Stability, Cellular Uptake, and in Vivo Tracking of Zwitterion Modified

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Stability, Cellular Uptake and in Vivo Tracking of Zwitterion Modified Graphene Oxide as Drug Carrier Jing Zhang, Liqun Chen, Jiada Chen, Quan Zhang, and Jie Feng Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01995 • Publication Date (Web): 08 Aug 2018 Downloaded from http://pubs.acs.org on August 15, 2018

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Stability, Cellular Uptake and in Vivo Tracking of Zwitterion Modified Graphene Oxide as Drug Carrier Jing Zhanga,*·Liqun Chena ·Jiada Chena·Quan Zhangb·Jie Fenga,* a

College of Materials Science & Engineering, Zhejiang University of Technology,

Hangzhou, Zhejiang 310014, China b

Key Laboratory of Microbial Technology for Industrial Pollution control of Zhejiang

Providence, College of Environment, Zhejiang University of Technology, Hangzhou, Zhejiang 310012, China *Corresponding authors at: College of Materials Science & Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China. E-mail addresses: [email protected] (J. Zhang), [email protected] (J. Feng).

ABSTRACT: In this paper, a novel kind of zwitterion modified graphene oxide (GO) for promoting stability and reducing aggregation of GO as drug carrier was proposed and demonstrated. Specifically, the GO was functionalized with a kind of zwitterion based silane, 3-(dimethyl(3-(trimethoxysilyl)propyl)-ammonio)propane-1-sulfonate (SBS). After zwitterion modification, the SBS functionalized GO (GO-SB) shows significantly enhanced stability in both serum-free and serum-containing solution, especially after loading doxorubicin hydrochloride (DOX). According to drug release profiles, the drug-loaded GO-SB exhibits thermosensitive and sustained release behavior. Meanwhile, in vitro studies show that the DOX loaded GO-SB could be easily internalized by HepG2 cells and exhibit obvious cytotoxicity on the cells. And in vivo studies demonstrate that the GO-SB drug carrier is capable of being taken by the larvae of zebrafish and can be eliminated from the body within several days. KEYWORDS: graphene oxide, zwitterion, drug carrier, stability, cellular uptake, in vivo tracking

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INTRODUCTION Graphene, a two-dimensional highly ordered lattice of sp2 hybridized carbon atoms, has attracted great interest of researchers since it has been reported in 2004 firstly.1 Because of its prominent mechanical, electronic and optical properties,2-4 it has been widely used in various fields. Unfortunately, the hydrophobic property of pure graphene and its tendency to agglomerate due to Vander Waals interaction and strong π-π stacking between the nanosheets hinder application of graphene in biological field, such as application in loading antineoplastic drugs and genes. As a graphene derivative, graphene oxide (GO) also has large two-dimensional plane which provides ultra-high specific surface area to load drugs through surface adsorption, π-π interaction, hydrogen bonding and so on. 5

Furthermore, it has been well documented that GO is biocompatible and

nontoxic, making GO a promising candidate for construction of drug carrier.6-10 GO can yield stable suspensions in water initially after being exfoliated into monolayer sheets, but it suffers from aggregation with time. And GO may aggregate immediately in bio-medium, especially after loading with anticancer drugs ， which would strongly limit exploration of GO based material in biological environment. Therefore, GO may need to be properly functionalized. Fortunately, hydrophilic oxygenated functional moieties on basal planes and edges of GO11, 12 not only provide it with water solubility, but also enable GO to be further functionalized.13, 14 In the past decade, various hydrophilic polymers have been utilized to modify GO to promote its stability and reduce its aggregation, including natural and synthetic ones. For example, some research groups have used dextran, cyclodextrin,15-17 sodium alginate (SA),18,

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chitosan,20,

21

albumin,22 R9

peptide23 and PEG24 to modify GO. The results have showed that these methods could enhance the solubility of GO based materials in water. But long-term stability of the GO carriers in bio-media or after drug loading has rarely been investigated

in

the

above

works.

