A Dual-sensitive Graphene Oxide Loaded with Proapoptotic Peptides

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A Dual-sensitive Graphene Oxide Loaded with Proapoptotic Peptides and Anti-cancer Drugs for Cancer Synergetic Therapy Jing Zhang, Liqun Chen, Biao Shen, Lingdong Chen, Jiaying Mo, and Jie Feng Langmuir, Just Accepted Manuscript • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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A Dual-sensitive Graphene Oxide Loaded with Proapoptotic Peptides and Anti-cancer Drugs for Cancer Synergetic Therapy Jing Zhang*, Liqun Chen, Biao Shen, Lingdong Chen, Jiaying Mo, Jie Feng* College of Materials Science & Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, 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: A dual-sensitivity drug delivery system (DDS) based on graphene oxide (GO) which simultaneously loads with proapoptotic peptides and anticancer drugs was rationally designed and fabricated for cancer synergetic therapy. Specifically, a kind of cell apoptosis peptide (KLAKLAK)2 (KLA) was anchored on surface of GO via disulfide bond to obtain GO-SS-KLA. Then aromatic anticancer drug doxorubicin (DOX) was loaded on the GO through π-π conjugation and hydrogen bonding interaction. Finally, bovine serum albumin (BSA) was used to coat the GO carrier to obtain a biological media stable GO based DDS, DOX@GO-SS-KLA/BSA. The results show that the KLA and DOX can be released responding to reductive and pH stimulus inside the cells, respectively, and achieve synergetic therapy of cancer. Moreover, the results of stability studies show that DOX@GO-SS-KLA/BSA could be stably dispersed in water for more than 8 days and in 10% fetal bovine serum (FBS) for at least 6 days. The constructed DOX@GO-SS-KLA/BSA exhibits great potential as drug carrier for co-delivery of various therapeutic agents. KEYWORDS: graphene oxide; pro-apoptotic peptide; biological medium stable; synergetic therapy

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INTRODUCTION Cancer, one of the major causes of human death, is always a thorny problem in medical field. In the world, the incidence of cancer is increased by 3-5% annually. And it is estimated that 20 million cancer patients will be added globally in 2030 1. Currently, most of the anticancer drugs are non-selective drugs. Due to their poor water solubility, nonspecific delivery and severe side effect, chemotherapeutic drugs have many limitations in fighting with cancer in clinic 2.Thus, delivery method of anticancer drugs plays a very important role in cancer therapy. Nanocarriers as drug delivery systems have attracted tremendous interest 3. Because of their unique surface characteristics and nano-scale, the nanocarriers that load with anticancer drug may reach a much better effect than the drug itself. For example, the nanocarriers can help to improve the watersolubility of hydrophobic drugs, improve the drug absorption and bioavailability, control the release of anticancer drugs in the microenvironment of tumor site, and improve the distribution of drugs in body for some extent 4, 5. In recent years, various nanomaterials of different sizes, shapes and chemical composition have been explored, including polymer micelles 6, liposomes 7, polymer vesicles

8

and inorganic nanoparticles 9, and so on

10-12

. Among them, graphene and

graphene oxide (GO) have been investigated as potential carriers for drug delivery 13, 14

, due to their advantageous properties, such as ultrahigh specific surface area (2630

m2/g), good biocompatibility and delocalized electrons with a high capacity for loading of aromatic drugs via π-π stacking and hydrophobic interactions 15-18. A research found that the loading ratio of DOX/GO (loading drug-carrier weight ratio) can reach 200% 19

. Furthermore, there are abundant oxygen-containing functional groups like hydroxyl

(–OH), epoxide (–O–) groups and carboxyl (–COOH) on the surface and edges of GO, which are conducive to surface functionalization 20, 21. Currently, many graphene and GO nanocarriers have been designed to be pHresponsive 22-24. It is well-known that the pH value in tumors (i.e. 6.5-6.8) is lower than that in normal tissues (i.e. 7.4), while the pH values can even decrease to 5.0-5.5 in lysosomes and endosomes

25

. Owing to the varying pH conditions, pH-responsive

nanocarriers can obviously improve bioavailability of drugs in anticancer therapy, showing extremely low cytotoxicity to normal cells

26, 27

. Always, the interactions

between DOX and GO based drug carriers mainly rely on π-π stacking, hydrogen


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bonding and hydrophobic interactions 28-30. Thus the pH-sensitive release of DOX could be easily achieved by GO based drug carriers. On one hand, π-π bonds can be interrupted in the low pH environment and hydrogen bonds between drug molecules and GO will be weakened at the low pH of the lysosome, which will result in release of the loaded DOX. On the other hand, protonation of the amino groups (-NH2) on DOX will also promote DOX release at low pH 31. In addition, a single treatment is often difficult to achieve the desired effect. Therefore, synergistic treatment systems with multiple drugs co-loading into the same nanocarrier and double/multiple stimuli-responsive systems have been developed to achieve enhanced therapeutic effect

32-34

. For instance, a polydopamine (PDA)

nanoparticle loading with photosensitizer (ZnPc12+) and cell cycle inhibitor nocodazole 35

