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Accepted Article Title: The Fabrication of rGO/(PLL/PASP)3@DOX Nanorods with pHSwitch for Photothermal Therapy and Chemotherapy Authors: Xiangming Li, Yihe Zhang, Zequn Ma, Meng Fu, and Qi An This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article. To be cited as: Chem. Eur. J. 10.1002/chem.201801884 Link to VoR: http://dx.doi.org/10.1002/chem.201801884

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FULL PAPER The Fabrication of rGO/(PLL/PASP)3@DOX Nanorods with pHSwitch for Photothermal Therapy and Chemotherapy Abstract: The development of well-controlled drug carriers that are stable and highly effective for the delivery of anticancer agents is challenging. Herein, we report a novel pH-controlled drug delivery system, utilizing reducing graphene oxide (rGO) -polymer selfassembly films as carriers, for the preparation of effective drug nanorods and nanoparticles. In this system, the rGO-polymer carriers were constructed by the alternating assembly of poly-L-lysine (PLL) and polyaspartic acid (PASP) around the rGO sheets. Furthermore, the rGO-polymer cores, which possess a positively charged surface as the desired template, could assemble with negatively charged doxorubicin (DOX) via electrostatic interactions. The DOX embedding efficiency and the morphology of the drug nanocomposites could be controlled by the number of rGO-polymer bilayers and concentration of the rGO-polymer bilayers and the initial DOX concentration. Importantly, the release of DOX could be regulated by controlling the pH and using a NIR laser. Under acidic conditions, the interactions between the PASP layer and DOX molecules can be broken, resulting in the gradual release of the DOX molecules. Upon NIR irradiation, the release of DOX could be further accelerated and a photothermal effect from rGO induced. Cellular uptake and cytotoxicity experiments indicated that the drug nanocomposites possessed effective anticancer activity. Thus, in this work, we present a useful strategy for the fabrication of pH-responsive drug nanocomposites for combined photothermal and chemical therapy. The nanocomposite can be used as a potential drug delivery system for practical cancer treatment.

Introduction Cancer is one of the most prolific threats to human health resulting in tens of millions of deaths per year.[1] The development of effective cancer treatments thus remains an important challenge. Traditional chemotherapy is one of the most common cancer treatments and has been widely employed; however, multidrug resistance (MDR) of cancer cells often hampers its anticancer efficacy and leads to therapeutic failure. [2-3] Recently, nanoscale drug carriers have been reported as an effective method to circumvent MDR and offer a remarkable intracellular drug delivery mechanism to improve anticancer efficacy.[4-10] Reduced graphene-based drug delivery systems have attracted the most attention owing to their unique physical, electrical and chemical properties, and they have been widely used as biomedical

X. Li, Prof. Dr. Y. Zhang, Z. Ma, M. Fu, Dr. Q. An. Beijing Key Laboratory of Materials Utilization of Nonmetallic Minerals and Solid Wastes, National Laboratory of Mineral Materials School of Materials Science and Technology, China University of Geosciences, Beijing, 100083, China. E-mail: [email protected]; [email protected] Supporting information for this article is given via a link at the end of the document.