Liu

and

co-workers

reported

naphthalene-terminated PEG (NP) modified GO nanosystem.25 In their study, they found PEG could improve stability of the GO nanosystem in serum-free and serum-containing mediums. But PEGylated materials are found out to 2


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generate anti-PEG antibodies when used in vivo,26, 27 which causes worry about future of the PEGylated therapeutics. To overcome deficiency of PEG, zwitterionic materials emerged as a class of biomaterials with super-hydrophilic characteristics, mediated by electrostatic interactions between zwitterions and surrounding water molecules.28, 29 Zwitterionic materials bear simultaneously a pair of oppositely charged ions in the same moiety while maintain overall neutral charge. Here, we tried to use zwitterion to modify GO in order to prolong the stability of GO based drug carrier. As shown in Figure 1, we synthesized a kind of zwitterion based silane and anchored the zwitterionic ligand on planes and edges of the GO. The results demonstrated that zwitterion coated on the GO greatly enhanced stability of the GO in serum-containing solution even after drug loading, and zwitterion modified GO exhibited favourable biocompatibility both in vitro and in vivo. We hope that the zwitterion modified GO would be applicable for most hydrophobic aromatic drugs other than anticancer drugs and thereby promote development of graphene based nanomaterial in biomedical application.

Figure 1. (A) Synthesis of the DOX@GO-SB and GO-SB-FITC, (B) In vitro cellular uptake of the DOX@GO-SB complex, (C) In vivo tracking of the GO-SB-FITC in zebrafish embryos and larvae.

MATERIALS AND METHODS Materials.

3


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Doxorubicin hydrochloride (DOX) was brought from Beijing HVSF united chemical materials Co., LTD. Fluorescein isothiocyanate (FITC) was obtained from Sigma-Aldrich. The embryos were purchased from Zhejiang University. Sample Preparation Synthesis of the GO, GO-SB, DOX@GO-SB, GO-SB-FITC. The details were described in the supporting information. In Vitro Drug Release from the DOX@GO-SB 6 mL of the above DOX@GO-SB solution was divided into three parts equally, and was transferred to three dialysis tubes (MWCO: 8000-14000 Da), respectively. After that, the tubes were immersed into 10 mL of 0.1 M PBS solution (pH=7.4) and incubated at 37 oC, 40 oC and 45 oC, respectively. At predetermined time intervals, 2 mL of dialysate was withdraw from the solution, and another 2 mL of fresh PBS solution was added to keep the volume invariably. The accumulative drug released was determined by fluorescence spectroscopy (FL-4600, Hitachi) with an excitation wavelength of 470 nm, and a slit width of 5 nm. Stability Tests of the GO, GO-SB, DOX@GO and DOX@GO-SB The concentrations of all the samples were determined by fluorescence spectroscopy, and all of them were diluted to 1 mg/mL. For each sample, 4 mL of solution was transferred into a vial and subjected to ultrasonic oscillation for 10 min. Meanwhile, equivalent volume of DI water or 20% fetal bovine serum (FBS) was added to the vial, respectively. Thus, final concentration of all the samples was 0.5 mg/mL. Results about stability of these samples were recorded by digital camera. In Vitro Cellular Internalization of the DOX@GO-SB To check cellular uptake of the DOX@GO-SB, HepG2 cells were seeded in a 24-well plate (8000 cells per well) and incubated with 1 mL of DMEM containing 10% FBS for 24 h (37

o

C, 5% CO2). Subsequently, the

DOX@GO-SB was added into culture medium at a concentration of 50 µg/mL. 4


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The cells were co-incubated with the DOX@GO-SB for 30 min, 1 h and 2 h respectively. Then, after the culture medium was removed, the cells were washed with PBS for several times and observed by fluorescent inverted microscope (Axio Observer A1, Carl Zeiss). In Vitro Cytotoxicity Study In vitro cytotoxicity of the GO-SB, DOX@GO-SB and free DOX were measured by studying viabilities of HepG2 cells with MTT assay.30 In brief, the cells were seeded in 96-well plates with a density of 5000 cells each well and incubated with 100 µL of DMEM containing 10% FBS for 24 h (37 oC, 5% CO2). After that, 100 µL of culture medium with various concentrations of the samples were added separately. The cells were incubated for another 48 h. Then, all the culture mediums were refreshed, followed by the addition of 20 µL of MTT (5 mg/mL) in each well. And the cells were further incubated for 4 h. Finally, culture medium in each well was replaced by 150 µL of DMSO. The optical density (OD) was determined by microplate reader (DG5033A, Huadong electronics) at 490 nm. The relative cell viability was calculated by following formula:

where ODsample was obtained in presence of the samples, ODcontrol was obtained in absence of the samples and ODblank was obtained in blank well. All the samples were sterilized previously. In Vivo Tracking and Biosafety Evaluation of the GO-SB-FITC in Zebrafish Embryos and Larvae Fertilized eggs were obtained from natural mating of adult zebrafish (Danio rerio AB line) bred at Animal Center of Zhejiang University. Embryos were collected within 2 h after spawning. Newly fertilized eggs, approximately at stage of 12 cells, were exposed to the GO-SB-FITC (100 µg/mL) for 5 days. One egg was placed in each well of a 96-well plate. During the exposure, 50% of solution in each well was displaced with fresh GO-SB-FITC solution every day. After 5 days of static exposure, the larvae or embryos were washed with water and incubated in aerating water for further observation, meanwhile, 90% 5