(NOC) exhibits a synergistic therapy effect

. The loaded NOC can not only inhibit

cell proliferation, but also facilitate nuclear uptake of ZnPc12+ in MCF-7 cells to improve photodynamic therapy (PDT) efficacy. For stimuli-responsive drug carriers, pH- and redox-responsive drug carriers can not only efficiently control the drug release in acidic and highly reductive environments

36, 37

, but also show excellent therapeutic

efficacy both in vitro and in vivo 38. Besides, the combination of proapoptotic peptides and chemotherapeutic agents is also a new strategy for cancer treatment. In this paper, we designed and constructed a dual-sensitive cancer combination treatment system utilizing GO to load with proapoptotic peptides and anticancer drugs. First, GO contained abundant hydroxyls was prepared by optimized Hummer's method. Afterwards, the proapoptotic peptide KLA was modified to the surface of GO through disulfide bonds. The proapoptotic peptide KLA, an amino acid sequence of (KLAKLAK)2, can induce mitochondrial-dependent apoptosis while remaining relatively non-toxic in extracellularly

39, 40

. For most tumor cells, the intracellular

concentration of glutathione (GSH) is about 2~10 mM, which is 100 to 1000 times higher than the extracellular concentration of GSH

41

. Therefore, the disulfide bond

would rupture under the reduction of GSH and the KLA could be released when the GO carrier entries cancer cells. Then the aromatic anticancer drug DOX was loaded on the GO by π-π conjugation and hydrogen bonding interaction. Finally, because of the poor hydrophilicity of the GO carrier after drug loading, BSA was used to coat GO carrier to enhance the stability of the system

42, 43

. The constructed drug carrier is

expected to release KLA and DOX in the microenvironment of tumor cells to achieve


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synergistic anticancer therapy. Specifically, the DOX is released responding to the low pH in the endosome while the KLA is released responding to the intracellular GSH. At the same time, the departure of KLA will also accelerate the release of DOX. MATERIAL AND METHODS Materials Graphite (100 mesh), (3-mercaptopropyl)trimethoxysilane, triethylamine (TEA), propargyl bromide were purchased from Aladdin Reagent and used directly. Doxorubicin hydrochloride (DOX) was acquired from Beijing Hua Lian Bo Technology Co. Fluoresceinamine isomer (FITC) was obtained from Acros and used directly. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and Dulbecco’s phosphate buffered saline (PBS) were purchased from Invitrogen Corp. All other reagents and solvents were provided by Shanghai Reagent Chemical Co. (China) and used without further purification. Synthesis of Graphene Oxide (GO) The graphene oxide (GO) with a large amount of hydroxyl groups was produced via an optimized Hummers method according to previous literature 44. Specifically, 12 mL of de-ionized (DI) water was mixed with 46 mL of ice-cooled concentrated sulfuric acid with mechanical agitation at 200 rpm in ice bath. Then 1.0 g of graphite was added and kept the temperature of the suspension below 10 oC. It is noteworthy that 3.0 g of KMnO4 needs to be added slowly. Subsequently, the reaction was heated to 40 oC with vigorously stirring at 300 rpm and maintained for 2 h. Next, the product was slowly poured into 300 mL of ice/water mixture to keep the temperature less than 10 oC, and stirred for 15 min. At last, 5 mL of H2O2 (30 % w/w in water) was then added dropwise. Afterward, the suspension was kept undisturbed overnight. After filtration, the crude product was washed with 50 mL of HCl aqueous solutions (10 % v/v in water) (3 times) to remove metal ions. The resulting solid was dispersed in 500 mL of DI water and purified by dialysis for one week using a dialysis bag with a molecular weight cut off of 10,000 Da. Finally, the GO was obtained by Freeze-drying method. Synthesis of Thiolated Graphene Oxide (GO-SH)


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120 mg of GO was dispersed in 40 mL of DI water by ultrasound, and adjusted to pH 9~10 with 0.1 M NaOH solution. GO dispersion was reacted with 1 mL of (3mercaptopropyl)trimethoxysilane by stirring intensely at room temperature for 24 h. After centrifugation (10000 rpm, 10 min), the resulting GO-SH was washed with DI water and methanol, and dried under vacuum. Synthesis of Disulfide Functionalized Graphene Oxide (GO-SS-NH2) First, S-(2-aminoethylthio)-2-thiopyridine hydrochloride was synthesized manually according to previous literature 45. Then, 180 mg of GO-SH was scattered in 30 mL of methanol under ultrasound with ice bath. To this solution, 180 mg of S-(2aminoethylthio)-2-thiopyridine hydrochloride was added and stirred intensely at room temperature for 24 h. After centrifuged (10000 rpm, 10 min) and washed with DI water and methanol, the GO-SS-NH2 was obtained by dried under vacuum. Synthesis of Alkyne-Modified Disulfide-Functionalized Graphene Oxide (GOSS-Alkyne) 100 mg of GO-SS-NH2 was dispersed in 30 mL of methanol. To this solution, 1 mL of propargyl bromide and 1 drop of triethylamine were added and stirred for 24 h. Then the mixture was centrifuged (8000 rpm, 10 min), washing with DI water and methanol. After vacuum drying, alkyne-modified disulfide-functionalized graphene oxide (GOSS-alkyne) was acquired. Synthesis of Proapoptotic Peptide Functionalized Graphene Oxide (GO-SSKLA) According to standard (Fmoc)-chemistry through solid-phase peptide synthesis 46, the peptide azide-KLA (N3-KLA) was synthesized. And the detail of the method is described in the Supporting Information. 50 mg of GO-SS-alkyne was dispersed evenly in 10 mL of methanol. Then 50 mg of N3-KLA, 50 mg (0.2 mmol) of CuSO4 • 5H2O and 80 mg (0.4 mmol) of sodium ascorbate were added to the solution. The reaction was carried out under the protection of nitrogen atmosphere at room temperature for 3 days. After centrifugation (8000 rpm, 5 min) and washing with DI water and methanol several times, GO-SS-KLA was obtained.


