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Design and development of graphene oxide nanoparticle/ chitosan hybrids showing pH-sensitive surface chargereversible ability for efficient intracellular doxorubicin delivery Xubo Zhao, Zhihong Wei, Zhipeng Zhao, Yalei Miao, Yudian Qiu, Wenjing Yang, Xu Jia, Zhongyi Liu, and Hongwei Hou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16910 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 26, 2018

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ACS Applied Materials & Interfaces

Design and development of graphene oxide nanoparticle/chitosan hybrids showing pH-sensitive surface charge-reversible ability for efficient intracellular doxorubicin delivery

Xubo Zhao*#, Zhihong Wei#, Zhipeng Zhao#, Yalei Miao#, Yudian Qiu#, Wenjing Yang§, Xu Jia†, Zhongyi Liu*#, Hongwei Hou#

#

College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, China.

§

Department of Anesthesiology, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450002, China.

†

School of materials and Chemical engineering, Zhongyuan University of Technology, Zhengzhou 450007, China.

ABSTRACT: A novel GON-based drug delivery system containing graphene oxide nanoparticles (GON) as carriers of anticancer drugs and chitosan/dimethylmaleic anhydride-modified chitosan (CS/CS-DMMA) as surface charge-reversible shells is fabricated via the classic self-assembly of the deprotonated carboxyl of graphene oxide nanoparticles (GON) and the protonated amine of CS backbone by electrostatic interaction, and the CS-DMMA serves as outmost layer. In this GON-based drug delivery system, the GON cores as desired carriers might adsorb doxorubicin hydrochloride (DOX) via the π−π stacking interaction between the large π conjugated structures of GO and the aromatic structure of DOX. Meanwhile, the chitosan-based polyelectrolyte shells served as smart protection screen to evade the premature release of as-loaded DOX in normal extracellular condition, and then the release of DOX was accelerated due to the detachment of chitosan coating at low pH. Furthermore, the re-exposure of amino groups after hydrolysis of CS-DMMA endowed the drug delivery system with positive surface charge by taking advantage of the pH difference between physiological condition and tumor microenvironment to enhance the cellular uptake. And then pH-dependent site-specific drug release was realized. The in vitro investigations confirmed that this promising GON/CS/CS-DMMA hybrids with charge reversible character possessed various merits including excellent encapsulation efficiency, high stability at physiological condition, enhanced cellular uptake by HepG2 cells and tunable intracellular

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chemotherapeutic agent release profiles, proving its capability as an intelligent anticancer agent nanocarrier with enhanced therapeutic effects. This smart GON/CS/CS-DMMA vehicle with surface charge reversible character may be used as a significant drug delivery system for cancer treatment.

KEYWORDS: Graphene oxide nanoparticles, Chitosan, Charge reversal, pH-responsiveness, Drug delivery system

INTRODUCTION Recently, graphene and its derivatives have been researched extensively due to their unique mechanical, electrical, chemical, and physical properties deriving from a closely packed honeycomb two-dimensional lattice with sp2-bonded carbon atoms,1 which prompt it to serve as a promising candidate for a wide range of technological applications, such as supercapacitor, storage battery, biosensing, bioelectronics, and chemotherapeutic agent delivery.2-6 Especially for chemotherapeutic agent delivery, graphene oxide-based system has been attracted extensive attention in the past decade.7,8 It’s well known that graphene oxide possess favorable biocompatibility, large specific surface area, large π-conjugated structures, and abundant functional groups, which qualify it as a potential building material to fabricate desired drug delivery system.7,8 To further explore the biomedical application in cancer therapy, the graphene oxide must be modified by organic biomaterials. In most cases, the introduction of biocompatible polymer coating endowed the graphene oxide-based system with favorable biocompatibility and

highly stability.5 Meanwhile, the multi-functional polymer brushes not only prevented the premature leakage of DOX from graphene oxide-based vehicle in normal physiological medium but also enhanced its intracellular site-specific drug release at tumor tissue.7 As an ideal drug delivery system, size-dependence is of fundamental importance for deeply delivering chemotherapeutic agent into tumors to get rid of the reticuloendothelial system (RES).9-11 It’s well known that newly formed vessels contain enormous abnormal vascular structures with various pores with diameter of mean size of 400 nm on the vascular walls to promote extravasation of drug delivery system from the bloodstream into the tumor, namely enhanced permeability and retention (EPR) effect.12-15 Most importantly, it’s reported that the prolong blood circulation was a prerequisite for enhancing tumor accumulation based on the EPR effect.16 Nishiyama and coworkers found that the penetration and efficacy of the chemotherapeutic