treatments, particularly for use in photothermal therapy (PTT) and cell imaging.[11-22] PTT is one of the most prominent methods for the application of rGO-based nanodrug systems.[23] Compared with traditional chemotherapy, PTT takes advantages of direct heating of the cancer cells and results in minimal damage to surrounding tissue.[24] Hence, photo-chemotherapy is regarded as an efficient approach for cancer therapy. However, there are a number of limitations concerning the use of native rGO as a direct nanodrug carrier for PTT, such as, irreversible aggregation, high cytotoxicity, poor colloidal dispersibility, and the use of a limited range of functional groups due to its nonspecific binding with proteins or ions. These disadvantages hinder the wider adoption of rGO. Thus, further modification of native rGO is highly desired so that nanocomposites that are stable and possess low cytotoxicity can be synthesized and used as nanodrug carriers. [25-28] One of the most simple and useful methods utilizes polymers for assembly with rGO to form multilayer templates via supramolecular interactions (such as electrostatic interactions and hydrogen bonding). Compared with traditional covalent methodologies, supramolecular self-assembly allows for the facile modification of rGO. For example, Shi and co-workers reported an operationally simple procedure for the preparation of water-soluble graphene via π−π stacking between graphene and pyrenebutyrate. [29] The Justin group used chitosan to modify the rGO surface via hydrogen bonding, and then employed them the nanocomplex as drug delivery devices.[30] Another study from Yang’s group showed a simple approach which used PEG to coat rGO via noncovalent interactions, and then explored the remarkable performance of rGO-based composites in in vivo cancer treatment.[31] It's worth noting that self-assembled rGO-polymer nanotemplates possess many potential responsive functions when they are used for the loading of anticancer drugs, such as, exhibiting pH and NIR responses. In this study, we report a facile and efficient approach for the preparation of pH responsive self-assembled (electrostatic interactions) rGO-polymer-DOX nanocomposites for photothermal and chemical therapy. As shown in Scheme 1, in this system, the rGO/(PLL/PASP)n self-assembled multilayers were prepared by using PLL and PASP that alternately assemble around the rGO template via the use of electrostatic interactions. The positively charged DOX molecules could then be loaded by the rGO/(PLL/PASP)n nanotemplate via electrostatic selfassembly or absorption to form the rGO/(PLL/PASP)n@DOX nanocomposite. One of the most interesting observations was that the DOX molecules could penetrate the interior of the rGO/(PLL/PASP)n bilayers and the DOX embedding efficiency could be improved by increasing the number of the bilayers. In addition, the DOX embedding efficiency and morphology of the rGO/(PLL/PASP)n@DOX could be further regulated with different concentrations of the rGO/(PLL/PASP)n@DOX dispersions and the initially added DOX. Next, the release of DOX from the rGO/(PLL/PASP)n@DOX could be regulated by pH control and a

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Xiangming Li, Yihe Zhang*, Zequn Ma, Meng Fu, Qi An*

10.1002/chem.201801884


Scheme 1. Schematic of A) PLL and PASP alternate self-assembly on a rGO template; B) rGO/(PLL/PASP)3@DOX fabrication, and pH and photo-thermal controlled drug release resulting in cancer cell death.

NIR laser. The PASP was protonated under acidic conditions resulting in the interactions between PASP and DOX, and PLL and PASP, being broken. Thus rGO/(PLL/PASP)n@DOX nanocomposites were able to disassemble under acidic conditions and DOX was gradually released. Furthermore, the disassembled PLL destroys the membrane of the cancer cells allowing the DOX molecules to diffuse into the cells more readily. NIR irradiation further enhanced the rate of the DOX embedding efficiency and induced a photothermal effect from the rGO component to kill cancer cells. Cytotoxicity experiments indicated that the rGO/(PLL/PASP)n@DOX nanocomposites show toxicity towards HeLa cells, with further cell death resulting from the photothermal effect of the rGO/(PLL/PASP)n@DOX nanocomposites. In addition, intracellular uptake experiments showed that the rGO/(PLL/PASP)n@DOX nanocomposites possessed the ability to enter Hela cells. Thus, rGO/(PLL/PASP)n@DOX nanocomposites exhibit enhanced biocompatibility in addition to in vitro cancer cell inhibition, suggesting that they have the potential for application in cancer therapy.

Results and Discussion Characterization of rGO/(PLL/PASP)n The successful formation of LbL polymer multilayers around rGO nanosheets was determined using UV-vis spectroscopy, zeta potentials and AFM. The characteristic peak at 270 nm in the UVvis spectrum confirmed the successful reduction of GO to rGO (Figure 1A). The rGO-polymer complex showed alternative positive and negative potentials at approximately +40 mV and -25 mV, respectively (Figure 1B). The initial potential of rGO was negative. Following assembly of PLL, the potential changed from a negative to a positive value. When PASP was assembled, the potential changed from a positive to negative value again. These results indicated the successful assembly of PLL and PASP