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of the water was replaced daily. The embryos and larvae were incubated at approximately 27 oC for 14 h under daylight and 10 h under dark each day. Each experiment was repeated three times. At the same time, embryos and larvae incubated in aerating water were used as control. FITC as a fluorescent tracer, which helps us understand the distribution and metabolism of the GO-SB-FITC in the body.31 All observations were done by Axio Observer A1 fluorescent inverted microscope. Ethics Statement on the Use of Animals The research animals are provided with the best possible care and treatment and are under the care of a specialized technician. All procedures were approved by Zhejiang University of Technology and were conducted in accord with the Guiding Principles for the Care and Use of Laboratory Animals. RESULTS AND DISCUSSION In this work, a kind of zwitterion based silane, SBS is used to modify GO to improve its stability. First, GO was synthesized via modified Hummer method and characterized by XRD. As shown in Figure S1, diffraction peak at about 2θ = 26o is graphite’s characteristic d002 diffraction peak of graphite. After graphite is oxidized, the peak disappears completely whereas a new, sharp diffraction peak appears at 2θ = 8.5o, confirming that GO is prepared successfully. In fact, diffraction peak of GO is usually near 2θ = 10o. 32 The difference between them may result from preparation process. During washing process of the GO, water molecules penetrate into and thereby increase space between the GO sheets via ultrasound. And structure of the GO is fixed by freeze-drying which leads to the left shifting of diffraction peak of the GO. Afterwards, the GO was reacted with SBS to obtain GO-SB. Zeta potential of the materials was characterized. Compared with zeta potential of graphite (-18.3 mV), the one of the GO decreases a lot to -33.64 mV (Table S1). During oxidation process, a large amount of hydroxyl and carboxyl groups are generated on planes and edges of the GO, resulting in a significant decrease of its zeta potential.33 Meanwhile, it should be note that these hydrophilic oxygenated groups are basis for further function of the GO. After the GO is modified with SBS, its zeta potential changes to -23.28 mV. Considering that the SBS is neutral charged, zeta potential of the resulted GO-SB increases after part of negative charged hydroxyl groups on the GO are reacted with 6


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the SBS. This suggests that modification of the GO is successful. Amount of zwitterion ligand in the GO-SB was determined by element analysis. Results are exhibited in Table S2. The amount of SB in the GO-SB is calculated via following formulas: or Where

represents weight percentage of SB in the GO-SB, while

represent weight percentage of N and S in the GO-SB. And

,

and and

represent molar mass of SB, N and S. The results calculated by the above two formulas are 67.3% and 67.5% respectively, which strongly prove that the zwitterion ligand is modified on the GO successfully. Then we utilized high resolution transmission electron microscopy (HRTEM) and atomic force microscopy (AFM) to study morphology of the GO-SB. As shown in Figure 2A, we find that the GO-SB is almost transparent when observed by HRTEM, implying that the GO-SB nanosheets are extremely thin. Their thickness was about 1.0 nm measured by AFM (Figure 2B and Figure 2C). As reported, thickness of monolayer graphene is approximately 0.335 nm.1 This indicates that the GO-SB prepared in this study contains 2-3 layers.

Figure 2. HRTEM image (A) and AFM images (B, C) of the GO-SB.

Meanwhile, all functionalization steps were monitored by FTIR to verify their success. As presented in Figure 3A, a sharp band at 1730 cm-1 corresponding to stretching vibrations of C=O in carboxyl groups indicates that graphite is oxidized successfully. After modification of the GO with the SBS, a new absorption band at 1037 cm-1 attributed to stretching vibrations of Si-O appeared (Figure 3B), confirming that the zwitterion based silane is connected to the GO sheet. Then, a kind of aromatic drug, DOX, which is often used for cancer treatment, was loaded on the GO-SB though π-π interaction to obtain 7


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DOX@GO-SB. According to spectrum of the DOX@GO-SB (Figure 3C), absorption peaks at 1211 cm-1 and 1281 cm-1 prove existence of the DOX on the GO-SB.