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agents in the tumors depended on the size of nanomedicines.16 Meanwhile, pH difference between microenvironment of normal tissues and tumor tissues have been utilized to fabricate the drug delivery system. In particularly, the intracellular acidic pH inside lysosomes and endosomes (pH 5.0−5.5) in the cytoplasm and extracellular slight acidic media (pH 6.5−6.9) in tumor tissues could be applied to trigger the release of the anticancer agent compared to normal tissues.17 Wu’s group introduced an acid-sensitive Schiff base bond to render the polymeric micelles with typical pH-responsive property upon changes in chemical environment. They believed that the polymeric micelles might serve as an effective pH-responsive nanoparticle to improve drug delivery and enhance the antitumor efficacy.18 So the reasonable drug delivery system must possess smaller size and tumor microenvironment-responsiveness to efficiently deliver chemotherapeutic agent. In addition, the surface charge to the drug delivery system also is of fundamental importance for controlling in vivo fate.19 It is expected that the drug delivery system exhibit negative surface charge to endow DDS with high stability and a prolonged circulation time during blood circulation. On the other hand, for acidic extracellular medium of solid tumor, the surface charge of nanocarriers might display positive surface charge to easily enhance internalized by the typical cells, namely surface charge reversible property by pH-trigger.20,21 It’s well known that the nanocarriers with positive charge display higher affinity to cell membranes with negative charge and thus cellular uptake can be easily accomplished.22 When the nanocarriers possess the surface charge-reversal property, the drug delivery system could be rendered a stealth character to maintain stability during blood circulation and spontaneously enhance cell internalization toward acidic extracellular environment of solid tumor. Zhao and coworkers synthesized a tumor extracellular microenvironment-sensitive drug platform based on cisplatin(IV) prodrug-loaded charge-convertible carbon dots (CDs) for imaging-guided drug delivery. Owing to the introduction of charge-convertible CDs, the slightly acidic extracellular microenvironment of solid tumor could induce charge conversion of the nanocarriers to enhance internalization.23 In addition, negatively charged nanocarriers have various disadvantages, such as clearance from blood circulation, drug degradation in lysosomes, low therapeutic efficiency, and undesirable side effects.23 It is noteworthy that thayumanavan's group designed a lot of interesting drug delivery vehicle with surface charge variation features to determine its biological fate.24-26 Owing to surface charge variation features in response to slight changes in pH, these drug delivery vehicle exhibited



rapidly cellular uptake.24,26 Although the drug delivery system have the potential to enhance the stability and reducing the side effects of chemotherapeutic drug, yet it is extremely important to note that the fate of the nanocarriers after deposition within the human/animal body is essential for biomedical application.27,28 To alter the fate of nanocarriers within the human/animal body, the biodegradability of nanocarriers should be considered emphatically in the field of drug delivery. It is interesting that nanocarriers coated with typical polymer and natural biomaterials with biodegradability were more unstable than uncoated nanocarriers, such as polyethylene glycol, polysaccharides, and so on.

27,29

Among them, chitosan (CS) has been applied extensively to serve

as a building material in the biomedical field due to biodegradability and biocompatibility.30 On the other hand, traditional nanomaterials (such as conjugates) were cleared rapidly by the reticuloendothelial system and fail to deliver the chemotherapeutic drug to solid tumor.31 Compared with these nanomaterials, graphene oxide presented the low uptake in reticuloendothelial system and the long blood circulation time at 1 mg kg-1 body weight within days.32 Therefore, graphene oxide have been attracted extensively to serve as desired carrier for loading the chemotherapeutic drug.5,7 Meanwhile, spherical graphene oxide exhibited favorable biocompatibility and larger surface area than flaky graphene oxide for biomedical application. 5,7 With the above results taken into consideration, the chitosan and GON were chosen as building materials to determine the fate of GON-based nanocarriers after deposition within the human/animal body to effectively deliver chemotherapeutic drug. To solve the above-mentioned problems, our groups reported a series of GON-based nanocarriers to deliver anticancer drug.5,7 Herein, a charge reversible GON-based drug delivery system was fabricated via the classic self-assembly between the deprotonated carboxyl of GON nanoparticles and the protonated amine of chitosan backbone, and the outmost layer was dimethylmaleic anhydride (DMMA) modified chitosan as shown in Scheme 1. Owing to the introduction of DMMA modified chitosan, the GON-based nanocarriers displayed negative charge during blood circulation and could reverse to be positively charged at tumor extracellular microenvironment.23 The charge reversible character of the GON-based platforms was endowed by the re-exposure of amino groups after hydrolysis of dimethylmaleic acid (DMMA) at tumor extracellular condition by means of the pH difference between normal physiological condition (pH 7.4) and tumor


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extracellular microenvironment (pH 6.5-6.9) for controlling drug release.

17

Meanwhile, the

GON-based nanocarriers possessed highly DOX-loading capacity and excellent encapsulation efficiency because of the π-conjugated structures and the large specific surface area of GON.33-36 In addition, the chitosan coating was also detached from the GON-based nanocarriers upon slight acidic environment to further accelerate the DOX release. Thus, this GON-based drug delivery system displayed enhanced favorable biocompatibility as well as tumor-inhibition efficacy in vitro investigation, exhibiting significant potential to cancer therapy.

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Scheme 1. Schematic illustration of the fabrication, charge reversal and DOX release of the GON/CS/CS-DMMA by pH trigger.

