Pluronic F127 self-assembled MoS2 nanocomposites as an effective glutathione responsive anticancer drug delivery system

In this study, bio-responsive polymeric MoS2 nanocomposites were prepared for use as a drug carrier for cancer therapy. Herein, we report the synthesis and demonstrate the self-assembly of pluronic F127 (PF127) on a cystamine–glutathione–MoS2 (CYS–GSH–MoS2) system, which can be used for GSH-triggered drug release under biological reducing conditions. The reduction-sensitive disulfide bond containing CYS was incorporated between the amphiphilic copolymer PF127 and GSH–MoS2 to achieve feasible drug release. Percent drug loading capacity and encapsulation efficiency were 51.3% and 56%, respectively. In addition, when the MoS2–GSH–CYS–PF127 nanocomposite was incubated in a GSH environment, the morphology of the nanocomposite tended to change, ultimately leading to drug release. The drug-loaded PF127–CYS–GSH–MoS2 polymeric nanocomposites efficiently released 52% of their drug content after 72 h of incubation in a GSH reduction environment. The HeLa cells treated with DOX loaded MoS2–GSH–CYS–PF127 showed 38% toxicity at drug concentration of 40 μg, which indicated that the successfully released of drug from carrier and caused the cell death. Further, fluorescence microscopy images of HeLa cells revealed the potential behavior of the MoS2–GSH–CYS–PF12 nanocomposite during the 2- and 4 h incubation periods; the nanocomposite was only found in the cytoplasm of HeLa cells. Interestingly, after 6 h of incubation, the drug was slowly released from the nanocomposite and could enter the nucleus as confirmed by fluorescence imaging of HeLa cells. Altogether, our synthesized PF127-coated MoS2 nanocomposite could be effectively adopted in the near future as a GSH-sensitive drug carrier.