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Preparation of FITC@GO-SS-KLA 50 mg of KLA-N3 was dispersed in 20 mL of PBS solution with pH 7.4. Then 2.5 mg of FITC was dissolved in 5 mL of DI water and slowly added dropwise to the KLA-N3 solution. FITC@KLA-N3 was obtained by stirring of the above mixture for 4 h. And free FITC in the resulting solution was removed by dialysis (Mw cutoff: 500 Da) for several days. Afterward, 50 mg of GO-SS-alkyne was dispersed evenly in 10 mL of methanol. And 10 mL of FITC@KLA-N3 solution (5 mg/mL), 50 mg (0.2 mmol) of CuSO4 • 5H2O and 80 mg (0.4 mmol) of sodium ascorbate were added to the GO-SSalkyne dispersion to react for 3 days under the protection of nitrogen atmosphere. After centrifugation (8000 rpm, 5 min) and washing with DI water and methanol several times, FITC@GO-SS-KLA was obtained. All the above reactions were performed at room temperature in the dark. Preparation of DOX@GO-SS-KLA Complexes 30 mg of GO-SS-KLA was dissolved in 30 mL methanol under ultrasound with ice bath. DOX@GO-SS-KLA was obtained by adding dropwise 5 mL (1 mg/mL) of anticancer drug DOX, stirring vigorously at room temperature overnight, and dialyzing (Mw cutoff: 3500 Da) against 500 mL of DI water for 3 days. Fluorescence intensity of the dialysate was measured by a fluorescence spectrophotometer (FL-4600, Hitachi) at the given time intervals (λex = 480 nm; slit width = 5 nm), the amount of unloaded DOX was calculated. The drug loading efficiency (DLE) of DOX@GO-SS-KLA was determined via the following equation: DLE(wt%) =

𝑚𝑡𝑜𝑡𝑎𝑙 −𝑚𝑢𝑛𝑙𝑜𝑎𝑑𝑒𝑑 𝑀

× 100%

(1)

𝑚𝑡𝑜𝑡𝑎𝑙 represents the total weight of DOX, 𝑚𝑢𝑛𝑙𝑜𝑎𝑑𝑒𝑑 represents the weight of unloaded drugs, and M represents the weight of DOX@GO-SS-KLA. Preparation of DOX@GO-SS-KLA/BSA 10 mg of DOX@GO-SS-KLA was dissolved in 10 mL of DI water. Then 10 mL of bovine serum albumin (BSA) solution (1 mg/mL) was added and stirred at room temperature overnight. After centrifugation and washing, DOX@GO-SS-KLA/BSA was obtained.


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Preparation of Complexes in Water and 10% Serum 12 mg of DOX@GO-SS-KLA was dissolved in 5.4 mL of DI water with ultrasound. After the treatment of ultrasonic oscillation for 0.5 h, half of the dispersion (2.7 mL) was placed in a 5 mL bottle and 300 μL of DI water was added. At the same time, 300 μL of pure fetal bovine serum (FBS) was added to the remaining dispersion. The obtained solutions were further treatment of ultrasonic oscillation for 15 min. The DOX@GO-SS-KLA/BSA in water and 10% serum were prepared in an identical way as described above. At last the solution volume and concentration in every bottle was maintained at 3 mL and 2 mg/mL. Characterization of GO-based Nanocomposites The infrared spectra of the GO, GO-SH, GO-SS-NH2, GO-SS-alkyne and GO-SS-KLA were obtained with a Fourier Transform Infrared (FTIR) Spectrometer (Nicolet 6700) using KBr pellets at room temperature. The spectra were obtained in the spectral region of 400-4000 cm-1 at a resolution of 4 cm−1 and 32 scans per sample. The size distribution and zeta potential of these samples in aqueous medium were determined with a new NanoBrook Omni particle size and zeta potential analyzer (Brookhaven). The signal processing of size and zeta potential were Dynamic Light Scattering (DLS) and true Phase Analysis Light Scattering (PALS), respectively. And the pH values of all the samples were adjusted to 7.0 using 0.1 M NaOH before measuring. When measuring, repeated 3 times for each sample and the test temperature was controlled at 25 °C and the scattering angle was 90 °. Transmission electron microscopy (TEM) and Atomic force microscope (AFM) were used to observe the morphology and thickness of the GO on a Tecnai G2 F30 STwin electron microscope operated at 200 kV and a Bruker Dimesion micrographs, respectively. The scan ranges of the AFM images were 4 μm by 4 μm. The X-ray diffraction (XRD) spectrum of the GO and graphite were determined by X-ray diffractometer (Rigaku Ultima IV) at 40 kV and 40 mA using Cu-Ka radiation (k = 0.15405 nm). In Vitro Release Experiments In Vitro Redox-responsive Drug Release