Figure 3. FTIR spectra of the GO (A), GO-SB (B) and DOX@GO-SB (C).

Since it has been reported that graphene has efficient fluorescence quenching effect,34 thus to further investigate preparation of the DOX@GO-SB complexes, we tried to measure fluorescence of the DOX@GO-SB complexes and free DOX of corresponding amount loaded on the DOX@GO-SB. Firstly, amount of the DOX in the DOX@GO-SB complexes was calculated. Results show that DLE of the DOX@GO-SB is about 23.8%. Then, we prepared solution with equivalent amount of free DOX as control. As shown in Figure S2, fluorescence intensity of the DOX@GO-SB complexes decreases about 59.3% compared with that of free DOX. It further confirms that the DOX has been successfully attached onto surface of the GO-SB. Afterwards, size distribution of the GO, GO-SB and DOX@GO-SB were characterized by dynamic light scattering (DLS, Zetasizer Nano ZS90) and results are displayed in Figure S3. As shown, average hydrated sizes of the GO, GO-SB and DOX@GO-SB are 190.1 nm, 164.2 nm and 220.0 nm, respectively. Correspondingly, the PDI of GO, GO-SB, and DOX@GO-SB are 0.387, 0.365, and 0.475, respectively. According to the results, we find that after the GO was modified with zwitterion ligand, its hydrated particle size is slightly reduced. This indicates that dispersion of the GO in water is improved after modifying of GO with zwitterion. And it should be noted that because of 8


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the zwitterion coating, a hydration layer is formed around the GO-SB. And this may cause the obtained hydrated size of the GO-SB given by DLS to become larger than its actual size. In addition, we find that size of the DOX@GO-SB complexes increases a bit after the DOX is loaded. To some extent, the DOX plays a role of bridge and narrowed distance between the nanosheets. Consequently, the nanosheets are easy to aggregate, resulting in increase of average size of the DOX@GO-SB complexes.

Figure 4. Digital photos of dispersion of the GO, GO-SB, DOX@GO and DOX@GO-SB in water and 10% FBS solution.

Then, to study effect of the zwitterion modification, we tested stabilities of the GO, GO-SB, DOX@GO and DOX@GO-SB in aqueous solution. As shown in Figure 4A, at the beginning, the GO and GO-SB solution are stable while their colour darkens and light transmittance decreases with time due to their gradual agglomeration. But till 24th day, no precipitate is found from both samples. In general, there is no remarkable difference between stability of the GO and the GO-SB in water. However, situation is quite different after the GO and the GO-SB load with DOX. We find that the DOX@GO starts to precipitate immediately at the very beginning (Figure 4B). 30 min later, the precipitation is obvious and the DOX@GO has precipitated completely after being placed statically for 1 day. Conversely, the DOX@GO-SB solution is still stable. As shown, colour of the DOX@GO-SB solution becomes darker at the 6th day. Nevertheless, till the 24th day, no obvious precipitation is observed in 9


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the DOX@GO-SB solution. Hence, we come to conclusion that after modified with zwitterion, GO would be more stable in water as drug carrier. Previous studies have reported that a protein coating termed “protein corona” would be around surface of nanomaterials after the nanomaterials are absorbed by human or other mammalian organisms,35, 36 which may lead to immune response or cause damage to cells and tissues bringing about health risks. Since drug carriers need to be used in bio-medium, we subsequently tried to investigate stability of the GO and the GO-SB in 10% FBS solution. As Figure 4C shows, the GO couldn’t be well dispersed in 10% FBS solution initially and its dispersion is very turbid. Not surprisingly, the GO in 10% FBS solution starts to precipitate at the 3rd day. Poor stability of the GO in 10% FBS solution is due to inherently high adsorption capacity of the GO.37 On the one hand, diameter of the GO becomes bigger because of absorption of protein. On the other hand, similar to DOX, serum components in FBS act as bridge between separate GO sheets, which decrease distance between them and induce them to aggregate. On the contrary, the GO-SB could be well dispersed in 10% FBS solution at the beginning. And the solution kept clear for several days. With time it becomes a little turbid at the 7th day and it is not until the 12th day, a little precipitate is observed at the bottom of the vial. Whether these samples could be stable in FBS solution or not depends on their anti-fouling properties. Zwitterionic materials could bind water molecules via electrostatic induction to form a hydration layer which could separate proteins from surface of materials,

37

and thus distance between the GO-SB sheets is also widened.