Introduction
In the past decade, there has been a growing interest for the construction of a remarkable reduction-responsive drug delivery system (DDS). 1 Recently, various biomolecules and DDSs have been synthesized as stimuli-responsive nanocarriers toward light, pH, magnetic eld, ultrasound, and redox potential by changing their surrounding environment. For example, Kim et al. reported that by changing the physicochemical properties of their delivery systems to cleave the disulphide linkage in the carrier to deliver the gene, the systems could be utilized to perform effective delivery and control cargo release at the target site. 2 In gene delivery systems, stimuli-responsive carriers have shown great potential by overcoming many obstacles in cellular gene delivery such as cellular uptake, escape from endosomes, and cargo release of biomolecules at the targeted intracellular location. 3,4 Generally, researchers have focused on multifunctional stimuli-responsive nanocarriers that can release drugs in response to internal or external stimuli such as pH, 5 redox, 6 temperature, 7 enzyme, 8-10 magnetic, and light [11][12][13][14][15][16] in the environment. Such nanocarriers have recently been engineered as smart DDS. 17 Simultaneous exposure to inherent or external stimuli-sensitive DDS activates activities that regulate drug release or adequately facilitate intracellular uptake or diffusion. 18 Moreover, drug release following administration may be achieved via structurally modifying the microcontroller class of DDS transporting the drug or breaking down the chemical components of the nanocarrier. The unique biochemistry of various engineered stimuli-sensitive DDS under specic conditions can result in specic temporal and spatial DDSs. [19][20][21] Nanocarriers are commonly designed to respond to a single external stimuli that activates drug release. 22,23 Usually, these include intracellular GSH, [24][25][26][27] ROS, 28,29 and lysosomal activity. 30 For GSH-triggered drug release, GSH acts as a reducing agent in the intracellular region by cleaving the sensitive disulde bond in the nanocarrier to initiate drug release. The intracellular concentration of GSH is approximately 0.5 to 10 mM. 31 Hence, such concentration can be attained in an in vitro study without difficulty as a GSH-rich cancer cell environment triggers drug release. 32,33 Two-D nanomaterials have uniquely been considered for biomedical application due to their outstanding electronic, physical, and chemical properties. 34 As the large surface area of 2D nanomaterials provides adequate area for loading responsive cargo biomolecules, they are favorable materials for stimuliresponsive biomedical applications. 35 In particular, MoS 2 , WS 2 , MoSe 2 , and WSe 2 have been studied as a replacement for graphene due to some limitations of graphene such as loss of its native properties when exfoliated using n-butyllithium (n-BuLi) method, zero band gap, structural defects and so on. [36][37][38][39][40] As molybdenum (Mo) is an essential element for individual enzymes in cells and S is a universal biological element, 41,42 MoS 2 was recently examined to elucidate its potential biomedical applications. Functionalization of the MoS 2 surface is simple and easy because of the readily available sulfur vacancy on MoS 2 . 43 The biological applications of MoS 2 -based nanocarriers have also been studied in a variety of therapeutic and diagnostic applications such as bioimaging, drug delivery, gene delivery, phototherapy, combined therapy, theranostic, and biosensing. However, limitations and drawbacks were found in the reported study. An analysis of some future aspects for the long-term development of MoS 2 -based nanocomposites as a potential nanomedicine has highlighted its limits. 44 Most of the recent studies on MoS 2 focused on its therapeutic application to mainly target tumors via passive release of drug molecules. A stimuliresponsive nanocarrier system with MoS 2 is therefore essential to achieve an on-demand DDS. 45 In this study, a GSH-responsive MoS 2 -GSH-CYS-PF127 nanocomposite was prepared for effective drug delivery in a GSH-rich environment. To prepare these nanocomposites, MoS 2 was rst exfoliated using GSH as a surfactant via sonication and this was followed by the addition of a disulde-containing CYS. Finally, PF127 was introduced to derive the GSH-responsive MoS 2 -GSH-CYS-PF127 nanocomposite. The tailored MoS 2 nanocomposite system exhibited sensitivity in the GSH environment as conrmed by TEM and DLS. To further evaluate the GSH-sensitive property of the MoS 2 nanocomposite, the anticancer drug, doxorubicin (DOX), was loaded onto the carrier and its release in a GSH environment was assessed. This GSH reduction-responsive drug release assessment was performed with phosphate buffer pH ¼ 7.4 and GSH ¼ 5 mM. We found that 52% of the drug was released aer 72 h. Subsequently, uorescence microscopic images revealed that a 6 h incubation of the MoS 2 nanocomposite in HeLa cells effectively killed the cells relative to the 2 h and 4 h periods. To add, the images revealed that the nanocomposite was located in the cell membrane alone. Therefore, this MoS 2 nanocarrier opens a window for its use as a stimuli-responsive nanocarrier for drug delivery (Fig. 1).

Materials
MoS 2 (10-30 mm) was purchased from Rose Mill Company, reduced L-glutathione (GSH), cystamine dihydrochloride (CYS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), pluronic F127 (PF127) and biological water were acquired from Sigma-Aldrich. Phosphotungstic acid N-hydrate (PTA) were purchased from J.T. Baker, (India), doxorubicin hydrochloride (DOX) were purchased from Cayman Chemical Company (USA), anhydrous dimethyl sulfoxide was obtained from Macron Fine Chemicals. Dulbecco's Modied Eagle Medium (DMEM), penicillin, sodium pyruvate, trypsin and sterilized fetal bovine serum (FBS), were purchased from Gibco (Carlsbad, CA). Human cervical carcinoma (HeLa) cells were obtained from the Bio Resource Collection and Research Center (Hsinchu, Taiwan). Regenerated cellulose tubular membrane with a nominal value of 1000 and 6000-8000 Da was purchased from Orange Scientic. Unless otherwise noted, all other reagents and solvents were obtained from Sigma-Aldrich, Alfa-Aesar, and TCI chemicals. Other reagents, and buffer solution components were of analytical grade. Distilled and deionized water (DI) were used in all experiments. All solutions and reagents were used without further purication. Transmission electron microscopy (HR-TEM) images were obtained with a Tecnai-F20 FEI-TEM system. Ten mL of the sample solution was placed on a 300 mesh carbon-stabilized, formvarcarbon-coated grid. Following a 2 min adsorption, the sample was washed with distilled water and air-dried. Each grid was then stained with 0.2% (w/v) PTA for 2 min. Excess staining solution was removed, and the sample was allowed to air-dry. Aer complete drying, grids were observed using HR-TEM and photographed with a CCD camera.
UV-visible images were captured with a Jasco V-730 spec-trouorometer. Particle size and zeta potential of the prepared DOX-loaded nanocomposites were determined by dynamic light scattering (DLS) and their zeta potential was also measured using a zeta potential analyzer. Data are presented as the average of three measurements. The amount of DOX in DOXloaded MoS 2 -GSH-CYS-PF127 composites was determined by UV-visible spectroscopy using Jasco V-730 spectrouorometer.