EXPERIMENTAL SECTION Reagents and Materials. Dimethylmaleic anhydride (DMMA) was obtained by J & K Chemical Ltd. Chitosan (viscosity-average molecular weight of 1.0 × 106 and 2.0×103) were purchased from Zhejiang Yuhuan Marine Biotechnique Company. Original graphite powder material was provided by Huatai Chemical Reagent Co. Ltd. Doxorubicin hydrochloride (DOX) was purchased from Beijing Huafeng United Technology Co. Ltd. Cytotoxicity assay kits and WST-1 cell proliferation were supplied by Melonepharma. Dimethyl sulfoxide (DMSO), acetic acid, triethylamine (TEA), and sodium hydroxide (NaOH) were provided by Tianjin Chem. Co., Ltd. During the course of the experiment, deionized water was chosen to complete the related procedures. Preparation of GON Nanoparticles. The preparation of graphene oxide nanoparticles (GON)



were completed according to our group’s previous research with natural graphite powder as the raw material.5 Synthesis of CS-DMMA. The DMMA modified chitosan was synthesized by referring to the previous Liu group’s paper.20 500 mg of CS (viscosity-average molecular weight of 2.0×103) and 400 mg of DMMA were mixed into 50 mL of DMSO. Subsequently, 50 µL of TEA was transferred into the above mixture. And the reaction was performed by magnetic stirring at the room temperature for 24 h. The mixtures then were transfer into dialysis tube (MWCO = 1000 Da) against abundant water with pH value of 8-9 (adjusted by NaOH) for 72 h to remove excess DMMA. The solution of resulting CS-DMMA was freeze-dried and stored below -20 °C. Preparation of GON/CS/CS-DMMA. The polyelectrolyte layer-coated graphene oxide nanoparticles (GON) were crafted by the self-assembly of CS and GON. In brief, 0.2 g of CS was added into the aqueous dispersion (200 mL) containing 0.05 g of GON to complete the assembly of CS at pH 5.8 (adjusted by acetic acid) under magnetic stirring for 8 h. Subsequently, the centrifugation was performed to obtain the GON/CS nanoparticles through rinsing with abundant water. Then the GON/CS nanoparticles were added into the aqueous dispersion (150 mL) containing CS-DMMA (0.2 g) under magnetic stirring with the aid of slight ultrasound at pH 7.0 and the reaction was performed at the room temperature for 8 h. Finally, the above mixtures were centrifuged and rinsed with abundant water to obtain the desired polyelectrolyte layer-coated graphene oxide nanoparticles GON/CS/CS-DMMA with pH-activated charge reversible properties. Drug Loading and Triggered Release. Drug-loading and release of the GON/CS/CS-DMMA hybrid nanocarriers were carried out by referring to the our previous work.5 10.0 mg of GON/CS/CS-DMMA or GON nanoparticles were added into 5.0 mL of 2.0 mg mL−1 DOX solution at pH 7.4 with the aid of slight magnetic stirring for the DOX-loading. The typical condition (pH 5.0) was chosen to evaluate the DOX release from the DOX-loaded GON/CS/CS-DMMA hybrid nanoparticles or DOX-loaded GON nanoparticles to simulate the tumor microenvironment. As compared with the tumor intracellular microenvironment, the drug release performance of the DOX-loaded GON/CS/CS-DMMA hybrid nanocarriers and the DOX-loaded GON nanoparticles were investigated in PBS (pH 7.4 or pH 6.5) with the same procedure as the pH 5.0.


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Cell Toxicity Assays. The biocompatibility of GON/CS/CS-DMMA was investigated via the general WST-1 assay using HepG2 cell. Furthermore, chemotherapeutic agent DOX was utilized to conduct the cell toxicity assays of the drug delivery system using HepG2 cell. In the typical steps, the HepG2 cells were transferred into 96-well plates at densities of 1× 105 cells per well for 24 h. Subsequently, the GON/CS/CS-DMMA and DOX-loaded GON/CS/CS-DMMA with different concentrations were transferred into the related well and incubated for 24 h. Finally, WST-1 assay was utilized to estimate the cell viability of HepG2 cells. Intracellular uptake. The confocal laser scanning microscopy (CLSM) (LYMPUS FV-1000) was utilized to investigate the intracellular uptake of DOX-loaded GON/CS/CS-DMMA hybrid nanocarriers. After the incubation for 24 h using HepG2 cells, the characteristic excitation wavelengths of 405 nm and 480 nm were chosen to locate the Hoechst and DOX, respectively. Analysis and Characterization. The JEM-1200 EX/S transmission electron microscope (TEM) was chosen to evaluate the morphologies of GON and GON/CS/CS-DMMA. These above samples were dispersed in water by magnetic stirring with ultrasound-assisted procedure and then deposited on a copper grid covered with a perforated carbon film, dried at 45 °C in vacuum, respectively. To evaluate the organic polyelectrolyte contents adsorbed onto the GON nanoparticles, the thermogravimetric analysis (TGA) was applied to conduct at nitrogen atmosphere from 25 to 800 °C at 10 °C/min. 1

H NMR spectra of CS and CS-DMMA were recorded on 400 MHz with Bruker ARX 400

spectrometer (Bruker, Germany) using D2O as solvent, FT-IR spectra of GON, GON/CS, and GON/CS/CS-DMMA were recorded in the range of 400−4000 cm−1 with a resolution of 4 cm−1. To determine the average hydrodynamic diameter (Dh) and the stability of GON/CS/CS-DMMA, dynamic light scattering (DLS) measurements were carried out with a Light Scattering System BI-200SM device (Brookhaven Instruments). The ζ potentials of GON, GON/CS, GON/CS/CS-DMMA, and charge reversible property of GON/CS/CS-DMMA in unique pH value (pH 6.5) were investigated using a Zetasizer Nano ZS. Lambda 35 UV−vis spectrometer was utilized to validate the drug-loading and pH-triggered release behavior of GON/CS/CS-DMMA hybrid nanoparticles. The encapsulation efficiency and



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DOX-loading capacity were estimated according to: DOX encapsulation efficiency (DEE) (%) =

DOX-loading capacity (DLC) (mg mg-1) =

× 100%

The cumulative release (%) of DOX at a particular time (t) can be calculated according to: Cumulative release (%) =

∑

× 100%

!"#

where, Mtotal indicates the loading capacity of DOX within the nanocarriers, V0 and V′ indicate the initial volume of release media and the volume of collected release media at speciﬁc time intervals (V0 = 150 ml and V′=5 ml), respectively; Ct indicates the concentration of DOX in release media at the scheduled time interval t.

