HRTEM; Figure 1c). They show superior optical properties to previously reported aminoGQDs

37

including strong excitonic absorption bands that extend to ~500 nm, bright

photoluminescence (PL) at 510 nm, a wide PLE spectrum, and high photo and chemical stability (Figure 1d and Figure S1a,b,d and S2a-d,g-i). The surface charge of sulfonic-GQDs is pH-dependent (Figure S2e), but the FL intensity is stable between pH 3-8 (Figure S1c). a

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Figure 1. Preparation and characterizations of sulfonic-GQDs. (a) Synthetic procedure. (b) TEM image and size distribution. (c) High-resolution TEM image and FFT pattern. (d) Ultraviolet (UV) absorption spectrum (ABS), PL, and excitation (PLE). (e) FT-IR spectrum showing the functionalization by both SO3and OH- groups.

In vitro cell nucleus targeting of sulfonic-GQDs. To study the cell imaging ability of sulfonic-GQDs, we conducted experiments with the 4T1 breast cancer cell line as the model. In our published work, we showed that positively charged amino-GQDs can easily pass through the cell membrane and concentrate in the cytoplasm.37 Unlike the positively charged amino-GQDs, the negatively charged sulfonic-GQDs did not enter cells even after coincubation for 48 h in vitro, most likely because negatively charged sulfonic-GQDs are usually repelled by the cell membranes. Confocal imaging of 4T1 cells showed that sulfonic6 ACS Paragon Plus Environment

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GQDs, fluorescing green under 405 nm excitation, accumulate on the cell membranes stained red with SYTO17 (Figure 2a), indicating that they failed to pass through the cell membranes of 4T1 cells. However, we noticed that the targeting process can be stimulated by mild heating or laser irradiation of cells cultured in an ultrathin film. An ultrathin layer of culture medium was generated with mild water-bath heating and laser-irradiation treatments (see the supporting information methods section for details). Once the thickness of the culture medium was shallow enough, e.g., less than 0.2 mm (Figure S3a,d), to enable 4T1 cells to be immersed only in the ultrathin layer of culture medium achieved both by mild treatments with water-bath heating (Figure 2b) and laser irradiation (Figure 2c), sulfonic-GQDs showed specific and selective nuclear targeting. This was confirmed by real-time dynamic in situ confocal merged images of sulfonic-GQD entry into cells and their nuclei during a period of 15 minutes (Figure 2d). Only a 6-minute mild irradiation dose was sufficient to initiate the nuclear targeting process of sulfonic-GQDs to cross the cell membranes in the irradiated region (Figure 2d and Figure S3b, see other doses required for different medium thicknesses in Figure S3a). We also investigated the cells cultured at 25, 37, 38°C with ultrathin film. It can be investigated that nuclear targeting of sulfonic-GQDs is quicker in the higher incubation temperature (Figure S4). During the subsequent several minutes, sulfonic-GQDs are rapidly and preferentially enriched in the nuclei, likely as a result of a strong electrostatic attractive force between the negatively charged sulfonic-GQDs and positively charged histones in the chromatin (Figure S5d).1, 45, 46 The mild water-bath heating and laser irradiation processes did not induce obvious acute cytotoxicity in the cells (Figure S5a-c). In addition, sulfonic-GQDs show superior photostability, since no photobleaching is evident, even after continuous laser irradiation for 40 minutes (Figure S2g-i). In addition to the 4T1 cancer cells, the in vitro nuclear targeting of the sulfonic-GQDs was also observed in normal cells such as GES-1 cells (gastric epithelial cell line), and NSCs cells (neural stem cell line; Figure S3c). We have thus established a universal in vitro nuclear 7 ACS Paragon Plus Environment


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targeting protocol by inducing an ultrathin ‘artificial’ mesoscopic fluid that provides a tension-like driving force as the transmembrane targeting trigger. The results above indicate the high sensitivity of the targeting process of sulfonic-GQDs on the microscopic surface tension (FMST) in the immediate vicinity of cultured cells. This nuclear targeting is specific for sulfonic-GQDs, as amino-GQDs can fail to accumulate in the nuclei even after being treated with a laser (Figure S6). Figure S3d sketches a simple mechanical principle that underlies the nanoparticle transmembrane nuclear targeting of sulfonic-GQDs. To overcome multiple biological barriers to the nuclear targeting without the need of constructing complicated bioconjugates, conventional receptor-mediated endocytosis pathways are replaced by a tension-triggered, transmembrane targeting pathway that involves tension-triggered insertion and rapid diffusion of sulfonic-GQDs within the cell membrane, followed by free diffusion in the cytoplasm, barrier-free penetration into the nucleus, and finally electrostatic absorption by positively charged histone in chromatin.1 Two seemingly antagonistic forces exerted on the extracellular nanoparticles are harnessed synergistically. First, the outward electrostatic repulsive force between the cells and sulfonic-GQDs (FR). Second an FMST that can drive the repelled nanoparticles toward tumor cells overcome the electrostatic barrier and initiate transmembrane transport. Heating and/or laser treatment were used to promote the ultrathin film medium to be thinner, thus increase the FMST and make it much higher than FR. Finally, sulfonic-GQDs could enter into cells and the cell nuclei targeting process occurred.


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Figure 2. Pressure-sensitive in vitro nuclear targeting process of sulfonic-GQDs. (a) Confocal imaging of 4T1 cells incubated in normal volume culture medium with sulfonic-GQDs (~2 mm-thick, as a control) for 48 h after both treatments with water-bath heating and laser irradiation for 15 min. Left, sulfonic-GQDs (excited at 405 nm, green), Middle, cell membranes stained with the membrane dye Cell Mask Orange (red), in red, and Right, merged. The merged image shows no sulfonic-GQDs crossing through the cell membranes. (b, c) Nuclear targeting of 4T1 cells immersed only in an ultrathin layer of culture medium (~0.2 mm-thick) with mild heating and laser treatments. Cells were fixed with 4% paraformaldehyde for 10 min. Left, sulfonic-GQDs (excited, green); Middle, nuclei stained with SYTO 17 (red); and Right, merged. The color superposition (yellow) in the merged images shows the nuclear targeting of sulfonic-GQDs. The mild heating treatment in (b) corresponds to placement into a 38 °C water bath for 15 min, and the laser irradiation treatment in (c) was continuous 405-nm excitation for 15 min. (d) Real-time dynamic confocal merged images of sulfonic-GQDs entry into cells and nuclei during 405 nm laser irradiation for 0-15 min. The colors are the same in (a). Scale bar, 10 µm.