Preparation of MoS 2 -GSH nanoparticles
Eight hundred mg of MoS 2 and 800 mg of L-GSH (1-1 ratio) were added to 40 mL of dimethyl sulfoxide (DMSO) and probe sonicated for 8-10 h with power density of 650 W. Aer sonication, the resulting greenish-black solution was allowed to settle for 24 h. Aer 24 h, centrifugation was conducted to remove the unexfoliated MoS 2 nanoparticles. The supernatant was collected and dialysis was performed using a 1000 Damembrane for 4 h to remove excess ligand. Moreover, the anhydrous DMSO assisted MoS 2 shows good exfoliation with long-term stability. In addition, many researchers revealed that anhydrous DMSO assisted exfoliation can provide effective method to obtained single and few-layer MoS 2 sheets. 46,47 Aer sonication, the resulting greenish-black solution was allowed to settle for 24 h. Aer 24 h, centrifugation was conducted to remove the unexfoliated MoS 2 nanoparticles. The supernatant was collected and dialysis was performed using a 1000 Damembrane for 4 h to remove excess ligand.

Preparation of MoS 2 -GSH-CYS-PF127 nanocomposite
Twenty mg of CYS and PF127 were added to 5 mL of MoS 2 -GSH nanoparticle solution. The nanocomposite was formed during dialysis and this was allowed to proceed to remove excess CYS. Prior to the preparation of the MoS 2 -GSH-CYS-PF127 nanocomposite, MoS 2 -GSH-PF127 and MoS 2 -GSH-CYS were synthesized via simple procedures. Twenty milligrams of PF127 was then added to 10 mL of MoS 2 -GSH nanoparticle solution during dialysis to prepare MoS 2 -GSH-PF127. Meanwhile, 20 mg of CYS was added to 10 mL of MoS 2 -GSH nanoparticle solution during dialysis to prepare MoS 2 -GSH-CYS.

Drug loading
The anticancer drug, DOX, was loaded into multifunctional nanocarriers. Briey, 50 mg of MoS 2 -GSH-CYS-PF127 was added to 50 mL of PBS and 5 mL of DOX solution (5 mg dissolved in 5 mL of DMSO) via an ultrasonic bath. The mixture was sealed and stirred for 24 h in the dark at room temperature. Thereaer, the DOX-loaded nanocomposite was dialyzed against PBS to remove unbound DOX molecules.

Drug release experiment in a reduction-sensitive environment
The in vitro reduction-sensitive release behavior of the drug was investigated by placing the DOX-loaded MoS 2 -GSH-CYS-PF127 nanocomposite (1.5 mL) in a dialysis bag. The dialysis bag was soaked in 15 mL of PBS with or without 5 mM GSH and placed on shaker set at 37 C and 180 rpm. At predetermined time intervals, 3 mL of the sample solution was removed and replaced with fresh PBS solution. For reduction-sensitive drug release, an equivalent amount of fresh PBS containing 5 mM GSH was continuously supplemented for 3 days. The amount of cumulative drug release (DOX) was measured via absorption at a wavelength of 480 nm.