That’s why the GO-SB is much more stable than pure GO in serum-containing solution. As shown in Figure 4D, stabilities of the DOX@GO and the DOX@GO-SB in 10% FBS solution are also studied. More obvious distinction is found between them. The DOX@GO has precipitated completely at the 1st day. As for the DOX@GO-SB, although colour of the solution is very dark, no precipitate is observed at the bottom until the 7th day. And the precipitate is extremely little. Additionally, situation does not become worse till the 12th day, implying that most of the DOX@GO-SB still keeps dispersed in 10% FBS solution. From these results, we find that after loading of drugs, stability of the DOX@GO-SB in 10% FBS solution is decreased compared with that of the GO-SB without drugs. As already stated, we speculate that the GO sheets approach to each 10


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other through DOX which act as a bridge. In spite of this, generally speaking, modification of GO with zwitterion indeed effectively improves stability of GO in serum owing to anti-fouling property of zwitterion.

Figure 5. In vitro drug release profiles of the DOX@GO-SB at different temperatures.

In this study, to investigate release behaviour of DOX from the DOX@GO-SB, in vitro drug release experiment was carried out at 37 oC, 40 oC and 45 oC, respectively. As represented (Figure 5), profiles show sustained DOX release at all three temperatures whereas release rates of the drugs at 40 o

C and 45 oC are higher than that of 37 oC during observing period. After about

80 hours, drug release rate is dramatically decreased for all temperatures and only a very small amount of drug was released. Over six day’s period, about 70% of the drug could be released at 40 oC and 45 oC while 54% at 37 oC. According to previous literature,38 there are many hydrogen bonds between the DOX and the GO sheets besides π-π stacking. Virtually π-π stacking interaction decreases distance between the GO sheets and drug molecules thus make them easy to form hydrogen bonds. Herein, both interactions of π-π stacking and hydrogen bonds make DOX molecules to be tightly attached on surface of the GO sheets. Since hydrogen bonding would be weakened with increase of temperature, the drug molecules could move easier and diffusion rate of the solution also become faster, resulting in difference in release rate of the DOX at different temperatures. These results indicate that the DOX@GO-SB complexes are sensitive to temperature and more drug can be released when temperature is slightly higher. Furthermore, many studies have reported that 11


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graphene and GO have strong near-infrared absorption with excellent photo-thermal effect,39-43 thus the synthesized DOX@GO-SB complexes hold potential for synergistic therapy. With a view to demonstrate potential biomedical application of the DOX@GO-SB complexes, firstly in vitro uptake experiments were carried out. As Figure 6 shows, red fluorescence (due to the DOX) could clearly be observed for HepG2 cells after 0.5 h incubation with the DOX@GO-SB complexes. Meanwhile, intracellular red fluorescence intensity increases and distribution of DOX in the cell changes obviously with incubation time. For example, after 0.5 h co-incubation, the DOX is evenly distributed inside the cell. However, with time the DOX begins to concentrate in nuclei. And after 2 h, almost no red fluorescence of the DOX could be observed outside nuclei. All these results strongly demonstrate that the GO-SB could be efficiently taken by tumor cells when used as drug carrier.

Figure 6. Fluorescent inverted microscope images of HepG2 cells after incubation with the DOX@GO-SB for different time: bright field images (left), fluorescence field images (mid) and overlap of bright field and fluorescence field (right). (The scale bar is 20 µm)

To evaluate tumor therapy efficacy of our graphene based drug carrier, in vitro cell viability was studied in HepG2 cells by MTT assays and corresponding data are displayed in Figure 7. As shown, the unloaded GO-SB doesn’t exhibit obvious inhibition effect on the cells for all concentration range. However, after the DOX is loaded, the DOX@GO-SB shows pronounced cytotoxic effect on HepG2 cells. That is to say that cytotoxicity of the 12


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DOX@GO-SB is mainly due to the DOX loaded. And the IC50 of the DOX@GO-SB was calculated to be 1.675 µg/mL (containing 0.05 µg/mL DOX). Furthermore, from Figure 7, it can be seen that the DOX@GO-SB is more toxic than free DOX at the same DOX concentration. It may be caused by the proton pump effect of the cells. When intracellular DOX concentration is too high, the DOX would be pumped out by the cells.44 Since DOX loaded on the GO-SB could be released gradually in the cells, effective time of the DOX is greatly extended, which therefore achieves better inhibition effect on the cells. Another possibility is that free DOX is easier to be degraded by intracellular enzymes than that is loaded on the drug carriers. Thus, cytotoxic effects of the free DOX are reduced.