RESULTS AND DISCUSSION Preparation of GON/CS/CS-DMMA. As illustrated in Scheme 1, the biocompatible GON/CS/CS-DMMA nanocarriers was crafted via the classic self-assembly between the deprotonated carboxyl of graphene oxide nanoparticles and the protonated amine of chitosan backbone, and the outmost layer was DMMA modified chitosan. They could present the negative charge in neutral or basic media. Whereas, the re-exposure of amino groups after hydrolysis of CS-DMMA endowed the drug delivery system with positive surface charge in slight acidic media, revealing their potential application for enhancement of intracellular uptake. Meanwhile, the introduction of organic polyelectrolyte endowed the biocompatible GON/CS/CS-DMMA nanocarriers with excellent stability during the blood circulation. On the other hand, the polyelectrolyte layer prevented the premature leakage of anticancer drug from the drug-loaded GON/CS/CS-DMMA nanocarriers in the physiological condition. Furthermore, the chitosan coating could be detached from the GON/CS/CS-DMMA hybrids due to the instability of electrostatic interaction upon slight acidic environment to accelerate the DOX release. The new chemical shift at d = 3.08 ppm (b) was ascribed to the typical protons of DMMA in the 1

H NMR spectrum of CS-DMMA with respect to that of CS as illustrated in Figure 1A. In addition,

chemical shift at d = 1.96 ppm (a) was ascribed to the original acetamide group of chitosan. Meanwhile, based on the area ratio between peaks of b and a, the substitution values of DMMA in CS-DMMA could be evaluated. The substitution values of DMMA conjugated with chitosan were approximately 68.54% of the total amine functions in chitosan. After adsorption of CS, the new


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typical peak at 1556 cm-1 of C-N stretching vibration in amine group of skeleton of chitosan was found in the spectrum of the GON/CS as shown in Figure 1B.37 It indicated that the GON/CS hybrids were successfully fabricated by self-assembly. And then the CS-DMMA was self-assembled onto the surface of GON/CS via hydrogen bond interaction at pH 7.0 under magnetic stirring. In the spectrum of GON/CS/CS-DMMA, the typical characteristic absorbance at 1556 cm-1 of C-N stretching vibration in amine group of backbone of chitosan was obviously enhanced.37 Furthermore, in the spectra of GON/CS and GON/CS/CS-DMMA, the C-H stretch peak at 2935 cm−1 was also obviously enhanced (Figure 1B).7 These results demonstrated that CS-DMMA was adsorbed onto GON/CS hybrids via hydrogen bond interaction between the amine group, and hydroxyl in the skeleton of chitosan and the carboxyl, hydroxyl, and amine groups in the backbone of DMMA modified chitosan.

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Figure 1. 1H NMR spectra of CS-DMMA and CS (2k) (400 MHz) in D2O (A); FTIR spectra of GON, GON/CS, and GON/CS/CS-DMMA (B). Furthermore, the trends of zeta potentials were traced during the process of self-assembly to assess the growth of the polyelectrolyte as shown in Figure 4B. The negative sign of the GON was found due to the deprotonated carboxyl, which served as suitable templates for the anchoring of the protonated CS polycation via electrostatic interaction. With the deposition of the protonated CS polycation, the zeta potential value changed from -42.3 mV to 36.4 mV. This positive sign demonstrated that the protonated CS was anchored onto the surface of GON nanoparticles. After adsorption of the CS-DMMA, the resultant GON/CS/CS-DMMA hybrids exhibited negative sign with a zeta potential value of −26.5 mV, which revealed the existence of the DMMA. The successive process of self-assembly displayed a full reversal of every time adsorption. These



results indicated that GON/CS/CS-DMMA hybrids were fabricated successfully via self-assembly. GON was synthesized according to the published study.5 TEM was applied to trace the morphologies and sizes of samples as shown in Figure 2. The graphene oxide (GO) exhibited lamellar structures with few thin layers. As-prepared GON displayed the spherical morphology with rough surface and near-monodisperse size with a diameter of around 84 nm, which could be used to adsorb the polyelectrolyte by electrostatic interaction. As-prepared GON had similar morphology, surface, and size compared with previous studies.5,7 After the adsorption of chitosan and DMMA modified chitosan, GON/CS/CS-DMMA hybrids exhibited the similar morphology compared to the GON nanoparticles as presented in Figure 2C. Aside from the morphology, however, the diameter of GON/CS/CS-DMMA increased to approximately 114 nm due to the adsorption of CS and CS-DMMA. Meanwhile, the polyelectrolyte coating onto GON was obviously found in the vision, and the GON/CS/CS-DMMA displayed irregular surface compared to TEM images of GON. More importantly, the SEM images were also applied to trace the process of preparation of the GON/CS/CS-DMMA hybrids. As shown in Figure 2E, the GON/CS/CS-DMMA hybrids have much coarser surface and denser polyelectrolyte layers than smooth surface of GON, consistent with the TEM results. These results therefore suggested that the graphene oxide nanoparticles with polyelectrolyte coating were successfully fabricated via self-assembly.