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The in vitro release behavior of FITC@GO-SS-KLA was investigated. 15 mg of FITC@GO-SS-KLA was dispersed in 3 mL of DI water and evenly divided into three parts (1 mL, for each) and transferred to dialysis bags (Mw cutoff: 3500 Da). Next, the dialysis bags containing FITC@GO-SS-KLA dispersion were immersed in 5 mL of PBS buffer at pH 7.4 with dithiothreitol (DTT) (0, 0.5, and 5 mM) and placed in a shaker (SHZ-82A) at 37 oC under oscillating slowly (150 rpm) to release drug. 2 mL of dialysate was withdrawn and 2 mL of the corresponding fresh buffer solution was added after each sampling. The amount of KLA released was determined by a fluorescence spectrophotometer (FL-4600, Hitachi). The fluorescence intensity of the medium at 516 nm was measured by the fluorescence spectrophotometer at the set time intervals (λex = 492 nm; slit width = 5 nm). The cumulative drug release is calculated by the formula: Cumulative release dose (%) = (Mt / M∞) ×100%

(2)

where Mt is the amount of KLA released during the time, and M∞ is the total amount of KLA loaded in the graphene oxide material. In Vitro pH-responsive Drug Release The in vitro drug release behaviors of DOX@GO-SS-KLA/BSA under different conditions were studied. At first, 15 mg of DOX@GO-SS-KLA/BSA was dispersed in 3 mL of DI water and evenly distributed into three dialysis bags (Mw cutoff: 3500 Da). Afterwards, 300 μL of α-chymotrypsin was added to each dialysis bag to accelerate the degradation of the BSA 47.The dialysis bags were immersed in 5 mL of buffer solutions at different pH values (5.5, 6.8, and 7.4) and then placed on a shaker (SHZ-82A) at 37 o

C under oscillating slowly (150 rpm). The solutions used in this work are PBS buffer

solutions (6.8 and 7.4) and acetic acid-sodium acetate buffer solution (5.5), respectively. 2 mL of dialysate was withdrawn from the solution periodically at given time intervals, and subsequently, the same volume of the corresponding fresh buffer solution was added after each sampling. The amount of released DOX was determined by fluorescence spectrophotometer (FL-4600, Hitachi) at 592 nm at the set time intervals (λex = 488 nm; slit width = 5 nm). The cumulative drug release is calculated by the formula: Cumulative drug release (%) = (Mt / M∞) ×100%


(3)

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where Mt represents the amount of drug released during the time, and M∞represents the total amount of drug loaded in the graphene oxide material. In Vitro Cell Cytotoxicity Assays The in vitro cytotoxicity of graphene oxide materials to HeLa cells was investigated by MTT assay. HeLa cells were first seeded into a 96-well plate at a density of 5×103 cells/well, and then 200 μL of DMEM medium containing 10% FBS and 1% chainpenicillin was added to each well for cultivating in an incubator for 24 h (37 oC, 5% CO2). Thereafter, graphene oxide materials with a set concentration gradient (from 0 to 24 μg/mL) were added to each well and further cultured for 48 h in the above medium. After completion of the incubation, the culture medium was removed and 200 μL of fresh medium and 20 μL of MTT solution (5 mg/mL) were added and the cells were incubated for another 4 h in the incubator. Then, the MTT medium was carefully removed from the wells and 100 μL of dimethyl sulfoxide (DMSO) was added. Subsequently, the optical density (OD) of each well at 492 nm was recorded on a microplate reader (DG5033A). The cell viabilities were calculated as follows: Cell viability =

𝑂𝐷𝑡𝑟𝑒𝑎𝑡𝑒𝑑 −𝑂𝐷𝑏𝑙𝑎𝑐𝑘 𝑂𝐷𝑐𝑜𝑛𝑡𝑟𝑜𝑙 −𝑂𝐷𝑏𝑙𝑎𝑐𝑘

× 100%

(4)