GSH-responsiveness of DOX-loaded MoS 2 -GSH-CYS-PF127 nanocomposites
The GSH-responsive behavior of DOX-loaded MoS 2 -GSH-CYS-PF127 nanocomposites was investigated in the presence of 5 mM GSH and PBS buffer by DLS. Briey, GSH (5 mM) was mixed with DOX-loaded MoS 2 -GSH-CYS-PF127 nanocomposites in different environments. The solution was then incubated on a shaking table at 37 C and changes in size of the nanocomposites were investigated at different time intervals by DLS.

MTT assay
HeLa cell line was cultured at density of 2 Â 10 4 in a 96-well plate and maintained in DMEM (Wisent Inc., USA) supplemented with 10% fetal bovine serum (Wisent Inc.) and 1% sodium pyruvate (Wisent Inc.) at 37 C in a humidied atmosphere with 5% CO 2 . Aer a 24 h incubation, the cells were exposed to 200 mg mL À1 to 6.25 mg mL À1 of MoS 2 -GSH-CYS-PF127, and 40 mg mL À1 to 1.25 mg mL À1 of free DOX and DOXloaded MoS 2 -GSH-CYS-PF127 nanocomposites. Aer a 24 h incubation in a humidied atmosphere, the old medium was removed and fresh medium with MTT (5 mg mL À1 ) was added followed by an additional incubation for 2-4 h for cytotoxic evaluation using an ELISA reader.

Cellular uptake and intracellular localization of DOX
HeLa cells were seeded in a confocal dish (35 mm Â 10 mm; Corning Inc., New York, USA) at a density of 1 Â 10 5 cells per well and incubated in DMEM for 24 h. Culture medium was removed and fresh DMEM containing free DOX and DOXloaded MoS 2 -GSH-CYS-PF127 nanocomposites (5 mg mL À1 ) were added and incubated for 2 h, 4 h, and 6 h in a humidied atmosphere. The cells were washed three times with PBS to remove excess nanoparticles. DAPI in PBS (100 nanomolar) was added to cells prior to incubation at room temperature for 15 min followed by rinsing with PBS and observation by uorescence microscopy.

Particle size and zeta potential
In the present study, we aimed to synthesize a GSH-responsive MoS 2 -based DDS. MoS 2 was rst exfoliated in DMSO via sonication and this was followed by the introduction of GSH as a surfactant. A disulde-containing CYS was then introduced followed by PF127 via self-assembly using the dialysis method. The synthesized GSH-responsive nanocarrier system was then examined using a variety of techniques such as UV-visible spectroscopy, TEM, DLS, and zeta potential, and drug release was conrmed by UV-visible spectroscopy and uorescence microscopy. For MoS 2 -GSH nanoparticles and MoS 2 -GSH-CYS, particle size was $61.6 nm and $110.2 nm, respectively. However, aer the formation of the self-assembled PF127 on the surface of MoS 2 -GSH, the size of the nanocarrier increased to 71.7 nm. Notably, MoS 2 -GSH had a smaller size before CYS loading; however, aer loading, particle size was $110.2 nm. The greater particle size might be due to the negative charge of MoS 2 -GSH which may have allowed a strong electrostatic interaction with the positive group of CYS. 48,49 In contrast, PF127 polymer coating reduced the size of the nanocarrier from size of MoS 2 -GSH-CYS particle from 110.2 nm to 82.3 nm. 50,51 Zeta potential values for MoS 2 -GSH and MoS 2 -GSH-PF127 were À12.2 mV, and À15.7 mV, respectively. As shown in Table 1, the zeta potential of MoS 2 -GSH-CYS and MoS 2 -GSH-CYS-PF127 increased toward positive values of 0.5 mV and 1.7 mV.