Figure 2. The TEM images of GO (A), GON (B), and GON/CS/CS-DMMA (C); and the SEM images of GON (D) and GON/CS/CS-DMMA (E). To evaluate the organic polyelectrolyte contents adsorbed onto the GON nanoparticles, the TGA technique was applied to conduct using the GON nanoparticles and GON/CS/CS-DMMA hybrids as shown in Figure 3. The obvious small mass loss of around 12 wt% from two samples was ascribed to the release of free moisture and removal of the water at temperatures lower than


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160 °C .38,39 At temperatures of 230 °C, the GON nanoparticles exhibited large mass loss of 14 wt%, which attributed to the functional groups (-COOH and -OH).38 Overall, the TGA data for the GON/CS/CS-DMMA hybrids showed a weight loss of 54 wt %, whereas the GON nanoparticles had only a weight loss of 33 wt %. Based on these above-mentioned data, it could be evaluated that the GON/CS/CS-DMMA hybrids contain 48 wt% organic polyelectrolyte. The result therefore indicated that GON/CS/CS-DMMA hybrids were successfully fabricated by self-assembly between chitosan, and DMMA modified chitosan and GON nanoparticles. 100

GON 90

GON/CS/CS-DMMA

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Figure 3. TGA curves of GON and GON/CS/CS-DMMA. As a desired DDS, the nanocarriers size plays an important role in cancer therapy, namely getting away from the reticuloendothelial system (RES) to efficiently deliver anticancer drugs into tumors.9-11 Meanwhile, hydrodynamic diameter (Dh) also is a significant parameter to evaluate the size of DDS. So the DLS analysis was applied to evaluate the Dh of the GON, GON/CS, and GON/CS/CS-DMMA hybrids. As presented in Figure 4A, the typical Dh of GON, GON/CS, and GON/CS/CS-DMMA hybrids increased from around 113 nm, 160 nm to 181 nm with the increasing of number of layer, consistent with the TEM results. Obviously, all of the samples presented a narrow size distribution in Figure 4A. This phenomenon indicated that the GON/CS/CS-DMMA hybrids were successfully fabricated via self-assembly. Especially for GON/CS/CS-DMMA hybrids, the sample maintained the stable hydrodynamic diameter after dispersion over 24 h, indicating that the sample displayed desired stability in phosphate buffer solution. Basis on the results of TEM images and DLS, the GON/CS/CS-DMMA hybrids with reasonable size, hydrodynamic diameter, and favorable stability could be obtained. Charge reversal. It’s well known that the amide bond between DMMA and CS could be cleaved



under slight acidic conditions (such as pH 6.8).20 Owing to the instability, the GON/CS/CS-DMMA hybrids were dispersed into slight acidic media (pH 6.8) for 3 h, the zeta potential changes from −26.5 mV to 18.6 mV (Figure 4B), revealing the acid-sensitive detaching of DMMA from this hybrids. For comparison, the zeta potential of GON/CS/CS-DMMA hybrids maintained negative sign at neutral phosphate buffer solution over 6 h. This result indicated that the GON/CS/CS-DMMA hybrids were successfully prepared via self-assembly. But even more crucial, the detachment of DMMA from the GON/CS/CS-DMMA hybrids were confirmed by zeta potentials at related phosphate buffer solution.20,21 Owing to the surface charges reversal by triggering in slight acidic media, the GON/CS/CS-DMMA hybrids display potential application to enhance the cellular uptake for effectively delivering chemotherapeutic agents upon the extracellular slight acidic pH of the tumor environment against cancer.

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a

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Dh(nm)

Figure 4. The typical Dh distribution of the GON (A-a), GON/CS (A-b), and GON/CS/CS-DMMA (A-c), as measured by DLS; and zeta potentials of the GON (B-a), GON/CS (B-b), and GON/CS/CS-DMMA (B-c) at pH 7.4 and GON/CS/CS-DMMA (B-d) at pH 6.5. Biocompatibility and cell toxicity. As a DDS, the biocompatibility acts a significant role in biomedical application. To confirm the significance, the biocompatibility of GON/CS/CS-DMMA hybrids was investigated using WST-1 assay after the incubation of HepG2 cells. After incubation for 24 h, the viability of the HepG2 cells with treatment of the GON/CS/CS-DMMA hybrids was over and above around 96% during all the testing concentrations (Figure 5), implying that the GON/CS/CS-DMMA hybrids displayed favorable biocompatibility on the HepG2 cells. Such favorable biocompatibility is mainly attributed to the biocompatible chitosan and graphene oxide nanoparticles.7, 40


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Concentrationof GON/CS/CS-DMMA (µg/mL) 50 100 150 200 250 300 c

100

Cell Viability (%)

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80 60

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a 40 20 0 2

3 4 5 6 7 DOX equivalent dose (µg/mL)