Figure 7. Viabilities of HepG2 cells after incubated with different concentration of the GO-SB, DOX@GO-SB, and free DOX for 48 h, respectively.

A lot of researches related to graphene as drug carriers have focused on their anti-tumor effect efficacy yet ignoring their metabolic problems which is crucial to fate of the drug carrier in body. Thus, bio-safety evaluation of the GO-SB in vivo was carried out and demonstrated in zebrafish model. Zebrafish is a kind of animal with similar physiological and pharmacological properties to humans, thus it is widely used in test of different biomaterials, genetics and auxology. Moreover, its tiny and transparent body along with strong fecundity is beneficial for zebrafish to be used as a model animal to study the properties of various materials.45, 46 FITC, a fluorescent label, was modified on the GO-SB for observation. In this study, zebrafish was treated with GO-SB-FITC for 5 days from embryo stage. Data show that viability of 13


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zebrafish incubated with the GO-SB-FITC is 86.7% while that of control group treated with water is 87.8% (n=30). There is no significant difference between them. Meanwhile, no deformed individual is observed in both of those groups. That means that GO-SB has no obvious toxicity on embryos and of zebrafish, indicating that the GO-SB is very biocompatible. And this is consistent with results given by the in vitro study of cytotoxicity on HepG2 cells. According to Figure 8, it could be seen that there was green fluorescence stuck on membrane of the embryos at the first day (Figure 8A1 and Figure 8A2). After the embryos were hatched at the 3rd day, green fluorescence was transferred into body of larvae, proving that the GO-SB could be taken by the larvae (Figure 8B1 and Figure 8B2). Subsequently, the larvae were transferred to fresh water at the end of the 5th day. By observing daily, we find out that fluorescence in body of the larvae fades gradually and almost disappears at the 9th day due to excretion (Figure 8C1 and Figure 8C2), suggesting that the GO-SB drug carrier can be eliminated from body of zebrafish within five days. This is favorable with view to drug release time of the DOX@GO-SB that about 70% of the drug loaded can be released within five days (Figure 5). More importantly, the current study shows that GO-SB could be removed from body after the drug was released.

Figure 8. Images of zebrafish embryo exposure to GO-SB-FITC (A1, A2), larva after treated with GO-SB-FITC for 5 days (B1, B2) and then treated with fresh water for 4 days (9th day) (C1, C2). (Left: fluorescence field, right: bright field)

CONCLUSIONS 14


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In this paper, we have proposed a novel kind of zwitterion modified GO for promoting stability and reducing aggregation of GO used as drug carrier. After SBS modification, the drug loaded GO-SB demonstrates significantly enhanced stability in serum-free and serum-containing solution compared with pure GO. The in vitro studies show that the DOX loaded GO-SB could be effectively internalized by HepG2 cells and exhibited obvious inhibition effect on the cells which is more effective than free DOX. Meanwhile, without drug loading, the GO-SB shows negligible cytotoxicity on HepG2 cells, revealing favorable biocompatibility of the GO-SB. And in vivo bio-safety evaluation of GO-SB demonstrates on zebrafish model indicates that the GO-SB has good biocompatibility in vivo and can be excreted from the body within several days. Moreover, it deserves to be mentioned that the GO-SB drug carrier has considerable capability to load various therapeutic substances. Such a facile,

efficient

yet

low-cost

approach

offers

extraordinary

potential

of

graphene-based nanosystem in biochemical applications. ASSOCIATED CONTENT Supporting Information Detailed preparation of the GO, GO-SB, DOX@GO-SB, GO-SB-FITC complexes, additional figures and tables. CONFLICTS OF INTEREST There are no conflicts of interest to declare. ACKNOWLEDGMENTS This work was funded by Zhejiang Provincial Natural Science Foundation of China (LY17E030005) and the National Natural Science Foundation of China (Grants 21404091 and 21404089). REFERENCES (1) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Zhang, Y.; Dubonos, S. V.; Grigorieva, I. V.; Firsov, A. A. Electric Field Effect in Atomically Thin Carbon Films. Science 2004, 306, 666-669. 15


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Stability, Cellular Uptake, and in Vivo Tracking of Zwitterion Modified

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