Smart self-cleaning function of sulfonic-GQDs. To investigate why sulfonic-GQDs target the nucleus after crossing the plasma membrane but analogous positively charged aminoGQDs prefer to stay in the cytoplasm (Figure S6),37 we studied detailed transmembrane properties at a high spatiotemporal resolution using molecular dynamics (MD) simulations.47 Figure 3 shows the models of the two representative charged amphiphilic GQDs functionalized with SO3-and NHNH3+ and their motion trajectories within a phospholipid bilayer during the MD simulations. When detached from the membrane, the positively charged amino-GQDs draw a layer of lipid molecules toward the graphene plane with their head groups attracted to the amino groups (Figure 3). In contrast, the negatively charged sulfonic-GQDs can avoid adhesion onto the NP surface through electrostatic exclusion of the lipid molecules from the surface when detached from the membrane (Figure 3; see Movies S1 and S2 for more details). The repulsive force and the ideal amphiphilic structure of the sulfonic-GQDs play a crucial role in driving intramembranous diffusion, avoiding trapping by the hydrophobic force, and self-cleaning the NP surface when the transmembrane NPs detach from the membrane. This electrostatic exclusion function can avoid the disturbance of nuclear targeting by absorbed biological molecules and reduce systemic toxicity and immune

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Figure 3. Interaction between the GQDs and cell membranes. Representative trajectories of the molecular dynamics (MD) simulations with sulfonic-GQDs and amino-GQDs detached from the lipid membranes. The amphiphilic GQDs were composed of a hydrophobic graphene core (2.21 × 2.41 nm2) and a hydrophilic periphery functionalized with SO3- or NHNH3+. The cell membrane is modeled as a negatively


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charged phospholipid bilayer for simplification. Water is shown in violet, the GQDs are shown as large spheres (carbon, green; sulfur, yellow; oxygen, red; nitrogen, blue; hydrogen, white) and the phospholipids are shown as tan lines with the hydrophilic charged atoms as small colored spheres (hydrogen, white; oxygen, red; nitrogen, dark blue; carbon, cyan; phosphorus, orange). The two phospholipids extracted from the membrane and adsorbed onto the amino-GQDs are shown as larger spheres with yellow tails. Projection views are in the final column.

In vivo tumor cell nucleus targeting performance of sulfonic-GQDs. To explore the targeting performance in vivo, we established four types of subcutaneous tumor mice models (breast cancer, hepatoma carcinoma, cervical carcinoma and lung adenocarcinoma). SulfonicGQDs were injected intravenously into tumor-bearing mice, and the fluorescence of sulfonicGQDs excited in vivo at 470 nm (Figure 4a). Both in vivo and ex vivo distribution results show that sulfonic-GQDs rapidly accumulated in tumors 0.5 h after injection and were cleared from the bodies 24 h later (Figure 4a,b and Figure S7a). Semi-quantitative ex vivo analysis shows sulfonic-GQDs were significantly present in the tumor tissues (54%) and had a much lower distribution in normal tissues (heart, 5%; liver, 11％; spleen, 4%; lung, 8%; kidney, 15% and brain, 3%) (Figure 4b). Confocal images of the frozen sections of tumor and normal tissues further demonstrate the exceptional in vivo cancer cell-nuclear targeting capability of sulfonic-GQDs (Figure 4c) with little or no accumulation in normal tissues (Figure 4d and Figure S7b,c).



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Figure 4. Bio-distribution of sulfonic-GQDs in a subcutaneous tumor model. (a) In vivo fluorescence images of tumor-bearing mice 0.5 and 24 h after intravenous injection of sulfonic-GQDs. (b) Average signals of sulfonic-GQDs from isolated organs of ex vivo images after sulfonic-GQDs injection (60 mg/kg) for 0, 0.5, 1, 2, 8 and 24 h. Mean±SEM; n = 5 per group. (c,d) Confocal images of frozen sections of cancer and normal tissues after the intravenous injection of sulfonic-GQDs for 0.5 h. Left, sulfonic-GQDs (excited at 405 nm, green); Middle, nucleus of cells stained with SYTO 17 (excited at 635 nm, red); and Right, merged. (c) Breast cancer; (d) Lung. (e) Confocal merged images of frozen sections showing the cancer cell nuclear targeting of sulfonic-GQDs for hepatoma carcinoma, cervical carcinoma and lung adenocarcinoma. Scale bar, 10 µm.

The cancer cell-nuclear targeting process of sulfonic-GQDs is also achieved in a wide range of cancer types, such as hepatoma carcinoma, cervical carcinoma and lung adenocarcinoma (Figure 4e), indicating the ability of sulfonic-GQDs to target a wide range of 12 ACS Paragon Plus Environment

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cancer types. Meanwhile, sulfonic-GQDs show perfect biocompatibility in vivo. There are no changes detected in the histological evaluations of the major organs and serum biochemical analysis results (Figure S8 and Table S1). To our knowledge, this is the first time that nanoparticles have been able to specifically target the cancer cell nuclei in vivo, with little intervention in cells of normal tissues.

High IFP sensitivity of sulfonic-GQDs in vivo. Higher IFP is a common feature of solid tumors and can be as high as 100 mm Hg.38, 39, 48 Based on the results showing that FMST triggered the in vitro transmembrane process, we inferred that high IFP in vivo triggered the transmembrane process and induced the specific tumor nuclear targeting of sulfonic-GQDs. Therefore, we verified whether the in vivo cancer targeting process of sulfonic-GQDs results from the intrinsically high IFP in tumors. Nicotinamide

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lower the IFP in tumor tissues (Figure 5d,e). After the treatments, we found that sulfonicGQDs failed to target the nuclei of 4T1 cells in breast cancer tissues with reduced IFP (6.5 mmHg and 7 mmHg; Figure 5b,c) compared with that of control cancer tissues (Figure 5a). These results provide strong support for our hypothesis that high IPF is the trigger for the specific in vivo targeting behavior of sulfonic-GQDs. The IFP-driven target in solid tumor of mice process is similar to the cell process. Higher IFP will trigger the sulfonic-GQDs enter into the cell membrane, and then target the cell nuclei.



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Figure 5. Verification of the in vivo cancer cell-nuclear targeting process of sulfonic-GQDs via IFP in tumors. (a-c) Confocal images of frozen sections of breast cancer tissues in control (a) and with the IFP in vivo reduced by the treatments with (b) nicotinamide and (c) imatinib, after intravenous injection of sulfonic-GQDs (60 mg/kg) for 0.5 h. Left, sulfonic-GQDs (excited, green); Middle, nucleus of cells stained with SYTO 17 (red); and Right, merged. Scale bars, 10 µm. (d,e) IFP in breast cancer tumors in control, and treated with nicotinamide and imatinib. Mean±SEM; n = 5 per group.