UV-visible spectroscopy
The synthesized MoS 2 nanocarrier was conrmed by UV-visible spectroscopy and the spectra for  52 The absorption peaks, namely A and B, were due to direct excitonic transitions at the K-point with energy difference caused by valence band spin-orbital coupling. Peaks C and D were assigned to the direct excitonic transition of the M-points between higher densities of state and region of the band structure. This study conrms the presence of synthesized exfoliated MoS 2 samples exhibits 2H MoS 2 .
Aer PF127 was coated onto the MoS 2 -GSH system, the wavelength decreased to 664 nm, 605 nm, 444 nm, and 390 nm. As the observed blue shi is usually caused by H-aggregates, this result suggests that the amphiphilic polymer results in dense parallel-aggregates of MoS 2 in the core of PF127. The wavelengths depicting the characteristic maximum absorption peaks shied from 669 nm (A), 606 nm (B), 445 nm (C), and 391 nm (D) to 675 nm, 617 nm, 457 nm, and 397 nm for MoS 2 -GSH-CYS, and 672 nm, 611 nm, 446 nm, and 395 nm for MoS 2 -GSH-CYS-PF127. As the observed red shi is usually caused by J aggregates, this result suggests that CYS induce the nanoparticle aggregation of MoS 2 -GSH and MoS 2 -GSH-PF127, which also proved in the DLS results ( Table 2). 49

Transmission electron spectroscopy (TEM)
PTA was used as the negative staining agent, thereby allowing a contrast between background and the MoS 2 core. Each sample was stained with 0.2% (w/v) PTA for 2 min and the morphology was investigated by TEM. As shown in Fig. 3a, MoS 2 -GSH nanoparticle had a spherical shape and a size of $80-90 nm;    this result was similar to that achieved by DLS. As shown in Fig. 3b, MoS 2 -GSH-PF127 displayed a homogenous and core shell that had a spherical structure and size of $50-60 nm. However, when CYS was coated on the surface of MoS 2 -GSH, the morphology changed to a core shell structure and its size increased to $100-150 nm. These results also support the DLS results (Fig. 3c). 48 MoS 2 -GSH-CYS-PF127 (Fig. 3d) had a core shell structure with additional small and larger-sized aggregate particles that resembled the nanocomposites.

Determination of drug loading of MoS 2 -GSH-CYS-PF127 nanocomposites
To determine drug loading and encapsulation efficiency, 5 mg of the freeze-dried nanocomposites were dispersed in 5 mL of PBS. The loading capacity of the MoS 2 nanocomposites were measured by UV-vis at an absorbance wavelength of 480 nm. Percent drug loading and encapsulation efficiency were calculated using the following equations: DOX concentration was calculated as: The calculated drug loading capacity and encapsulation efficiency were 51.36% and 56%, respectively. 3.5. UV-visible spectroscopy, particle size, zeta potential, GSH reduction sensitivity, and cumulative drug release studies of DOX-loaded MoS 2 -GSH-CYS-PF127