8

Figure 5. Cell viability assay in HepG2 cells treated with GON/CS/CS-DMMA (c), DOX-loaded GON/CS/CS-DMMA at pH 7.4 (b), and DOX-loaded GON/CS/CS-DMMA at pH 6.5 (a) for 24 h. In addition, the related values were exhibited as the mean ± standard deviation (SD; n = 5). To further evaluate the inhibition against cancer cells, we incubated DOX-loaded GON/CS/CS-DMMA hybrids with treatment of HepG2 cells at various conditions. As shown in Figure 5, the DOX-loaded GON/CS/CS-DMMA hybrids toward HepG2 cells displayed obvious cytotoxicity at pH 6.5 after incubation for 24 h compared with that of pH 7.4. The cytotoxicity toward HepG2 cells at pH 6.5 was apparently higher than that of pH 7.4, indicating that transformation of surface charges from negative at pH 7.4 to positive at pH 6.5 enhanced cellular uptake and promoted delivery of DOX to cellular nuclei, further killed HepG2 cells. In addition, the viability of HepG2 cells treated with DOX-loaded GON/CS/CS-DMMA hybrids at pH 7.4 also exhibited the similar trends. It likely may be due to the slight intracellular acidic media triggering the DOX delivery from the DOX-loaded GON/CS/CS-DMMA hybrids after cell internalization to further inhibit the growth of HepG2 cells. In particularly, the intracellular environment inside lysosomes and endosomes exhibit obviously acidic feature (pH 5.0−5.5)41, which can trigger the DOX release from the DOX-loaded GON/CS/CS-DMMA hybrids. Furthermore, the relevance of the morphologies of HepG2 cells and corresponding cultured samples could be directly demonstrated by fluorescence inversion microscope system (Figure 6). Surprisingly, nearly all of HepG2 cells died after incubation of the DOX-loaded GON/CS/CS-DMMA hybrids at pH 7.4 or 6.5 for 24 h, while a great amount of the HepG2 cells survived after incubation of the GON/CS/CS-DMMA hybrids. On the basis of above analysis, these results are consistent with design hypothesis of GON/CS/CS-DMMA hybrids, namely the selective cell internalization by



charge reversal at typical acidic environment.

Figure 6. Characteristic micrographs of the HepG2 cells treated with GON/CS/CS-DMMA (A), DOX-loaded GON/CS/CS-DMMA at pH 7.4 (B), and DOX-loaded GON/CS/CS-DMMA at pH 6.5 (C) at 37 °C for 24 h. The scale bar presented 100 µm. Drug-Loading and in Vitro Triggered Release. Drug encapsulation efficiency (DEE) and Drug-loading capacity (DLC) are two key parameters to assess the performance of nanocarriers. It’s well known that modal DOX was loaded onto graphene oxide via π−π stacking interaction between the large π conjugated structure of GO and the aromatic structure of DOX.7,42 Furthermore, the abundant amine groups and hydroxyl of the backbone of chitosan could form a hydrogen bond via interaction with the amine groups of DOX.40 DOX could be therefore noncovalently loaded on the GON/CS/CS-DMMA hybrids by a hydrogen bond interaction and a π−π stacking interaction at pH 7.4 under the magnetic stirring. After the loading of DOX, the mixture was centrifuged and rinsed with abundant deionized water to obtain the DOX-loaded GON/CS/CS-DMMA. The efficient drug encapsulation efficiency (89.35% ± 4.32% (mean ± standard deviation, n=3)) and high drug-loading capacities (0.8935 mg/mg ±0.4320 mg/mg (mean ± standard deviation, n=3)) of the drug delivery system resulted from combination of the hydrogen bond interaction between DOX and CS and the π−π stacking interaction between the DOX and GON/CS/CS-DMMA hybrids. Meanwhile, the GON nanoparticles also possessed similar DOX encapsulation efficiency (%) and high DOX-loading capacities of 91.24% ± 4.08% and 0.9124 mg/mg ±0.4080 mg/mg (mean ± standard deviation, n=3), respectively. As a biocompatible and pH-activated charge reversible vehicle, the GON/CS/CS-DMMA hybrids not only have a certain DLC and DEE but also possess a relatively stable structure. The zeta potentials of the GON/CS/CS-DMMA hybrids and DOX-loaded GON/CS/CS-DMMA hybrids were also


Page 14 of 25

Page 15 of 25

determined, and the values were −26.5 ± 1.08 and −12.43 ± 0.41 mV, respectively. The changes of values to these zeta potentials successfully revealed that the DOX molecules have been loaded

90

0.6 pH=7.4

80 70 pH 7.4 GON pH 5.0 GON pH 7.4 GON/CS/CS-DMMA pH 6.5 GON/CS/CS-DMMA pH 5.0 GON/CS/CS-DMMA

60 50 40 30 20 10

Log of cumulative release

Cumulative release of DOX (%)

onto the GON/CS/CS-DMMA hybrids.