Interface identification of tumor tissue with sulfonic-GQDs. To further confirm the IFPtriggered tumor cell nucleus targeting mechanism, we investigated the interface recognition and sensitivity of the IFP-triggered tumor cell nucleus targeting process of sulfonic-GQDs. Sulfonic-GQDs also exhibited good targeting in an orthotopic liver tumor model and showed high IFP sensitivity. The sulfonic-GQDs were significantly distributed in the orthotopic liver tumor tissues (68%) and had much lower distribution levels in normal tissues after 0.5 h injection (heart, 2%; spleen, 3%; lung, 7%; kidney, 14%; brain, 6%; Figure 6a,b and Figure S8d). From the results shown in Figure 6, we can see that sulfonic-GQDs has very good tumor cell nuclear targeting with very high IFP sensitivity, and the tumor tissue can be recognized very easily in early stage. The IFP of both the tumor and para-carcinoma tissues increased with the tumor volume (Figure S9a-c,e). The sulfonic-GQDs recognized and distinguished the interface of tumor tissue, para-carcinoma tissue, and normal tissue under IFP conditions as low as 7 mmHg in the tumor (36 mm3 of tumor volume), which indicates a 14 ACS Paragon Plus Environment

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very high sensitivity to IFP (Figure 6c). The IFP of normal tissues in normal, orthotopic and subcutaneous models were all below 3 mmHg (Figure S9f). In the ultra-low IFP group (tumor: 3 mmHg, para-carcinoma: 1 mmHg), the IFP was too low to initiate the transmembrane process that would allow the sulfonic-GQDs to target the tumor nuclei. In the low IFP group (tumor: 7 mmHg, para-carcinoma: 3 mmHg), the sulfonic-GQDs displayed a sensitive tumor nuclear targeting performance. When the IFP of the tumor increased to 12 mmHg, the IFP of the para-carcinoma tissue (6 mmHg) also increased. IFP-sensitive nuclear targeting of the sulfonic-GQDs also occurred in the para-carcinoma tissue. Similar results were found in the high IFP group (Figure S9e). However, the nuclear targeting efficient is much lower in para-carcinoma tissue of the high IFP group, with values IFP at 10 mmHg, which were higher than the lower IFP (IFP=7 mmHg) group. The IFP sensitivity in paracarcinoma tissues is much lower than in tumor tissues. The surface charge of sulfonic-GQDs is pH-dependent (Figure S2e) and decreases when the NPs are in the acidic TME, which lowers the repulsive force between the tumor cells and the sulfonic-GQDs.5 This is may be the reason that the tumor tissue nuclear targeting process is more sensitive to the IFP than the para-carcinoma tissue. The nuclear-targeting mechanism we report here shows potential to be a high-efficiency strategy to target tumor cell nuclei with minimal uptake in normal tissue cells, which has multiple applications in cancer therapy and cancer diagnosis. For example, imaging technologies have been developed to exploit the stiffness marker to enhance cancer detection, such as sono-elastography and magnetic resonance imaging elastography.51-53 Sulfonic-GQDs could be used as a fluorescent bio-maker to more easily recognize tumor cells at a very early stage and the interface of tumor and normal tissues without relying on biochemical markers (Figure 4 and 6). This new mechanism of targeted NP design also has promising potential to serve as a guiding principle to design targeted NPs for nanomedicine (Figure S11). Furthermore, the low toxicity both in vitro and in vivo (Figure S5 and S8 and Table S1), low 15 ACS Paragon Plus Environment


cost ($0.185 per gram, Figure S10), and good photo and chemical stability of sulfonic-GQDs, as well as the ability to mass produce the NPs (Figure 1 and Figure S1-2) make the clinical application of sulfonic-GQDs a reality, suggesting a promising future for their integration into tumor diagnosis and therapy. Control 0.5 h

b

96 h 1.5X10 10

Heart Liver+tumor

1.0X10 10

Spleen Lung Kidney

0.5X10 10

3500

Control 0.5 h 96 h

3000 2500 2000 1500 1000 500

Mid IFP

y

Br ai n

Ki dn e

Lu ng

He ar t Li ve r+ tu m or

High IFP

Sp le en

0

Brain

Ultra low IFP

Low IFP

27 mmHg

12 mmHg

7 mmHg

3 mmHg

10 mmHg

6 mmHg

3 mmHg

1 mmHg

Para

Tumor

c

Average signal (Counts)

a

Liver

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Figure 6. Bio-distribution of sulfonic-GQDs and confocal images of frozen sections in an orthotopic tumor model. (a,b) Bio-distribution of sulfonic-GQDs in an orthotopic tumor model 0.5 and 96 h after the intravenous injection of the sulfonic-GQDs. (a) Ex vivo fluorescence images of isolated organs. (b) Average signals of the sulfonic-GQDs from isolated organs. Mean ± SEM; n = 5 per group. (c) Confocal images of frozen orthotopic hepatoma tumor and para-carcinoma sections in the four IFP groups (the mid and low IFP groups) after in situ injection for 0.5 h of sulfonic-GQDs (60 mg/kg).

CONCLUSION In this study, we demonstrated the exceptional ability of sulfonic-GQDs to target the nuclei of cancer cells in vivo, with minimal uptake by cells in normal tissues. The key selective factor for such specific tumor targeting is the high IFP in tumor tissues, which allows the penetration of sulfonic-GQDs into the cell membrane in a variety of cancer types. MD simulations 16 ACS Paragon Plus Environment

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suggest that once transported into the membrane, the negatively charged functional groups of these GQDs will leave the membrane, which allows the GQDs to retain a small enough size to achieve penetration through the nuclear membrane into the nucleus with a self-cleaning function. The specific high IFP in solid tumors is usually regarded as a physiological barrier in cancer treatments, but here, from a new perspective, it has played an important enabling role for the specific nuclear targeting of cancer cells by sulfonic-GQDs. The exceptional ability for specific nuclear targeting of cancer cells in vivo holds enormous promise for tumor diagnosis without specificity of cancer types and for potential new drug- and even genedelivery therapies in clinical medicine. To our knowledge, this is the first time that nanoparticles have been able to exactly target the cancer cell nuclei in vivo, with no intervention in cells of normal tissues.