Where ODtreated, ODcontrol, ODblank are the absorbance values from the sample wells, the positive control wells and the background wells. The OD value is measured on the basis of four independent parallel samples, and the results are expressed as mean ± standard deviation (SD). In Vitro Cell Co-Culture The experiment of co-culture of graphene oxide materials with cells was performed on HeLa cells. Firstly, HeLa cells were seeded into 24-well plates at a density of 5×104 cells/well, and 1000 μL of DMEM medium containing 10% FBS and 1% penicillin was added to each well. And the 24-well plates placed in a 5% CO2 incubator at 37 oC for 24 h. Then, 1000 μL of different concentrations of DOX@GO-SS-KLA/BSA dispersed in DMEM medium containing 10% FBS and 1% chain-penicillin were added into the cell culture plate, respectively. After co-cultivation for 4 h, the medium in the wells was discarded and the nucleuses of cells were stained with 100 μL of Hochest 33258 staining solution. After incubating for 20 min in the incubator, the staining solution in


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the wells was discarded and the cells were washed with 200 μL of PBS. Inverted fluorescence microscope (Nikon Ti) was used to observe the phagocytosis of the DOX@MSN-SS-KLA/BSA complex by HeLa cells. RESULTS AND DISCUSSION Preparation and Characterization of GO-based Nanocomposites

Scheme 1. (A) Schematic illustration of the fabrication of GO complexes and (B) a dualsensitivity cancer combination treatment system accumulates at tumor sites via the EPR effect and releases drugs in the microenvironment of tumor cells to achieve synergistic anticancer therapy. (a) The GO nanocomposites penetrate cell membrane and are uptaken by tumor cells; (b, c) the low pH leads to release of DOX in endosomes; (d) KLA is released via the cleavage of disulfide bonds by the concentrated GSH and the departure of KLA promotes the release of DOX; (e) the released KLA and DOX synergistically enhance therapy effect.

In this work, a dual-sensitive cancer synergetic therapy system that simultaneously delivers proapoptotic peptide and anti-cancer drug was fabricated based on GO (shown as Scheme 1). The proapoptotic peptide KLA was modified to the surface of GO through reductive sensitive disulfide bonds while DOX was loaded on GO by π-π conjugation and hydrogen bonding interaction. Then, the biological stability of the GO carrier was enhanced by the coating of BSA. It has been reported that disulfide bond would cleave in presence of GSH

48

. So KLA can be released from the GO

nanocomposites responding to concentrated GSH existing inside tumor cell. And then


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KLA could be transported to mitochondria to induce mitochondrial dysfunction. At the same time, intracellular release of DOX will occur in the low pH environment of endosomes. The released KLA and DOX are expected to realize synergetic therapy of cancer. Specifically, the synthesis route of GO-SS-KLA is shown in Scheme 2A. First, GO was synthesized according to the Hummer’s method. And the yield of GO was 68.2%. Surface functionalization of the GO was monitored by Fourier transform infrared spectroscopy (FTIR) (Figure 1). For GO, broad peaks appear at 3600 cm-1 and 3300 cm1

(Figure 1A). According to the characteristic peaks (C=O: 1730 cm-1, C–O (epoxy):

1230 cm-1, C–O (alkoxy): 1066 cm-1, O–H: 3600 ~ 3300 cm-1) of oxygen-containing functional groups that may be generated after the graphite was oxidized, we think the broad infrared peaks at 3600 cm-1 and 3300 cm-1 are attributed to the hydroxyl groups on the GO. And as reported in the literature, the bend vibration peak of adsorbed water molecules appears at 1634 cm-1 49. This is also consistent with our expectation that the prepared GO contains a large amount of hydroxyl groups, which facilitates the modification of the siloxane in the next step.

Scheme 2. The schematic illustration of the fabrication of GO-SS-KLA nanocomposite (A) and structure of peptide KLA with the (KLAKLAK)2 sequence (B).


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Figure 1. The FTIR spectra of different samples: (A) GO, (B) GO-SH, (C) GO-SS-NH2, (D) GO-SS-alkyne, (E) GO-SS-KLA

The GO was then reacted with (3-mercaptopropyl)trimethoxysilane to obtain GOSH (yield: 80.5%). Compared with the GO, the characteristic peak of thiol groups (-SH) at 2550cm-1 proves the existence of thiol groups (-SH) on the surface of GO-SH (Figure 1B). At the same time, the peak at 1100 cm-1 represents the infrared characteristic peak of the siloxane bond (-Si-O-), and the characteristic peak of methylene (-CH2-) also appears at 2900 cm-1 50, which further confirms that GO-SH is synthesized successfully. Afterwards, the GO-SH was further modified with S-(2-aminoethylthio)-2-thiopyridine hydrochloride to produce GO-SS-NH2 (yield: 82.4%). In the FTIR spectrum of GO-SSNH2, it is found out that the peak of thiol groups (-SH) in the reactant GO-SH disappears while the characteristic peak of disulfide bond (-S-S-) appears at 466 cm-1 (Figure 1C). In addition, referring to Zeta potential analysis, we find out that the surface potential of GO-SH changes from -57.0 mV to 13.2 mV after amino modification (Table 1). Since the thiol groups are negative charged while the amino groups are positively charged, the increase of the zeta potential proves the successful modification of amino group. Next, in the FTIR spectrum of GO-SS-alkyne, we can see that the typical absorption peak of alkyne groups (C ≡ C) appears at 2122 cm−1 (Figure 1D), indicating the successful modification of alkyne moieties which can be used for the subsequent modification of KLA.