around the rGO sheet. TEM images of the rGO/PLL/PASP, rGO/(PLL/PASP)/PLL and rGO/(PLL/PASP)3 showed that the rGO-polymer complex maintains a quasi-2D-morphology with a rougher surface (Figure 1C-D & Figure S1). Compared with rGO/PLL/PASP, the thickness of rGO/(PLL/PASP)/PLL was greater, indicating that the polymer PLL successfully assembled around the rGO/PLL/PASP. The accurate thickness of the rGOpolymer complexes was verified by AFM; the thickness of the rGO/(PLL/PASP)/PLL was measured to be 6.3 nm, whereas the thickness of the rGO/PLL/PASP was measured to be 5.2 nm (Figure 1E-F). On the basis of the above morphological results, it could be determined that the rGO-polymer complex could be prepared by a facile self-assembly using electrostatic interactions; the nanocomposites were nicely dispersed with minimal aggregation. Fabrication of rGO/(PEG/PAA) n@DOX nanorods The rGO/(PLL/PASP)n@DOX complex was obtained via classic self-assembly between the carboxyl group of PASP and the protonated amine of DOX. While the pure DOX could not selfassemble into nanorods, even it is at the same condition of the rGO/(PLL/PASP)3@DOX. The UV-vis spectrum of the free DOX and the rGO/(PLL/PASP)3@DOX was shown in Figure 2A; the characteristic absorption peak at 480 nm indicated the successful loading of DOX by rGO/(PLL/PASP)3. And the characteristic absorption peak at 940 nm confirmed the near-infrared absorption of the rGO/(PLL/PASP)3@DOX. The loading effect is 0.078 mg/mL (the original concentration of DOX is 0.25 mg/mL). ATRFTIR spectroscopy was used to further confirm that the DOX successfully assembled with the rGO/(PLL/PASP) 3. After assembly with DOX, a new peak at 1708 cm−1 corresponding to a C=O stretching vibration of the DOX was observed in the spectrum of the rGO/(PLL/PASP)3@DOX complex, as shown in Figure S2. Furthermore, the C−H stretching peak at 2935 cm−1 was also obviously enhanced, indicating the presence of DOX. The inside images, (a) and (b) in Figure 2A, represented the


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Figure 1. A) UV-vis spectra of rGO/(PLL/PASP)3; B) Zeta potential of different rGO/(PLL/PASP)n layers; TEM images of C) rGO/PLL/PASP and D) rGO/(PLL/PASP)/PLL; AFM images and height prolifes of E) rGO/PLL/PASP and F) rGO/(PLL/PASP)/PLL.

aqueous dispersions of rGO/(PLL/PASP) 3 and rGO/(PLL/PASP)3@DOX, respectively. It can be seen that the dispersions both possessed good stability in water. As shown in Figure 2B, TEM was used to explore the morphologies and sizes of the rGO/(PLL/PASP)3@DOX nanorods. TEM showed the small size of the rod-like structures of the rGO/(PLL/PASP)3@DOX nanocomposites with a length of approximately 60 nm and a diameter of approximately 10 nm. The nanorods were embedded onto a larger nanosheet, and the large size of the sheets should facilitate multivalent interaction with cell membrane and lead to effective adhesion, which should contribute to the high effect of the therapy effect. This size is beneficial to uptake by the cancer cells.[32-35] For example, Medintz and co-worker demonstrated that the smaller gold nanoparticles (about 10 nm) enter into the cells more easily.[36] SEM showed that most of the DOX was inside of the rGO/(PLL/PASP)3 film (Figure S3). It is worth mentioning that the DOX embedding efficiency of the rGO/(PLL/PASP)n@DOX can be regulated by changing the number of bilayers of rGO/(PLL/PASP)n. As shown in Figure 2C, the intensity of the characteristic absorption peak at 480 nm was higher when the number of bilayers was increased, indicating that the greater the number of bilayers, the higher the DOX embedding efficiency. This phenomenon can be explained as follows: The carboxyl groups in PASP were negatively charged following protonation and were thus able to assemble with the positively charged DOX molecules via electrostatic interactions. Furthermore, due to the polymer layer of the rGO/(PLL/PASP) n having a not very high density, many small gaps were present these layers. The DOX molecules were able to enter the interior