EXPERIMENTAL SECTION Material synthesis. Sulfonic-GQDs were synthesized via a modified hydrothermal molecular fusion method,37 which we previously used to synthesize single-crystalline GQDs functionalized with NHNH3+ and OH groups. Pyrene (1 g, TCI, purity 98%) was nitrated into 1,3,6- trinitropyrene in hot HNO3 (50 ml) at 80 °C under refluxing and stirring for 24 h. After being cooled to room temperature, the mixture was diluted with deionized (DI) water (250 ml) and filtered through a 0.22-µm membrane to remove the acid. The resultant yellow 1,3,6trinitropyrene was dispersed in an Na2SO3 solution of DI water (100 ml, 0.5 M) under stirring for 0.5 h. The suspension was transferred to a 150 ml ceramic autoclave and heated at 130 °C for 12 h. After being cooled to room temperature, black GQD colloids were obtained without insoluble byproducts. Sulfonic-GQDs were made at 200 °C for 12 h in a polytetrafluoroethylene (Teflon)-lined autoclave (100 ml) filled with a suspension (50 ml) of 1,3,6-trinitropyrene containing 0.5 M Na2SO3 solution. The colloids were dialyzed in a dialysis bag to remove unreacted sodium salt and dried at 80 °C for structural characterization. 17 ACS Paragon Plus Environment


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For cell and animal experiments, the resultant GQDs were used without purification. Material characterization. TEM observations were performed on an aberration-corrected TEM (Titan 80–300, FEI, USA) operating at 80 kV acceleration voltage, and a chargecoupled device camera (2k×2k, UltraScan 1000, Gatan, USA) was used for image recording with an exposure time of 1~2 s. Absorption and fluorescence spectra were recorded at room temperature

on

a

spectrophotometer

(Hitachi

3100,

Japan) and

a

fluorescence

spectrophotometer (Hitachi 7000, Japan), respectively. FT-IR spectra of dried samples were recorded with a Bio-Rad FTIR spectrometer FTS165 (USA). XPS spectra were collected using a Kratos Axis Ultra DLD X-ray photoelectron spectrometer (Japan). XRD patterns were obtained with a Rigaku 18 KW D/max-2550 using Cu Kα radiation. The Zeta potentials of sulfonic-GQDs aqueous solution at various pH values were measured on a Nanosizer (Zetasizer 3000 HS, Malvern, UK). The QYs of dilute sulfonic-GQD aqueous solutions were determined by comparing the integrated PL intensities (excited at 360 nm) and the absorbency values (at 360 nm) using quinine sulfate (QY: 0.55) in sulfuric acid (0.1 mol/l, η = 1.33) as the reference. Cell culture. 4T1 cells (mouse breast cancer cell line), HEPG2 cells (human hepatoma carcinoma cell line), SMMC-7721 cells (human hepatoma carcinoma cell line), HeLa cells (human cervical carcinoma cell line), A549 cells (human lung adenocarcinoma cell line), GES-1 cells (gastric epithelial cell line), and NSCs cells (neural stem cell line) were ordered from the Chinese Academy of Sciences. Roswell Park Memorial Institute-1640 (RPMI 1640) cell culture medium, Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gino Biological Pharmaceutical Technology Co., Ltd. (Hangzhou, China). Cells were maintained in the appropriate culture medium (high-glucose RPMI 1640 for SMMC-7721, DMEM for GES-1, NSCs, HeLa, HEPG2, 4T1 and A549) supplemented with 10% FBS and incubated at 37 °C in a 5% CO2 atmosphere. All cell lines used in this paper were ordered from the Chinese Academy of Sciences and cultured follow the protocol. 18 ACS Paragon Plus Environment

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Nuclear targeting process in vitro. Heating in a water bath and laser irradiation were used to manipulate the stimuli-responsive in vitro nuclear targeting of sulfonic-GQDs using a confocal microscope (Olympus FM 1000, Japan) with 4T1 cells as the primary model. The 4T1 cells were seeded in confocal glass-bottom culture dishes (diameter: 40 mm) and cultured in RPMI 1640 (2 ml per dish) at 37 °C under 5% CO2/95% air for 24 h. To rapidly induce an ultrathin film on the cell monolayers, we used water-bath heating and laser irradiation to drive the nuclear targeting of sulfonic-GQDs. The used culture medium was removed and replaced with fresh culture medium containing sulfonic-GQDs (30 mg/L) at an ultrathin depth (~0.1 mm). Treatment of the cell monolayers incubated with the sulfonic-GQDs was completed using mild heating at 38 °C in a water bath or local heating with argon lasers (405 nm, 100 mW) for 7-15 min. The nuclear targeting process was observed in situ using the fluorescence of the sulfonic-GQDs excited with a 405-nm laser. Nuclear targeting in vivo. Female BALB/c mice and nude female BALB/c mice (5 weeks old) were provided by the Shanghai Slack Experimental Animal Center (Shanghai, China). All animal experiments were performed following “The National Regulation of China for Care and Use of Laboratory Animals” and approved by the Institutional Animal Care and Use Committee of Shanghai University. In the subcutaneous tumor models, breast carcinoma cells (4T1) were implanted in the flank of the mice. In the orthotopic tumor models, hepatoma carcinoma cells (HEPG2) were orthotopically implanted into the livers of nude mice. For each model, sulfonic-GQDs (60 mg/kg) were intravenously injected into experimental tumor-bearing mice via the tail vein. The mice were sacrificed 0.5 h after administration, and the tumors and other normal tissues were then collected. The IFP and tumor volume of each tumor was assessed. For the IFP-sensitivity study, twenty orthotopic tumor models were divided into four IFP groups: the high IFP group (>16 mmHg), mid IFP group (11-15 mmHg), low IFP group (7-10 mmHg) and ultra-low IFP group (3-6 mmHg). After administration, the tumors in the four IFP 19 ACS Paragon Plus Environment


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groups, the liver and other tissues were imaged ex vivo using a Maestro 500FL in vivo imaging system (Cambridge Research &. Instrumentation, Inc., USA). The imaging was performed at an excitation wavelength of 470 nm to excite the fluorescence in the sulfonicGQDs. The fluorescence signal was detected at 480 to 530 nm, and the contribution of autoﬂuorescence in the analyzed images was removed using commercial software (MaestroTM 2.4). To confirm the in vivo nuclear targeting of the sulfonic-GQDs, the frozen tissue slides were prepared and imaged by confocal microscopy. To determine the bio-distribution and metabolic processing of the sulfonic-GQDs in the subcutaneous tumor models, sulfonic-GQDs (60 mg/kg) were intravenously injected into the high IFP group tumor-bearing mice via the tail vein. After administration, the tumors, liver and other tissues were collected at different time intervals (0.5, 1, 2, 8 and 24 h) and imaged ex vivo using a Maestro 500FL in vivo imaging system. For the orthotopic tumor models, sulfonic-GQDs (60 mg/kg) were intravenously injected via the tail vein into the high IFP group of tumor-bearing mice. After administration, the tumors, liver and other tissues were collected at different time intervals (0, 0.5 and 96 h) and imaged ex vivo using a Maestro 500FL in vivo imaging system. In each time point, we plus all the organ signals as total signal. The signal intensity formula is as follow:

For the universal targeting studies, the other three types of subcutaneous solid tumors, hepatoma carcinoma, cervical carcinoma and lung adenocarcinoma, were established in the flanks of mice. When the tumor IFP was well established within the mice (high IFP group), sulfonic-GQDs (60 mg/kg) were intravenously injected into the tumor-bearing mice via the tail vein. After administration, the tumors, liver and other tissues were collected at 0.5 h. Frozen sections were generated from the tumor tissues. Before imaging with a confocal microscope, the cell nuclei were stained with SYTO 17. 20 ACS Paragon Plus Environment

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IFP sensitivity study by nicotinamide and imatinib treatment. Nicotinamide (Sigma, USA) and imatinib (STI571, Gleevec®, Selleck, USA) were dissolved in sterile saline solution (0.9% NaCl) before each experiment. The mid IFP group of subcutaneous tumorbearing mice were used in this experiment. For nicotinamide, the tumor-bearing mice were given i.p. injections at a dose of 500 mg/kg in a volume of 0.01 ml/g of body weight. For imatinib, the tumor-bearing mice were given a dose of 50 mg/kg once a day for 4 days through gavage. After the tumor-bearing mice were treated with nicotinamide or imatinib, the tumor IFP decreased by more than 40%. To detect whether lowering the IFP would prevent the GQDs from targeting the nucleus of the tumor, Sulfonic-GQDs (60 mg/kg) were intravenously injected into tumor-bearing mice (untreated, nicotinamide-treated and imatinib-treated) through the tail vein. The collected tumors with a relatively high GQD enrichment 30 min after administration were made into tissue slices using the methods described above. Before imaging under a confocal microscope, the cell nuclei within the tissue slices were stained ex vivo using a red fluorescence dye of SYTO17 (commonly used to stain the cell nucleus) to compare with the in vivo nuclear staining by GQDs. Fluorescence imaging was performed at 405-nm and 635-nm excitations to excite the fluorescence of the GQDs and SYTO 17, respectively. Molecular dynamics simulations. We performed MD simulations for both types of GQDs that detached from the cancer cell membranes. The molecular model for the sulfonic-GQDs was constructed as a monolayer graphene nanosheet (2.21 nm × 2.41 nm) with edge sites covalently bound to SO3- and OH groups, based on our FT-IR and XPS spectrum data, the averaged lateral size in TEM images and the group contents given by the XPS survey spectrum (Figure 1). Similarly, the model for the amino-GQDs was constructed as a monolayer graphene nanosheet with the same lateral size but with the edge sites covalently bound to NHNH3+ and OH groups. The negatively charged cell membrane that usually occurs 21 ACS Paragon Plus Environment


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in cancer cells 54 was modeled in our simulations as a negatively charged phospholipid bilayer by a mixture of neutral palmitoyloleoylphosphatidylethanolamine (POPE) and negatively charged palmitoyloleoylphosphatidylglycerol (POPG) at a 3:1 ratio. The Berger lipid force field, one of the most commonly used and extensively validated membrane force fields,55 was adopted in our simulations using the GROMACS software package version 4.5.4. The graphene force field parameters were the same as in previous studies

56, 57

with carbon atoms

modeled as uncharged Lennard-Jones particles with a cross-section of σCC = 3.58 Å and a potential well depth of εCC = 0.0663 kcal mol-1. The C-C bond length of 1.42 Å, C-C-C bending angle of 120°, and C-C-C-C planar dihedral angles were maintained by harmonic potentials with spring constants of 322.55 kcal mol-1 Å-2, 53.35 kcal mol-1 rad-2, and 3.15 kcal mol-1, respectively. These settings can generate a model graphene with flexural rigidity (0.194 nN nm) that is reasonably close to the ab initio results (0.238 nN nm) and other model studies (0.11, 0.225 nN nm).58 The parameters of the sulfonate, amine and hydroxyl edge groups were taken from the CHARMM generalized force field for benzenesulfonate, phenylhydrazine and phenol to form stable configurations of C-SO3-, C-NHNH3+ and C-OH at the graphene edges, respectively, except for the charges of C-NHNH3+, which were estimated using geometric optimizations at the B3LYP/6-31G (d) level

59

in the Gaussian-09 package [i.e., the charge

distribution of C-NHNH3+: 0.44 (C), -0.54 (N), 0.365 (H in NH), and 0.425 (H in NH3)]. Water was represented by the simple point-charge SPC/E model. We applied periodic boundary conditions in all directions. The NPT ensemble was used, with the temperature controlled at 310 K by a velocity-rescale thermostat with a coupling coefficient of τT = 0.5 ps, and the pressure at 1 bar by the Berendsen barostat with a coupling coefficient of τP = 1 ps. The particle-mesh Ewald method was used to calculate long-range electrostatic interactions, whereas the vdW interactions were treated with a smooth cutoff at a distance of 12 Å. A time step of 2.0 fs was used, and the data were collected every 1 ps. After 40 ns of initial equilibration of the solvated lipid systems, GQDs were introduced into the system by 22 ACS Paragon Plus Environment

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replacing overlapping water molecules at a vertical distance of ~3 nm away from the membrane and pulling them into the membrane with force applied to the corner atoms of the GQDs with a constant velocity of 10-3 nm ps-1 to its center of mass. Position restraints were then applied between the centers of mass of the GQDs and the membrane for 20 ns to allow the GQDs to adjust their initial postures before production runs. The achieved GQDs-inserted states mimicked the states of the former-half GQDs transmembrane movement initiated by the high IFP. The position restraints were released, and the MD simulations were allowed to run to investigate how both types of GQDs exit the membranes. Interaction between histones and sulfonic-GQDs. First, histones (Sigma, USA) were dissolved in water to form a homogeneous solution; the solution was then centrifuged at 20,000 rpm for 20 min, and the supernatant was collected for use. Next, the histone solution was mixed with sulfonic-GQDs to yield a final concentration of histones of 0.5 mg/mL or 2 mg/mL and GQDs at 150 mg/L. After co-incubation at 37 °C in PBS at pH 7.4 for different times (10 min, 30 min or 2 h), each sample was centrifuged at 20,000 rpm for 20 min to precipitate the adsorbed protein aggregates.1 Finally, the aggregates were redissolved, and the histone concentration was characterized using a Bradford assay (Beyotime Laboratories, China) according to the manufacturer’s instructions. IFP measurements. Tumor IFP was measured using the wick-in-needle technique according to previously described methods.60 Briefly, a 2–3-mm side hole was drilled 5 mm from the tip of a 23-gauge hypodermic needle. Five 6-0 Ethilon surgical sutures were threaded through the needle. Then, the needle, filled with nylon floss and saline supplemented with 50 IE/ml of heparin, was inserted into the center of the subcutaneous tumor and connected to a pressure transducer (Alcott Biotech Co. Ltd. Shanghai, China). This setup enabled continuous and stable recording of fluid pressure. Fluid communication between the needle and the transducer was confirmed by compression and decompression of the tubing during each measurement. Tumor IFP was determined by calculating the mean of the two readings. 23 ACS Paragon Plus Environment