0 0

5

10

15

20

0.4 0.2 0.0 -0.2 -0.4

Y=0.73X-1.30 R2=0.97

-0.6 -0.8 0.8

25

Time (hours)

1.2

pH=6.5

0.6 0.4 0.2 0.0

Y=0.92X-1.29 R2=0.99

-0.2 -0.4 1.6

2.0

2.4


0.8

1.2

2.0

2.4

pH=5.0

1.0

0.8

1.6

Log of time

2.0

1.2


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


1.6 1.2 0.8 0.4

Y=1.04X-0.83 R2=0.98

0.0 0.8

1.2

Log of time

1.6 2.0 Log of time

2.4

Figure 7. Cumulative drug release from the DOX-loaded GON and DOX-loaded GON/CS/CS-DMMA in simulated body fluids. Determination of DOX by UV−vis analysis at 480 nm. Each value represented the mean (SD < 1%, n = 3) (A); Curves of Korsmeyer-Peppas models to the DOX release from DOX-loaded GON/CS/CS-DMMA under different conditions: pH 7.4 (B), pH 6.5 (C), and pH 5.0 (D). Furthermore, for the Korsmeyer-Peppas models, the portion of the release curve where cumulative release of DOX(%) < 60% should be used. Considering the differences between extracellular pH in tumor tissues and normal tissues, the three conditions of pH 5.0, pH 6.5 and pH 7.4 were chosen to simulate the microenvironment of tumor tissues and the normal physiological media, respectively. The cumulative release ratio to DOX-loaded GON/CS/CS-DMMA was only 5.10% at phosphate buffer solution (pH 7.4) within 24 h, mimicking the extracellular trafficking pathway (Figure 7A). As shown in Figure 7A, pH value of release media even decrease to 6.5, the cumulative release ratio to DOX-loaded GON/CS/CS-DMMA was only 19.45%. By contrast, the cumulative release ratio to DOX-loaded



GON exceed 20.0% at phosphate buffer solution (pH 7.4) within 24 h as shown in Figure 7A. Difference of cumulative release between DOX-loaded GON/CS/CS-DMMA and DOX-loaded GON indicated that CS/CS-DMMA coating effectively prevented the premature release of DOX at phosphate buffer solution (pH 7.4). To further simulate the drug release behavior of DOX-loaded GON/CS/CS-DMMA at intracellular environment of tumor tissues, the typical release condition was conducted at pH 5.0. A rapidly cumulative release of 56.41 wt % came true within 6 hours, subsequently the cumulative release displayed a sustained release profiles in the last 18 hours, and then the cumulative release ratio of DOX further reached to 84.75 wt % (Figure 7A). It’s well known that solubility of DOX was improved in slight acidic media,43 which disturbed the hydrogen bond interaction and π−π stacking interaction between DOX and GON/CS/CS-DMMA hybrids, and further accelerated diffusion of DOX. Furthermore, the chitosan coating could be detached from the GON/CS/CS-DMMA hybrids due to the instability of electrostatic interaction upon slight acidic environment to accelerate the DOX release.40,44 DOX-loaded GON exhibited faster cumulative release than DOX-loaded GON/CS/CS-DMMA in the same time range as shown in Figure 7A. This phenomenon was attributed to DOX-loaded GON without CS/CS-DMMA coating, which directly exposed the surface of DOX-loaded GON to accelerate the cumulative release of as-loaded DOX. With increasing of release time, the cumulative release ratio of DOX further reached to 82.20 wt %, which was consistent with that of DOX-loaded GON/CS/CS-DMMA. We therefore speculated that combination of the solubility enhancement of DOX and the detachment of the chitosan from GON/CS/CS-DMMA hybrids synergistically controlled the DOX release profiles at slight acidic media, mimicking the intracellular trafficking pathway in the tumor tissues. As a novel DDS, GON/CS/CS-DMMA hybrids exhibited enhanced stability at physiological media to efficiently inhibit the premature leakage of DOX due to the introduction of CS/CS-DMMA coating. Especially in slight acidic media, most DOX could be quickly released to inhibit the growth of tumor cells. We believe that it offers these significant merits to reduce severe side effects and improve therapy efficiency. The accumulative release data at pH 7.4, pH 6.5, or pH 5.0 at 37 °C were analyze using the semiempirical Korsmeyer-Peppas equation (Figure 7B, C, and D). The Korsmeyer-Peppas equation for the DOX-loaded GON/CS/CS-DMMA hybrids resulted in especial release exponents (n) and linearity with coefficients of correlation (R2) of 0.73 and 0.97, 0.92 and 0.99, and 1.04 and


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0.98, respectively. It yielded n of approximately 0.73 and 0.92 with comparatively good linearity, implying that the accumulative release of DOX were anomalous at pH 7.4 and 6.5, respectively. This release mechanism of DOX could be corresponded to a Case III mechanism or pseudo-Fickian at pH 7.4 and 6.5.45,46 As for the neutral media (pH 7.4) or slight acidic media (pH 6.5), the polyelectrolyte layers of chitosan were shrunk, hindering the cumulative release of the DOX molecules through the chitosan coating of the hybrids to result in lower cumulative release of only 5.10% or 19.45%. Meanwhile, as for pH 5.0, release exponent n was around 1 with favorable linearity, implying that release mechanism of DOX could be regarded as swelling-controlled drug release.47 Considering the detachment of the polyelectrolyte layers of chitosan of the GON/CS/CS-DMMA hybrids, which efficiently exposed surface of hybrids to accelerate the swelling-release of DOX at low pH (pH 5.0). Meanwhile, the solubility enhancement of DOX was another drive force, which also accelerated accumulative release of DOX in simulative acidic tumor microenvironment. As a consequence, the solubility enhancement of DOX and the detachment of the chitosan from GON/CS/CS-DMMA hybrids therefore synergistically control the burst release of DOX at slight acidic media, mimicking the intracellular trafficking pathway in the tumor tissues. Intracellular uptake. To visualize the intracellular uptake, the DOX-loaded GON/CS/CS-DMMA vehicle was evaluated using HepG2 cells by a CLSM technique at pH 6.5 and pH 7.4 as shown in Figure 8. Considering the investigation form the Zeta potential and WST-1 assay, the pH value of 6.5 served as incentive to trigger the surface charge reversal of the GON/CS/CS-DMMA hybrids to further enhance cellular uptake, and then enhanced transformation of DOX to cellular nuclei of tumor tissues. The dazzling red fluorescence of DOX molecules were found in cell nuclei as shown in Figure 8A, meaning the effective cell internalization of the DOX-loaded GON/CS/CS-DMMA vehicle and efficient intracellular release of chemotherapeutic agent DOX compared with that of pH 7.4. Meanwhile, as shown in Figure 8B, the most of red fluorescence of DOX molecules were detected at outside the cellular nuclei. This results also are consistent with design hypothesis of GON/CS/CS-DMMA hybrids, namely the selective cell internalization by charge reversal at typical acidic environment to enhance the cellular uptake. Remarkably, it should be further noted that almost all of the tumor cells died as shown in Figure 8A. But ever more crucial, it’s well known that DOX could easily diffuse through the endolysosomal membrane, and