of the rGO/(PLL/PASP)n through these polymer gaps and assemble with the internal PASP layer. Thus, a greater number of bilayers allowed for a higher number of DOX molecules to assemble with the PASP, and thus more DOX molecules could enter the rGO/(PLL/PASP)n complex. The adjustability of the DOX embedding efficiency of the rGO/(PLL/PASP)n@DOX highlights the advantage of our preparation strategy which allows for the increased loading of DOX by increasing the number of bilayers of rGO/(PLL/PASP)n. Meanwhile, the morphology of the rGO/(PLL/PASP)n@DOX nanocomposite could be influenced by changing the number of layers of rGO/(PLL/PASP) n. When the outermost layer was a PASP layer, such as rGO/(PLL/PASP)1 or rGO/(PLL/PASP)2, a hybrid complex consisting of nanodots was formed, as shown in Figure S4A&C. When the outermost layer was a PLL layer, such as rGO/(PLL/PASP) 1.5 or rGO/(PLL/PASP)2.5, a hybrid complex was formed as spherical nanoparticles, as shown in Figure S4B&D. As many studies have reported,[37-39] the fluorescence of the DOX molecules could be quenched by rGO. To further confirm the assembly of the DOX molecules with the rGO/(PLL/PASP)n and that they can indeed enter the bilayer of the rGO/(PLL/PASP)n, the fluorescence of various rGO/(PLL/PASP)3@DOX nanocomposites prepared with different concentrations of rGO/(PLL/PASP)3 were explored. The suspension of the rGO/(PLL/PASP)3@DOX nanocomposite was equal to that of the DOX concentration. As shown in Figure 2D, the intensity of fluorescence of the rGO/(PLL/PASP) 3@DOX nanocomposite gradually decreased with increasing concentrations (20, 40, 80, 160 μg/mL) of rGO/(PLL/PASP)3. The results indicated that DOX molecules attached to the surface of the rGO nanosheets and successfully entered rGO/(PLL/PASP) 3. Additionally, it could be seen that the higher the concentration of rGO/(PLL/PASP)3, the greater the potential loading of the DOX molecules.

Figure 2. A) UV−vis spectra of free DOX and rGO/(PLL/PASP)3@DOX. The inset shows the stability images of: a) rGO/(PLL/PASP)3 and b) rGO/(PLL/PASP)3@DOX; B) TEM image of rGO/(PLL/PASP)3@DOX nanorods; C) UV−vis spectra of rGO/(PLL/PASP)n@DOX prepared with different number of layer; D) Fluorescence spectra of rGO/(PLL/PASP)3@DOX prepared with different concentrations of rGO/(PLL/PASP)3.


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Figure 3. A) Illustration showing the pH and NIR controlled release of rGO/(PEG/PAA)3@DOX and local heat of rGO/(PEG/PAA)3; Cumulative DOX release (B) and Korsmeyer-Peppas models curves of the DOX release (C&D) from the rGO/(PLL/PASP)3@DOX at pH 7.4 and 6; E) The photothermal performance of the rGO/(PLL/PASP)3@DOX nanorods; F) Cumulative release of DOX from the rGO/(PLL/PASP)3@DOX in the presence or absence of NIR laser.

The DOX embedding efficiency is not only affected by the bilayer number of the rGO/(PLL/PASP)3 but is also affected by the initial concentration of the added DOX. As shown in Figure S5, the intensity of the 480 nm peak increased when the initial concentration of the added DOX was increased from 0.05 mg/mL up to 0.35 mg/mL. When the initial concentration was 0.05 mg/mL, the DOX embedding efficiency was 8.46%, which represents low efficiency. However, when the initial concentration was increased to 0.35 mg/mL, the DOX embedding efficiency rose to 61.86%, whereas it was 33.48% and 30.56% for concentrations of 0.15 mg/mL and 0.25 mg/mL, respectively (Figure S6). These results indicated that the DOX embedding efficiency could be effectively controlled by altering the initial concentration of the added DOX. Further studies examining the concentration of added DOX and its effect on the morphology of the rGO/(PLL/PASP) 3@DOX nanocomposite, were conducted. As shown in Figure S7, when the concentration of added DOX was 0.05 mg/mL and 0.15 mg/mL, amorphous rGO/(PLL/PASP)3@DOX composites were formed. When the concentration of added DOX was 0.25 mg/mL and 0.35 mg/mL, rGO/(PLL/PASP)3@DOX nanorods or nanoparticles were respectively formed. In Vitro Triggered Release Controlled by pH and PhotoThermal Effect As illustrated in Figure 3A, a combination of pH response and photothermal effect of the rGO/(PLL/PASP)3@DOX