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Interstitial fluid pressure values in muscle and subcutaneous tissue served as controls. For the orthotopic tumor models, the IFP of the tumor, para-tumor and normal tissues were determined by this method. Additionally, for the human hepatoma tumor models, the IFP of the tumor, para-tumor and liver tissue were also determined using this method. Toxicity evaluation in vitro and in vivo. The cytotoxicity of sulfonic-GQDs was evaluated using the 4T1 cell line. Cell viability was expressed as the percentage of viable cells in the total number of cells. The 4T1 cells were seeded in 96-well plates (5 × 103 cells per well) and incubated in RPMI 1640 culture medium containing different concentrations of sulfonicGQDs for 12 or 24 h. The viability of the cells in each well was measured with a CCK-8 Kit (Dojindo Laboratories, Japan) according to the manufacturer’s instructions. We also evaluated the cytotoxicity of sulfonic-GQDs after driving nuclear targeting using mild heating and laser treatment. The 4T1 cells were co-incubated with 30 mg/L of sulfonic-GQDs in an ultrathin medium (~0.1 mm deep), followed by a heat treatment for 15 min and incubation at 37 °C for an additional 1 and 24 h before detection. For the laser treatment, 4T1 cells were co-incubated with 30 mg/L of sulfonic-GQDs, followed by 15 min of laser heating before analysis. For the in vivo toxicity evaluation, accumulation determination and toxicological assays, blood samples and major tissues were collected from mice treated with the sulfonic-GQDs (60 mg/kg and 300 mg/kg). Serum was collected by centrifuging the blood at 3,000 rpm for 10 min. Liver function was evaluated by assessing the levels of total bilirubin (TBIL), alanine aminotransferase (ALT; in ESI), aspartate aminotransferase (AST; in ESI), and alkaline phosphatase (ALP) in the serum. Blood urea nitrogen (BUN) and serum creatinine (Cr; in ESI) were evaluated to determine nephrotoxicity. All the biochemical parameters were determined according to the methods described in the references using commercial kits (Nanjing Jiancheng Bioeng Inst., China). All histological tests were performed using standard laboratory procedures. The tissues were fixed with 4% (v/v) formalin and then embedded in a paraffin block, sliced into 5-µm thicknesses and placed onto glass slides. After staining with 24 ACS Paragon Plus Environment

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hematoxylin-eosin (HE), the slides were examined by a pathologist using an optical microscope (Nikon U-III Multi-points Sensor System, USA).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. Additional figures of the characterization of sulfonic-GQDs, in vitro nuclear targeting of sulfonic-GQDs, cytotoxicity evaluation, transmembrane transport of amino-GQDs, biodistribution of sulfonic-GQDs, histology and serum biochemistry study to measure toxicity. (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *Email: [email protected] *Email: [email protected]

Author Contributions Y.L.W and M.H.W conceived and designed all the experiments. Y.L.W, C.J.Y, L. D, C. C. L, J. W. did the cell and animal experiments and imaging. K.K. Zhang, Y. N. H performed the material synthesis and characterization. Y.S. T and H.P. F performed MD; Y. L.W, Q. L, and 25 ACS Paragon Plus Environment


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M.H.W wrote the paper. All authors discussed the results and analyzed the data. +These authors contributed equally. Notes The authors declare that there are no competing financial interests. Readers are welcome to comment on the online version of the paper. ACKNOWLEDGMENT We thank Prof. Evelyn Hu from Harvard School of Applied Science and Electrical Engineering, Prof. Herman Suit from Massachusetts General Hospital and Prof. D.Y. Pan from Shanghai University for discussion. This work was supported by National Natural Science Foundation of China (Nos. 11575107, 21371115, 11025526, 21101104, 11422542, 21471097), the Innovation Program of Shanghai Municipal Education Commission (No. 13YZ017), Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13078).

REFERENCES (1) Zhang, X., Wang, H., Wang, H., Zhang, Q., Xie, J., Tian, Y., Wang, J., and Xie, Y. (2014) Single-layered graphitic-C(3)N(4) quantum dots for two-photon fluorescence imaging of cellular nucleus. Adv. Mater. 26, 4438-43. (2) Kanapathipillai, M., Brock, A., and Ingber, D. E. (2014) Nanoparticle targeting of anti-cancer drugs that alter intracellular signaling or influence the tumor microenvironment. Adv. Drug Deliv. Rev. 79-80, 107-118. (3) Stylianopoulos, T., and Jain, R. K. (2015) Design considerations for nanotherapeutics in oncology. Nanomed.-Nanotechnol. Biol. Med. 11, 1893-1907. (4) Cheng, Z. L., Al Zaki, A., Hui, J. Z., Muzykantov, V. R., and Tsourkas, A. (2012) Multifunctional nanoparticles: Cost versus benefit of adding targeting and imaging capabilities. Science 338, 903-910. (5) Li, C. (2014) A targeted approach to cancer imaging and therapy. Nat. Mater. 13, 110115. (6) Leserman, L. D., Barbet, J., Kourilsky, F., and Weinstein, J. N. (1980) Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A. Nature 288, 602-4.