Page 18 of 25

then entered the cell nuclei.5,24 Subsequently, the DOX molecules freely intercalated into single or double-strand of DNAs, resulting in the intranuclear accumulation of DOX to further disrupt the replication and transcription processes in tumor cells. This investigation was in accordance with the main mechanism of DOX’s antitumor activity as previous reports.5,7 It revealed that the GON/CS/CS-DMMA vehicle efficiently delivered the DOX to the cell nucleus. These results of DOX-loaded GON/CS/CS-DMMA vehicle to cancer cells kept consistent with previously reports.48-50 In addition, the result was also consistent with the conclusion of WST-1 assays as presented

in

Figure

5.

So

these

results

confirmed

engineering-hypothesis

of

the

GON/CS/CS-DMMA vehicle, namely the selective cell internalization at typical pH being induced through charge reversible behavior and acid activated release profiles by pH-trigger.

Figure 8. Intracellular uptake of the drug delivery system at pH 6.5 (A) and pH 7.4 (B) exhibited by CLSM in HepG2 cells after 24 h of incubation. Images from left to right presented the bright field, DOX fluorescence in cells (red), cell nuclei stained by Hoechst (blue), and the merged one. The scale bar represented 50 µm.

CONCLUSIONS In summary, the charge reversible GON/CS/CS-DMMA hybrids capable of responding to slight acidic microenvironment of solid tumor has been successfully fabricated to efficiently deliver DOX as well as enhanced cancer therapeutic efficiency. The GON/CS/CS-DMMA vehicle with charge reversal from negative charge during blood circulation (pH 7.4) to be positive charge at tumor extracellular microenvironment (pH 6.5) has enormous merits including excellent


Page 19 of 25 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


encapsulation efficiency, highly DOX-loading capacity, prolonged circulation time during blood circulation, enhanced cellular uptake by tumor cells, and tunable intracellular chemotherapeutic agent release profiles, which not only improved the drug delivery efficiency to overcome the limitation of GON-based nanocarriers with single function, but also reduced the side effects and further enhanced the tumor inhibition efficiency. Meanwhile, the Korsmeyer-Peppas equation implied that the release mechanism of DOX could be regarded as swelling-controlled DOX release at pH 5.0. The design of GON/CS/CS-DMMA vehicle with charge revisable character provides a novel route to develop an intelligent drug delivery system with enhanced therapeutic efficiency for promising tumor extracellular microenvironment-responsive vehicle in cancer therapy. AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]

[email protected]

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This project was granted financial support from the National Natural Science Foundation of China (Grant No. 21704093). REFERENCES 1. Akhavan, O.; Ghaderi, E. Toxicity of Graphene and Graphene Oxide Nanowalls against Bacteria. ACS Nano 2010, 4, 5731-5736. 2. Mohanty, N.; Berry, V. Graphene-based Single-Bacterium Resolution Biodevice and DNA Transistor: Interfacing Graphene Derivatives with Nanoscale and Microscale Biocomponents. Nano Lett. 2008, 8, 4469–4476. 3. Hong, W.; Bai, H.; Xu, Y.; Yao, Z.; Gu, Z.; Shi, G. Preparation of Gold Nanoparticle/Graphene Composites with Controlled Weight Contents and Their Application in Biosensors. J. Phys. Chem. C 2010, 114, 1822–1826. 4. Choi, B. G.; Park, H. S.; Park, T. J.; Yang, M. H.; Kim, J. S.; Jang, S. Y.; Heo, N. S.; Lee, S. Y.; Kong, J.; Hong, W. H. Solution Chemistry of Self-assembled Graphene Nanohybrids for High-Performance Flexible Biosensors. ACS Nano 2010, 4, 2910–2918.



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For Graphic Table of Contents Use Only

Design and development of graphene oxide nanoparticle/chitosan hybrids showing pH-sensitive surface charge-reversible ability for efficient intracellular doxorubicin delivery Xubo Zhao*#, Zhihong Wei#, Zhipeng Zhao#, Yalei Miao#, Yudian Qiu#, Wenjing Yang§, Xu Jia†,

R el

ea s

e

Zhongyi Liu*#, Hongwei Hou#

g

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


D ru

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chitosan

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