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Table 1 The results of the Zeta potential of different samples.

Samples

Zeta potential（mV）

Graphite

-18.1 ±0.2

GO

-24.6 ±0.3

GO-SH

-57.0 ±0.3

GO-SS-NH2

-13.2 ±0.8

GO-SS-alkyne

-25.7 ±0.1

GO-SS-KLA

-10.7 ±0.3

DOX@GO-SS-KLA

-15.2 ±0.5

DOX@GO-SS-KLA/BSA

-19.9 ±0.4

To connect KLA onto GO-SS-alkyne, KLA was synthesized manually employing a standard Fmoc chemistry through the solid phase peptide synthesis and terminated with azide group (-N3). And the yield of KLA-N3 was 34.4%. The structure of KLA is shown in Scheme 2B and the structure of KLA-N3 is confirmed by ESI-MS (Figure S4). By reaction of GO-SS-alkyne and KLA-N3 via "click chemistry", reductive responsive GO-SS-KLA was obtained (yield: 92.7%). As shown in Figure 1E, the characteristic peak of alkyne groups disappears while strong absorption peak characteristic peak of amide bonds appears at 1680 cm-1, confirming that KLA has been successfully anchored onto the GOs. The amide bonds are mainly derived from the proapoptotic peptide KLA. Due to the presence of many amino groups in the proapoptotic peptide structure, the surface potential of the GO based material changes from -25.7 mV to 10.7 mV after modification of the proapoptotic peptide (Table 1). It also demonstrated the successful synthesis of the GO-SS-KLA. Drug Loading and Release KLA Loading and in Vitro Reductive Responsive Drug Release


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In GO-SS-KLA, KLA are attached to the surface or edge of GO via disulfide bonds. As known, the disulfide bonds are reductive sensitive and can cleave in presence of GSH. To further understand the release behavior of KLA from the GO-SS-KLA under reductive conditions, we labeled KLA with FITC to obtain FITC@KLA-N3 and used it to prepare FITC-labeled GO-SS-KLA (FITC@GO-SS-KLA) 51. FITC is a fluorescein marker, which is widely used in cell biology, immunology, and drug research 50. FITC is obtained by introducing an isothiocyano group (-N=C=S) to the structure of fluorescein. Herein, the isothiocyano group of FITC can react with primary amine groups on KLA and form a thiourea bond to generate a strong fluorescent dye-peptide conjugate, thereby achieving fluorescent labeling of the drug carrier.

Figure 2. In vitro drug release profiles of FITC@GO-SS-KLA at different DTT concentrations (0, 0.5, and 5 mM) at 37 oC.

The drug loading content of KLA is calculated to be 61.2 wt% based on the equation (2) mentioned in the “in vitro redox-responsive drug release” section. In in vitro release studies, we selected the reductive agent DTT as an alternative to GSH to provide a reductive environment to disrupt disulfide bonds. As shown in Figure 2, in 5 mM DTT solution, the release rate of KLA from FITC@GO-SS-KLA is significantly faster than that under 0 mM and 0.5 mM DTT within 6 h. Similarly, the KLA release rate at the concentration of 0.5 mM DTT is also obviously higher than that without DTT. Theoretically, disulfide bond is stable in the absence of DTT, and FITC-labeled KLA should not be detected. However, a very small amount of FITC fluorescence was


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detected actually. This may due to the fact that a very small portion of FITC is adsorbed on the drug carrier instead of bonding to KLA. After 48 h, the amounts of released KLA are 5.03 %, 36.49 %, 64.32 % corresponding to DTT concentrations of 0 mM, 0.5 mM, 5 mM, respectively. With time, the release rates at all conditions are extremely slow. Actually, we observed partial precipitation of the dispersion in the dialysis bag after 12 h of dialysis. Because FITC@GO-SS-KLA is not very stable in the solutions, it will be aggregated after a period of time. That is why we use BSA to coat the GO-SS-KLA. In general, these results indicate that the release of KLA from FITC@GO-SS-KLA has reductive responsiveness and the release rate of KLA increases with the increase of DTT concentration. DOX Loading and in Vitro pH-Responsive Drug Release Due to the differences of microenvironment between tumors and normal tissues, pH responsiveness is a common stimulus for the controlled release of anticancer drugs. In order to investigate the pH-responsive release of DOX from DOX@GO-SS-KLA nanocarriers, the in vitro drug release experiments were carried out under acidic (pH 5.5 and 6.8) and physiological (pH 7.4) conditions. The drug loading content of DOX in the DOX@GO-SS-KLA is 33.75 wt%. Because of the poor water solubility of DOX@GO-SS-KLA, we coated the outer surface of the system with BSA. Previous studies have shown that modifying nanomaterials with BSA can significantly improves the stability and biocompatibility of the material