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Page 27 of 31

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


(7) Heath, T. D., Fraley, R. T., and Papahdjopoulos, D. (1980) Antibody targeting of liposomes: Cell specificity obtained by conjugation of F(ab')2 to vesicle surface. Science 210, 539-41. (8) Hood, J. D., Bednarski, M., Frausto, R., Guccione, S., Reisfeld, R. A., Xiang, R., and Cheresh, D. A. (2002) Tumor regression by targeted gene delivery to the neovasculature. Science 296, 2404-2407. (9) Farokhzad, O. C., Jon, S., Khademhosseini, A., Tran, T. N. T., Lavan, D. A., and Langer, R. (2004) Nanopartide-aptamer bioconjugates: A new approach for targeting prostate cancer cells. Cancer Res. 64, 7668-72. (10) Weissleder, R., Kelly, K., Sun, E. Y., Shtatland, T., and Josephson, L. (2005) Cellspecific targeting of nanoparticles by multivalent attachment of small molecules. Nat. Biotechnol. 23, 1418-1423. (11) Geng, Y., Dalhaimer, P., Cai, S. S., Tsai, R., Tewari, M., Minko, T., and Discher, D. E. (2007) Shape effects of filaments versus spherical particles in flow and drug delivery. Nat. Nanotechnol. 2, 249-255. (12) Hu, C. M. J., Fang, R. H., Wang, K. C., Luk, B. T., Thamphiwatana, S., Dehaini, D., Nguyen, P., Angsantikul, P., Wen, C. H., Kroll, A. V., et al. (2015) Nanoparticle biointerfacing by platelet membrane cloaking. Nature 526, 118-121. (13) Petros, R. A., and DeSimone, J. M. (2010) Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discov. 9, 615-627. (14) Salvati, A., Pitek, A. S., Monopoli, M. P., Prapainop, K., Bombelli, F. B., Hristov, D. R., Kelly, P. M., Aberg, C., Mahon, E., and Dawson, K. A. (2013) Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nat. Nanotechnol. 8, 137-143. (15) Wu, X. Y., Liu, H. J., Liu, J. Q., Haley, K. N., Treadway, J. A., Larson, J. P., Ge, N. F., Peale, F., and Bruchez, M. P. (2003) Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat. Biotechnol. 21, 41-46. (16) Pouton, C. W., and Seymour, L. W. (1998) Key issues in non-viral gene delivery. Adv. Drug Deliv. Rev. 34, 3-19. (17) Danhier, F., Feron, O., and Preat, V. (2010) To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J. Control. Release 148, 135-146. (18) Tong, R., and Langer, R. (2015) Nanomedicines Targeting the Tumor Microenvironment. Cancer J. 21, 314-321. (19) Martin, J. D., Fukumura, D., Duda, D. G., Boucher, Y., and Jain, R. K. (2016) Reengineering the tumor microenvironment to alleviate hypoxia and overcome cancer heterogeneity. Cold Spring Harb. Perspect. Med. 6. (20) Lu, Y., Aimetti, A. A., Langer, R., and Gu, Z. (2016) Bioresponsive materials. Nat. Rev. Mater. 1, 16075. (21) Bourzac, K. (2016) Cancer nanomedicine, reengineered After recent setbacks, researchers hope to find approaches more attuned to the complexities of cancer biology. Proc. Natl. Acad. Sci. USA 113, 12600-12603. (22) Pan, D. Y., Zhang, J. C., Li, Z., and Wu, M. H. (2010) Hydrothermal route for cutting graphene sheets into blue-luminescent graphene quantum dots. Adv. Mater. 22, 734-738. (23) Peng, J., Gao, W., Gupta, B. K., Liu, Z., Romero-Aburto, R., Ge, L. H., Song, L., Alemany, L. B., Zhan, X. B., Gao, G. H., et al. (2012) Graphene quantum dots derived from carbon fibers. Nano Lett. 12, 844-849. (24) Shen, J. H., Zhu, Y. H., Yang, X. L., and Li, C. Z. (2012) Graphene quantum dots: emergent nanolights for bioimaging, sensors, catalysis and photovoltaic devices. Chem. Commun. 48, 3686-3699. 27 ACS Paragon Plus Environment


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

(25) Ritter, K. A., and Lyding, J. W. (2009) The influence of edge structure on the electronic properties of graphene quantum dots and nanoribbons. Nat. Mater. 8, 235-242. (26) Li, Y., Hu, Y., Zhao, Y., Shi, G. Q., Deng, L. E., Hou, Y. B., and Qu, L. T. (2011) An electrochemical avenue to green-luminescent graphene quantum dots as potential electronacceptors for photovoltaics. Adv. Mater. 23, 776-780. (27) Shen, H., Zhang, L. M., Liu, M., and Zhang, Z. J. (2012) Biomedical applications of graphene. Theranostics 2, 283-294. (28) Feng, L. Z., and Liu, Z. A. (2011) Graphene in biomedicine: Opportunities and challenges. Nanomedicine 6, 317-324. (29) Liu, Z. G., Xiao, J. C., Wu, X. W., Lin, L. Q., Weng, S. H., Chen, M., Cai, X. H., and Lin, X. H. (2016) Switch-on fluorescent strategy based on N and S co-doped graphene quantum dots (N-S/GQDs) for monitoring pyrophosphate ions in synovial fluid of arthritis patients. Sens. Actuator B-Chem. 229, 217-224. (30) Bhatnagar, D., Kumar, V., Kumar, A., and Kaur, I. (2016) Graphene quantum dots FRET based sensor for early detection of heart attack in human. Biosens. Bioelectron. 79, 495-499. (31) Li, N., Than, A., Wang, X. W., Xu, S. H., Sun, L., Duan, H. W., Xu, C. J., and Chen, P. (2016) Ultrasensitive profiling of metabolites using tyramine-functionalized graphene quantum dots. ACS Nano 10, 3622-3629. (32) Nafiujjaman, M., Revuri, V., Park, H. K., Kwon, I. K., Cho, K. J., and Lee, Y. K. (2016) Enhanced photodynamic properties of graphene quantum dot conjugated Ce6 nanoparticles for targeted cancer therapy and imaging. Chem. Lett. 45, 997-999. (33) Jovanovic, S. P., Syrgiannis, Z., Markovic, Z. M., Bonasera, A., Kepic, D. P., Budimir, M. D., Milivojevic, D. D., Spasojevic, V. D., Dramicanin, M. D., Pavlovic, V. B., et al. (2015) Modification of structural and luminescence properties of graphene quantum dots by gamma irradiation and their application in a photodynamic therapy. ACS Appl. Mater. Interfaces 7, 25865-25874. (34) Li, S. H., Li, Y. C., Cao, J., Zhu, J., Fan, L. Z., and Li, X. H. (2014) Sulfur-doped graphene quantum dots as a novel fluorescent probe for highly selective and sensitive detection of Fe3+. Anal. Chem. 86, 10201-10207. (35) Dong, H. F., Dai, W. H., Ju, H. X., Lu, H. T., Wang, S. Y., Xu, L. P., Zhou, S. F., Zhang, Y., and Zhang, X. J. (2015) Multifunctional poly(L-lactide)-polyethylene glycolgrafted graphene quantum dots for intracellular microRNA imaging and combined specificgene-targeting agents delivery for improved therapeutics. ACS Appl. Mater. Interfaces 7, 11015-11023. (36) Qiu, J. C., Li, D. S., Mou, X. N., Li, J. H., Guo, W. B., Wang, S., Yu, X., Ma, B. J., Zhang, S., Tang, W., et al. (2016) Effects of graphene quantum dots on the self-renewal and differentiation of mesenchymal stem cells. Adv. Healthc. Mater. 5, 702-710. (37) Wang, L., Wang, Y. L., Xu, T., Liao, H. B., Yao, C. J., Liu, Y., Li, Z., Chen, Z. W., Pan, D. Y., Sun, L. T., et al. (2014) Gram-scale synthesis of single-crystalline graphene quantum dots with superior optical properties. Nat. Commun. 5, 9. (38) Cairns, R., Papandreou, I., and Denko, N. (2006) Overcoming physiologic barriers to cancer treatment by molecularly targeting the tumor microenvironment. Mol. Cancer Res. 4, 61-70. (39) Heldin, C. H., Rubin, K., Pietras, K., and Ostman, A. (2004) High interstitial fluid pressure - An obstacle in cancer therapy. Nat. Rev. Cancer 4, 806-813. (40) Zhu, S. J., Zhang, J. H., Qiao, C. Y., Tang, S. J., Li, Y. F., Yuan, W. J., Li, B., Tian, L., Liu, F., Hu, R., et al. (2011) Strongly green-photoluminescent graphene quantum dots for bioimaging applications. Chem. Commun. 47, 6858-6860.