nanocomposite allows for the control of drug release and thus cancer treatment. Under an ambient temperature and acidic conditions, the electrostatic interactions between PASP and DOX can be broken; the DOX molecules disassemble with the PASP layer and are released from the rGO/(PLL/PASP) 3@DOX nanocomposites thus resulting in cancer cell death. An NIR laser is able to further accelerate the release of DOX and induce the photothermal effect of rGO. Considering that the extracellular pH between cancer cells and normal cells is different, an acidic solution (pH 6.0) and PBS buffer solution (pH 7.4) were chosen to simulate the microenvironment of cancer cells and normal physiological media, respectively. As expected, a rapid cumulative release ratio of 48.5% of the rGO/(PLL/PASP)3@DOX nanocomposite was observed in acidic solution (pH 6) within 24 h, as shown in Figure 3B. By contrast, the cumulative release ratio in PBS solution (pH 7.4) was only 21.4% within 24 h. As shown in Figure S8, the rGO/(PLL/PASP)3@DOX nanocomposites incubated in acid solution for 2 days could disassemble into smaller nanoparticles, indicating that the electrostatic interactions in the rGO/(PLL/PASP)3@DOX nanocomposites could be broken in acid solution. The results indicated that the rGO/(PLL/PASP)3@DOX nanocomposite exhibited a sensitive response to pH. The semi-empirical Korsmeyer-Peppas equation[40-42] was used to further analyze the accumulative release data at pH 6 and pH 7.4, as shown below: (1) (2) Where Mt indicates the concentration of DOX which is released from the rGO/(PLL/PASP)3@DOX nanocomposite at the diffusion time t; M∞ indicates the total concentration of DOX which is released from the rGO/(PLL/PASP)3@DOX nanocomposites at equilibrium; k represents the apparent rate constant; and n is the diffusion index. Eq (1) is for Mt/M∞ ≤0.6. Eq (2) is a variant of Eq (1). The mechanism of release of the DOX molecules from the rGO/(PLL/PASP)3@DOX nanocomposites can be described as follows. For nanoparticles and nanorods, there are four distinguishable modes of diffusion which are dependent on the value of n: i) Case I, n = 0.5 refers to Fickian diffusion, which represents steady-state diffusion; ii) Case II, n = 1 refers to nonFickian diffusion or swelling-controlled drug delivery, which uses the hydrophilicity of the drug carrier to swell and then release the drug; iii) Case III, the value of n is between 0.5 and 1, suggesting a combination of Cases I and II; iv) Case IV, n ≤ 0.5 representing a pseudo-Fickian diffusion, where the sorption curves are similar to Fickian diffusion curves, but the approach to final equilibrium is very slow. The value of n and k can be easily obtained according to eq (2), as shown in Figure 3C&D. There are two strategies for the release of DOX from the rGO/(PLL/PASP)3@DOX complex at pH 6 or pH 7.4. For the first strategy, the special diffusion index (n) and linearity with coefficients of correlation R 2 for the release of the rGO/(PLL/PASP)3@DOX nanocomposite in solution at pH 6 or pH 7.4 were 0.57/0.99 and 0.3/0.95, respectively. The value


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of n was between 0.5 and 1, indicating that the mechanism of release of DOX from the rGO/(PLL/PASP)3@DOX nanocomposite at pH 6 was a combination of both Cases I and II. This signified that the polymers in the rGO/(PLL/PASP) 3@DOX nanocomposite were able to contribute to the release of DOX at pH 6. When the DOX molecules were released from the rGO/(PLL/PASP)3@DOX complex at pH 7.4, n < 0.5, indicating a mechanism corresponding to Case IV in which a slow release occurs until a final equilibrium was attained. In the second strategy, the n values of the rGO/(PLL/PASP)3@DOX nanocomposites at pH 6 and pH 7.4 were both below 0.1. This signified that the mechanism of release of DOX from the rGO/(PLL/PASP) 3@DOX nanocomposite with this strategy involves a pseudo-Fickian diffusion (Case IV), irrespective of the pH. In addition, the rGO/(PLL/PASP)3@DOX nanocomposites possessed a remarkable photothermal effect. The rGO/(PLL/PASP)3@DOX dispersion (0.25 mg/mL) resulted in an increase in temperature from 26.4 °C to 38.1 °C (the temperature almost increased 12 °C while it increased 4.6 °C and 7.6 °C when the concentrations of the rGO/(PLL/PASP) 3@DOX dispersion were 0.05 mg/mL and 0.15 mg/mL), indicating that our nanodrugs exhibited an effective photothermal effect, originating primarily from the rGO component (Figure 3E & Figure S9A). Figure S9BC showed the thermal profile of the rGO/(PLL/PASP)3@DOX dispersion. According to Roper's report, the photothermal conversion efficiency[43-45], η, could be calculated by using Eq (3):