42, 52

. Moreover, the biodegradability

of BSA may achieve sustainable drug release. From the drug release profiles shown in Figure 3, during the whole drug release process, the drug release rate at pH 6.8 is slightly faster than that at pH 7.4. At pH 5.5, the drug release rate is significantly faster than the other two (pH 6.8 and 7.4) and more than 70% of the loaded drug is released within 48 h. Contrastively, cumulative release amount of drugs in PBS solution at pH 6.8 and 7.4 are about 35.02% and 24.22% after 48 h, respectively. From the experimental results, the decrease of pH value increases the release rate of DOX from DOX@GO-SS-KLA/BSA. The reason is that pH affects the interaction between the GO and DOX. As already stated, DOX is loaded on the GO through π-π interaction and hydrogen bonding interaction. As the pH decreases, protonation of the amino groups on DOX is increased, thereby reducing the hydrogen bonding interaction and resulting in faster DOX release rate 31. In addition, at low pH, the π-π stacking interaction between DOX and GO also weakens, enhancing drug


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release

27

.Within the initial 9 h, the DOX is released very fast. After 72 h, the drug

release rates tend to be gentle. Finally, at 144 h (6 days), the corresponding cumulative release amounts of DOX are 83.96 %, 45.35 %, 36.08 % at pH 5.5, 6.8, 7.4, respectively. In short, these results show that the DOX@GO-SS-KLA/BSA is pH-sensitive, and the drugs can be released faster with the decrease of the pH value. Therefore, the selective release of anti-cancer drugs from the DOX@GO-SS-KLA/BSA in cancerous tissues can be achieved, which would reduce side effects.

Figure 3. In vitro drug release profiles of DOX@GO-SS-KLA/BSA at different pH (5.5, 6.8, and 7.4) at 37 oC.

In Vitro Cell Cytotoxicity The cell viabilities of HeLa cells in the presence of GO based materials were measured to evaluate the therapeutic effect of DOX@GO-SS-KLA/BSA. The cytotoxicity of GO/BSA, GO-SS-KLA/BSA, DOX@GO-SS-KLA/BSA, and free DOX with various concentrations (from 0 to 24 μg/mL) was tested on HeLa cells using MTT assay. It can be found out in Figure 4 that there is negligible cytotoxicity of the GO/BSA within the experimental concentration range. In other words, the carrier itself is relatively nontoxic to cells. Besides, all the other samples exhibited enhanced cytotoxicity to HeLa cells with increased dose (Figure 4). It has been reported that the proapoptotic peptide KLA would selectively distort the matrix of the mitochondrial phospholipid physically and prompt the mitochondria-dependent apoptosis 53-56. Thus,


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GO-SS-KLA/BSA shows higher cytotoxicity compared with GO/BSA. For example, when the concentration is 12 μg/mL, the cell viabilities of HeLa cells co-cultured with GO/BSA and GO-SS-KLA/BSA for 48 h are 86.8% and 64.6%, respectively (Figure 4). According to Figure 2, KLA can be released in the condition of reductive agent. Thus, due to the reductive environment in cytoplasm, part of KLA would be released from GO-SS-KLA/BSA after GO-SS-KLA/BSA enters the cells and lead to the mitochondria-dependent apoptosis of the cells.

Figure 4. Viabilities of HeLa cells incubated with GO/BSA, GO-SS-KLA/BSA, DOX@GOSS-KLA/BSA and free DOX for 48 h.

Furthermore, as shown in Figure 4, compared with free DOX, the cytotoxicity of DOX@GO-SS-KLA/BSA is much greater under the same dose of DOX. It is worth to note that the DOX loaded on DOX@GO-SS-KLA/BSA is not completely released during the 48 h. For instance, the cell viability of HeLa cells is 28.5% at 12 μg/mL of DOX@GO-SS-KLA/BSA (containing 1 μg/mL DOX). According to the drug release profiles shown in Figure 3, about 24.2% of DOX is released from the DOX@GO-SSKLA/BSA within 48 h (pH 7.4). However, it is impossible for the drug carrier to be under the environment of pH 7.4 for 48 h. When it enters the endosomes, the pH would decrease. If the release amount of the drug is calculated under the condition of pH 5.5, about 70% of DOX would be released. Therefore, the actual released DOX is between 25% and 70% of the total. That is, when the DOX concentration loaded in the material is 1 μg/mL, the actual released DOX concentration is 0.25-0.7 μg/mL. As represented


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in Figure 4, the cell viability is 55%-30% when the concentration of free DOX is 0.250.7 μg/mL, which is higher than the cell viability of the cells cultured with the DOX@GO-SS-KLA/BSA (28.5%). This result suggests that the DOX-loaded GO carrier has greater cytotoxicity than free DOX at the same DOX content. The increased cytotoxicity should be owed to KLA on DOX@GO-SS-KLA/BSA. From the above results, we can conclude that DOX@GO-SS-KLA/BSA has higher toxicity than single carrier GO-SS-KLA/BSA and single anticancer drug DOX. The synergetic effect of DOX and KLA would greatly improve the therapeutic effect. In Vitro Cell Co-culture

Figure 5. Fluorescent inverted microscope images of HeLa cells after incubation with

DOX@GO-SS-KLA/BSA for different time: bright field images (left), fluorescence field images (right). (The scale bar is 20 μm)