Page 28 of 31

Page 29 of 31

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


(41) Li, L. L., Wu, G. H., Yang, G. H., Peng, J., Zhao, J. W., and Zhu, J. J. (2013) Focusing on luminescent graphene quantum dots: Current status and future perspectives. Nanoscale 5, 4015-4039. (42) Zhu, S. J., Zhang, J. H., Tang, S. J., Qiao, C. Y., Wang, L., Wang, H. Y., Liu, X., Li, B., Li, Y. F., Yu, W. L., et al. (2012) Surface chemistry routes to modulate the photoluminescence of graphene quantum dots: From fluorescence mechanism to upconversion bioimaging applications. Adv. Funct. Mater. 22, 4732-4740. (43) Jin, G., Jiang, L. M., Yi, D. M., Sun, H. Z., and Sun, H. C. (2015) The influence of surface modification on the photoluminescence of CdTe quantum dots: Realization of bioimaging via cost-effective polymer. Chem. Phys. Chem. 16, 3687-3694. (44) Lee, B. T., Kim, H. A., Williamson, J. L., and Ranville, J. F. (2016) Bioaccumulation and in-vivo dissolution of CdSe/ZnS with three different surface coatings by Daphnia magna. Chemosphere 143, 115-122. (45) Shintomi, K., Iwabuchi, M., Saeki, H., Ura, K., Kishimoto, T., and Ohsumi, K. (2005) Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs. Proc. Natl. Acad. Sci. USA 102, 8210-5. (46) Mottet, D., and Castronovo, V. (2008) Histone deacetylases: target enzymes for cancer therapy. Clin. Exp. Metastas. 25, 183-9. (47) Tu, Y. S., Lv, M., Xiu, P., Huynh, T., Zhang, M., Castelli, M., Liu, Z. R., Huang, Q., Fan, C. H., Fang, H. P., et al. (2013) Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets. Nat. Nanotechnol. 8, 594-601. (48) Gutmann, R., Leunig, M., Feyh, J., Goetz, A. E., Messmer, K., Kastenbauer, E., and Jain, R. K. (1992) Interstitial hypertension in head and neck tumors in patients: correlation with tumor size. Cancer Res. 52, 1993-1995. (49) Lee, I., Boucher, Y., and Jain, R. K. (1992) Nicotinamide can lower tumor interstitial fluid pressure: mechanistic and therapeutic implications. Cancer Res. 52, 3237-3240. (50) Vlahovic, G., Rabbani, Z. N., Herndon, J. E., II, Dewhirst, M. W., and Vujaskovic, Z. (2006) Treatment with Imatinib in NSCLC is associated with decrease of phosphorylated PDGFR-[beta] and VEGF expression, decrease in interstitial fluid pressure and improvement of oxygenation. Br. J. Cancer 95, 1013-1019. (51) Butcher, D. T., Alliston, T., and Weaver, V. M. (2009) A tense situation: Forcing tumour progression. Nat. Rev. Cancer 9, 108-122. (52) Glaser, K. J., Felmlee, J. P., Manduca, A., Mariappan, Y. K., and Ehman, R. L. (2006) Stiffness-weighted magnetic resonance imaging. Magn. Reson. Med. 55, 59-67. (53) Garra, B. S. (2007) Imaging and estimation of tissue elasticity by ultrasound. Ultrasound Q. 23, 255-68. (54) Lin, J. Q., Zhang, H. W., Chen, Z., and Zheng, Y. G. (2010) Penetration of lipid membranes by gold nanoparticles: Insights into cellular uptake, cytotoxicity, and their relationship. ACS Nano 4, 5421-5429. (55) Benz, R. W., Castro-Roman, F., Tobias, D. J., and White, S. H. (2005) Experimental validation of molecular dynamics simulations of lipid bilayers: A new approach. Biophys. J. 88, 805-817. (56) Patra, N., Wang, B. Y., and Kral, P. (2009) Nanodroplet activated and guided folding of graphene nanostructures. Nano Lett. 9, 3766-3771. (57) Titov, A. V., Kral, P., and Pearson, R. (2010) Sandwiched graphene-membrane superstructures. ACS Nano 4, 229-234. (58) Lee, C., Yang, W., and Parr, R. G. (1988) Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 37, 785-789. (59) Becke, A. D. (1988) Density-functional exchange-energy approximation with correct asymptotic behavior. Phys. Rev. A, Gen. Phys. 38, 3098-3100. 29 ACS Paragon Plus Environment


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

(60) Fadnes, H. O., Reed, R. K., and Aukland, K. (1977) Interstitial fluid pressure in rats measured with a modified wick technique. Microvasc. Res. 14, 27-36.


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Graphical Table of Contents. T

Tumor cells

Orthotopic tumor model

Targeting to the tumor cell nuclei

Subcutaneous tumor model

Out of the normal tissue cells

Normal tissue cells

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Sulfonic-GQDs

In this study, we demonstrate the exceptional in vivo cancer cell nuclei targeting capability of sulfonic-GQDs with minimal uptake by cells in normal tissues. The key selective factor for such specific tumor targeting is the high interstitial fluid pressure in tumor tissues.


Tumor Cell-Specific Nuclear Targeting of Functionalized Graphene

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