(3) where h is heat transfer coefficient, S is the surface area of the container. The Tmax is the equilibrium temperature, Tsurr represents ambient temperature. (Tmax - Tsurr) is 12.1 °C according to Figure S9B. Qdis is the loss of heat in this system, and it is calculated independently to be 10.5 mW by using a quartz cuvette cell containing pure water without the rGO/(PLL/PASP) 3@DOX nanocomposites. I is the power of laser (1 W/cm2), A940 is the absorbance of the rGO/(PLL/PASP)3@DOX at 940 nm (Figure 2A, value is 0.364). The value of hS can be obtained from Eq (4) and the Figure S9C:

(4) where τs is the sample system time constant, mD and CD are the mass (1 g) and heat capacity (4.2 J g-1 °C-1) of deionized water used as solvent, respectively. Thus, the photothermal conversion efficiency of the rGO/(PLL/PASP)3@DOX (0.25 mg/mL) heated by 940 nm laser can be calculated to be 24.3%. Meanwhile, upon NIR irradiation (the power density is 1 W/cm2), the diffusion of the DOX molecules released from the rGO/(PLL/PASP)3@DOX composites could be accelerated (Figure 3F). The DOX embedding efficiency of the rGO/(PLL/PASP)3@DOX complex irradiated by the NIR laser reached 40.1% within 3.5 h; in the absence of irradiation, it was only 23.8%. This result showed that the photothermal property of

Figure 4. A-C) CLSM image of HeLa cells after incubation for 24 h with rGO/(PLL/PASP)3@DOX. Images from left to right show a DOX fluorescence in cells (red), cell nuclei stained by Hoechst (blue), and then merged one; Cell viability assay: D) HeLa cells incubated with free DOX, different dosage of rGO/(PLL/PASP)3 or rGO/(PLL/PASP)3@DOX for 24 h, E) HeLa cells incubated with rGO/(PLL/PASP)3 or rGO/(PLL/PASP)3@DOX in the presence or absence of NIR laser (the power is 1 W/cm2).

the rGO/(PLL/PASP)3@DOX was able to effectively regulate the cumulative release of DOX. Intracellular Uptake and Cell Toxicity To investigate the intracellular uptake, rGO/(PLL/PASP) 3@DOX nanorods incubated with Hela cells were evaluated using CLSM studies, as shown in Figure 4A-C. The images, from left to right, showed the autofluorescence of the DOX (red), cell nuclei with Hoechst stain (blue), and a combination of both. According to the CLSM measurements, the autofluorescence of the DOX molecules originating from the rGO/(PLL/PASP) 3@DOX nanorods was predominantly located in the nucleus of the cells, indicating that the rGO/(PLL/PASP)3@DOX nanorods were effectively adsorbed by the endosomes after endocytosis. To confirm the disassemble PLL from the rGO/(PLL/PASP)3@DOX nanocomposites could further destroy the membrane of Hela cells, Hela cells were incubated with pure PASP, free DOX and rGO/(PLL/PASP)3@DOX nanocomposites for 24 h. And then the trypan blue was used to detect cell membrane integrity. The trypan blue can only enter into the cells with broken membrane and display a blue mark under the optical microscope.[46-47] As shown in Figure S10, trypan blue could go into the Hela cells incubated with free DOX and rGO/(PLL/PASP)3@DOX while it could not go into the control group and the Hela cells incubated with pure PASP, indicating that the membrane of the Hela cells was destroyed by the free DOX or rGO/(PLL/PASP)3@DOX. On the other hand, compared with free DOX, more trypan blue entered into the Hela cells incubated with rGO/(PLL/PASP)3@DOX. The result indicated that the disassembled PLL could destroy the cell membrane allowing the DOX molecules to diffuse into the cells more readily to kill cancer cells. As a desired drug delivery system, resistance to cancer cells plays a significant role in many practical biological applications.