As cell internalization is a prerequisite for efficient drug delivery, cellular uptake of DOX@GO-SS-KLA/BSA was investigated by inverted fluorescence microscopy. The HeLa cells were incubated with 10 μg/mL of DOX@GO-SS-KLA/BSA for 0.5 h, 1 h and 2 h. As shown in Figure 5, with the increase of incubation time, the intracellular red fluorescence (due to the DOX) intensity increased and the distribution of intracellular DOX changed significantly. Meanwhile, the shape of the cells has also changed dramatically. Specifically, the DOX (red fluorescence) could be observed in the cell and predominantly located in cytoplasm within 0.5 h. However, with time, the DOX began to concentrate in nucleus and the cell morphology gradually deteriorated


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from shuttle type to circular. After 2 h, almost no red fluorescence of the DOX could be observed in cytoplasm. All these results strongly suggest that the DOX@GO-SSKLA/BSA can be efficiently uptaken by tumor cells. Study of the Effect of BSA Coating on the Stability of GO Complex in Water and in Serum-Containing Solution Due to the poor hydrophilicity of drug loaded GO-SS-KLA, we used BSA to improve the stability of the GO complex 42, 43. The stability of DOX@GO-SS-KLA in water and serum-containing solution was monitored over an 8-day standing still (Figure 6 and Figure 7). For DOX@GO-SS-KLA, a non-BSA-coated material, precipitation started to appear after standing for 6 h and obvious sediments were observed after 12 h in water (Figure 6A). In contrast, the DOX@GO-SS-KLA/BSA did not show any sediment after 8 days (Figure 6B). This remarkable difference confirms that the coating of GO complex with BSA could improve the stability of the material.

Figure 6. The digital photos of the dispersion of DOX@GO-SS-KLA and DOX@GO-SSKLA/BSA in water (2 mg/mL). (A) DOX@GO-SS-KLA, (B) DOX@GO-SS-KLA/BSA.


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Figure 7. The digital photos of the dispersion of DOX@GO-SS-KLA and DOX@GO-SSKLA/BSA in 10% FBS (2 mg/mL). (A) DOX@GO-SS-KLA, (B) DOX@GO-SS-KLA/BSA.

When using the designed drug nanocarriers in vivo, surface adsorption of blood components should be considered. According to the literature, a fundamental model about nanoparticle stability in serum has been used for the predictive evaluation on the in vivo stability

57

. Generally, the preliminary serum-tolerance assessment was

conducted in 10% serum solution, which is a typical serum concentration in most in vitro cell culture studies

58

. In this study, we also used 10% FBS as experimental

condition. Similar to the case in water, a small amount of sediments appeared after 6 h standing and abundant precipitation were detected after 12 h for DOX@GO-SS-KLA (Figure 7A). However, nearly no sediment was detected on the 6th day for DOX@GOSS-KLA/BSA. With time, aggregation of DOX@GO-SS-KLA/BSA was observed on the 8th day (Figure 7B). Nevertheless, the BSA coating greatly improves the stability of GO based drug carrier in serum-containing solution. CONCLUSION In summary, a dual-sensitive (pH- and redox-) cancer-combination treatment system DOX@GO-SS-KLA/BSA for co-delivery of anticancer drug DOX and proapoptotic peptide KLA in tumor cells was fabricated. The stability of the system is greatly enhanced by the coating of BSA. Once the DOX@GO-SS-KLA/BSA arriving at tumor site, the loaded DOX and KLA can be released quickly due to the weakening of π-π conjugation and hydrogen bonding interaction between DOX and GO and the cleavage of disulfide bonds, respectively. The in vitro experimental results of cytotoxicity prove


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that the DOX@GO-SS-KLA/BSA could be easily uptaken by tumor cells and exhibit synergetic effect. This drug delivery system demonstrated in this study shows great potential in tumor therapy. SUPPORTING INFORMATION Detailed preparation of the azide acetic acid, azide-KLA, and additional figures. 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) Bray, F.; Jemal, A.; Grey, N.; Ferlay, J.; Forman, D. Global Cancer Transitions According to the Human Development Index (2008-2030): A Population-Based Study. Lancet Oncol. 2012, 13, 790-801. (2) Kouranos, V.; Dimopoulos, G.; Vassias, A.; Syrigos, K. N. Chemotherapy-Induced Neutropenia in Lung Cancer Patients: the Role of Antibiotic Prophylaxis. Cancer Lett. 2011, 313, 9-14. (3) Cheng, Z.; Al, Z. A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A. Multifunctional Nanoparticles: Cost Versus Benefit of Adding Targeting and Imaging Capabilities. Science 2012, 338, 903-910. (4) Dan, P.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751-760. (5) Park, J. S.; Yi, S. W.; Kim, H. J.; Kim, S. M.; Shim, S. H.; Park, K. H. SunflowerType Nanogels Carrying a Quantum Dot Nanoprobe for Both Superior Gene


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A Dual-sensitive Graphene Oxide Loaded with Proapoptotic Peptides

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