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Conclusions In summary, we have developed a controllable, stable, and highly efficient methodology for the fabrication of rGO/(PLL/PASP)n@DOX nanocomposites possessed pH-switch and photothermal effect via electrostatic self-assembly. The release of DOX from the prepared hybrid nanocomplex can beregulated via adjustment of the pH and NIR laser. Additionally, the disassembled PLL could destroy the membrane of cancer cells allowing for easier delivery of DOX. Upon NIR irradiation, the rate of DOX release could be further increased and inducing an efficient photothermal effect from the rGO component of the nanocomposite. At pH 6.0, the mechanism of release of rGO/(PLL/PASP)n@DOX could be regarded as a combination of Fickian diffusion and swelling-controlled DOX release, according to the Korsmeyer-Peppas equation. In vitro cell experiments suggested that the rGO/(PLL/PASP)n@DOX nanocomposite possessed a remarkable ability to enter and kill cancer cells. In addition, NIR irradiation further reduced the viability of cancer cells incubated with rGO/(PLL/PASP) n@DOX. Therefore, the rGO/(PLL/PASP)n@DOX nanocomposite is a promising candidate that combines a pH response and NIR response for the chemical and photothermal treatment of cancer cells.

Experimental Section Materials. Graphene oxide was obtained from XFNANO (Jiangsu, China). Doxorubicin hydrochloride (DOX) and poly-L-lysine (2 KDa) was purchased from Aladdin of China. Polyaspartic acid was obtained from Yuan Ye Co., Ltd. (Shanghai, China). Hydrazine hydrate (80%) was purchased from Sinopharm Chemical Reagent Co. Ltd, (Beijing, China). Preparation of rGO/(PLL/PASP)n. The rGO/(PLL/PASP)n nanosheets were prepared via layer-bylayer assembly technology. Briefly, 20 mL of graphene oxide (0.25 mg/mL) was prepared and subjected to ultrasound cell smashing for 10 min to form a stabilized dispersion. The dispersion of graphene oxide was added into 20 mL of poly-L-lysine solution (5 mg/mL) with a low stirring rate to avoid agglomeration. The mixture was stirred for 20 min and then heated at 80 °C for 2 h to reduce the graphene oxide (using 15 μL of hydrazine hydrate (80%) as a reducing agent). The generated rGO/PLL was centrifuged and washed with deionized water three times. The product was then dispersed in 20 mL deionized water to give uniformly dispersed product, which was added dropwise into polyaspartic acid solution (5 mg/mL). The mixture was stirred with a low stirring rate for 20 min to allow for modification of PASP around the rGO/PLL cores. The generated rGO/PLL/PASP was centrifuged and washed with deionized water three times. The rGO/(PLL/PASP)n was obtained by controlling the desired number of bilayers using the same assembly method. Fabrication of rGO/(PLL/PASP)3@DOX nanorods. To prepare the drug loading nanorods, 1 mL of the rGO/(PLL/PASP)3 dispersion (0.25 mg/mL) was mixed with 1 mL of DOX solution (0.25 mg/mL) and stocked for 24 h at room temperature in the dark. The mixture was then centrifuged at 10000 rpm for 20 min to remove excess DOX and washed with PBS solution (0.1 M, pH 7.4) three times. The rGO/(PLL/PASP)n@DOX nanocomposites, with differing amounts of the bilayer and different concentrations of DOX, were prepared using the same method mentioned above. The DOX embedding efficiency (DEE) of rGO/(PLL/PASP)n@DOX was calculated using the following Eq (5).

(5) where Mtotal represents the initial amount of DOX initiator, and MDOX is the amount of DOX in the rGO/(PLL/PASP)n@DOX. Photothermal effect of the rGO/(PLL/PASP)3@DOX nanorods. The rGO/(PLL/PASP)3-DOX dispersions were irradiated by NIR light with a power density of 1.0 W/cm2 (940 nm) for 35 min. The temperature of the dispersion was recorded by an infrared thermometer at 5 min intervals. Cellular uptake and Distribution study. The in vitro cellular uptake and distribution studies were performed using confocal laser scanning microscopy (CLSM). Typically, Hela cells were seeded at a density of 4.0 ×104 per well into a confocal chamber and incubated overnight. Afterwards, 60 μL of rGO/(PEG/PAA)3@DOX was added into the chamber and


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As shown in Figure 4D, the Hela cells were incubated with free DOX (0.25 mg/mL), varying doses of the rGO/(PLL/PASP)3 nanofilm (0.25 mg/mL) and rGO/(PLL/PASP)3@DOX nanorods (0.25 mg/mL). The rGO/(PLL/PASP)3 nanofilm exhibited low cytotoxicity towards cells. Contrastingly, the rGO/(PLL/PASP)3@DOX nanorods incubated with Hela cells displayed obvious cytotoxicity after 24 h. The cell viability decreased to 47 % upon addition of 60 μL of the rGO/(PLL/PASP)3@DOX dispersion; cell viability was 78 % and 58 % for 20 μL and 40 μL of the rGO/(PLL/PASP)3@DOX nanocomposite dispersion, respectively. When compared with free DOX, the rGO/(PLL/PASP)3@DOX showed a superior effect to kill cancer cells at the same dosage. These results demonstrated that the cell viability could be controlled by altering the dosage of rGO/(PLL/PASP)3@DOX nanorods and further reduced by adding a small amount of the drug composites. To further evaluate the photothermal performance of the rGO/(PLL/PASP)3 nanofilm and the rGO/(PLL/PASP)3@DOX nanorods against Hela cells, NIR laser was used for inducing the photothermal conversion effect. Upon NIR irradiation, Hela cells apoptosis could be induced by local temperature change around the drug nanoparticles and the possibly generated active species from the rGO component.[48-50] The binding energy between rGO/(PLL/PASP)3 and DOX could be changed by the photothermal heating, leading to DOX release from the carrier and diffuse into the cancer cells. As shown in Figure 4E, following NIR irradiation, the cell viability could be further reduced from 95 % to 75%, and 48 % to 37 %, in the presence of rGO/(PLL/PASP)3 and rGO/(PLL/PASP)3@DOX, respectively. These results indicated that the rGO/(PLL/PASP)3@DOX nanodrugs can combine photothermal and chemical functions for the treatment of cancer cells.

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incubated for 24 h. The cells were then washed three times with PBS. After this step, the cells were fixed with 4% paraformaldehyde and stained with Hoechst 33258 (10 μgmL -1) over 15 min at 4 °C. The chambers were rinsed three times with PBS and measured by CLSM. In Vitro Cytotoxicity Assay. The in vitro cytotoxicity of rGO/(PLL/PASP) 3 and rGO/(PLL/PASP)3@DOX were investigated using MTT assays on Hela cells. Generally, Hela cells exhibiting exponential growth were seeded into 96-well plates at a density of 2.5 × 104 cells per well; the cells were then incubated overnight at 37 °C to allow cell attachment. Next, different volumes (20 μL, 40 μL, 60 μL) of the rGO/(PLL/PASP)3 and rGO/(PLL/PASP)3@DOX dispersions were added to each well. After 48 h, the medium was removed and DMSO (150 μL) was added. Cell viability was measured by the intensity of absorbance at 490 nm and presented as a percentage of the control values. Characterization. The ultraviolet-visible absorption spectra were measured by UV6100 double beam spectrophotometer. Fourier transform infrared spectra (FT-IR) was registered with a Bruker Spectrum TENSOR II. The zeta potential analysis was performed on Malvern Instruments Zetasizer Nano-ZS90. TEM image was recorded by transmission electron microscopy (H-8100, Hitachi). The thickness of samples was measured by Atomic force microscopy (AFM, Dimension 3100, Veeco, USA).

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FULL PAPER

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[50] A. Riedinger, P. Guardia, A. Curcio, M. A. Garcia, R. Cingolani, L. Manna, T. Pellegrino, Nano Lett. 2013, 13, 2399−2406.


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FULL PAPER Entry for the Table of Contents

FULL PAPER A facile and efficient approach for the preparation of pH responsive selfassembled (electrostatic interactions) rGO-polymer-DOX nanocomposites for photothermal and chemical therapy is reported.

Xiangming Li, Yihe Zhang*, Zequn Ma, Meng Fu, Qi An*

The Fabrication of rGO/(PLL/PASP)3@DOX Nanorods with pH-Switch for Photothermal Therapy and Chemotherapy


Accepted Manuscript

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