Concentration of DOX
As shown in Fig. 4a, the characteristic maximum absorbance for DOX was $286 nm and 483 nm, which are values similar to previous reports. 53,54 Furthermore, the DOX peak in the drugloaded MoS 2 -GSH-CYS-PF127 was $302 and 524; these were observed to shi from the native DOX peaks of $286 and 483. This shiing might be due to the electron donor-acceptor interaction between DOX and MoS 2 nanocomposites. 55,56 The size and zeta potential of the drug-loaded MoS 2 nanocomposites were signicantly increased ( Table 2). Before loading, the MoS 2 nanocomposites had a size of $82.3 nm and zeta potential value of $1.7 mV. Aer DOX loading, the size of the MoS 2 nanocomposites was $102 and zeta potential value was 9.4 mV. This result indicates that the nanoparticles were nearly electrically neutral which is benecial for prolonged circulation in blood without the possibility of elimination. 57 The stability of DOX-loaded MoS 2 -GSH-CYS-PF127 has been evaluated for one month, and the size of drug carrier showed around 99.7 nm (Table 3), which also proved that the PF127 has successfully covered on the surface of drug carrier. The GSH sensitivity of the drug-loaded MoS 2 nanocomposites was explored in the presence and absence of 5 mM GSH in PBS buffer by measuring the changes in particle size at different time intervals. As shown in Table 3, the particle size of the DOX-loaded MoS 2 -GSH-CYS-PF27 increased from 102 to 261 nm in 24 h, 479 nm in 48 h, and 778 nm in 72 h in the presence of GSH. This is due to the cleavage of the disulde linkage in the core of the MoS 2 nanocomposites, thereby leading to larger particle sizes. In contrast, dramatic size changes were not found in drug-loaded MoS 2 nanocomposites in the absence of GSH.
By using the dialysis method, we demonstrated the release of DOX from MoS 2 -GSH-CYS-PF127 nanocomposites at 37 C in PBS buffer (pH 7.4) in the presence and absence of 5 mM GSH. The DOX solution was removed from the drug release setup and UV was measured at predetermined intervals for 72 h. As shown in Fig. 4d, the 5 mM GSH-containing MoS 2 nanocomposites released more DOX (52%) relative to the nanocomposites without GSH (32%). Due to the disulde reducing CYS present in the core of the MoS 2 nanocomposites, introducing GSH  breaks the disulde bond, ultimately leading to drug release. [58][59][60] The introduction of 5 mM GSH to drug loaded MoS 2 system effectively reduce the disulphide bond of cystamine results the dissociation of ligands (GSH and CYS) and recombination of ligands (GSH and CYS) on the surface of MoS 2 . In addition, the disruption of cysteamine positive cystamine on the surface easily to make the aggregation of negative charge of MoS 2 with surface positive charge cystamine and then nally leading to increase the size in DLS measurement (Table 4). However, the particle size of DOX-loaded MoS 2 -GSH-CYS-PF127 is increased while absence of GSH environment which may occurred due to instability of PF127 during drug release environment. The fresh phosphate buffer has been added and make dilution of DOX-loaded MoS 2 -GSH-CYS-PF127 solution and resulted the particle size slightly increased at 48 and 72 hours. Moreover, a maximum cell death of almost 65% was observed at 40 mg mL À1 . Interestingly, the cell death ratio of DOX-loaded MoS 2 -GSH-CYS-PF127 was almost equal to that of free DOX. These results proved the potential of GSH to induce drug release from the MoS 2 nanocomposite system and the capability of this nanocomposite as a drug carrier. To differentiate between the toxicity of the nanocomposite and drug, the cytotoxicity of the leading MoS 2 -GSH-CYS-PF127 nanocomposites was initially resolved prior to the addition of free DOX and DOX-loaded nanoparticles. The cytotoxicity of free DOX and DOX-loaded MoS 2 -GSH-CYS-PF127 was assessed using HeLa cells at several concentrations aer a 24 h treatment. This assured that MoS 2 -GSH-CYS-PF127 could be used as a drug carrier. Herein, 95% of HeLa cells were viable aer a 24 h incubation, even at the high concentration of 200 mg mL À1 . From 100 mg mL À1 to 12.5 mg mL À1 , cell viability reached more than 100% as shown in Fig. 5a; (the free nanoparticle concentrations were 200, 100, 50, 25, 12.5, and 0 mg mL À1 ). We also found that DOX and DOXloaded MoS 2 -GSH-CYS-PF127 displayed low toxicity at the concentrations tested (free DOX and DOX-loaded nanocomposites: 40, 20, 10, 5, 2.5, and 0 mg mL À1 ). As shown in Fig. 5b, HeLa cells were treated with free DOX and DOX-loaded MoS 2 -GSH-CYS-PF127 nanocomposites for 24 h. The HeLa cells incubated with DOX loaded MoS 2 -GSH-CYS-PF127 showed 38% toxicity at drug concentration of 40 mg, which indicated that the successfully released of drug from carrier and caused the cell death. Moreover, DOX-loaded MoS 2 -GSH-CYS-PF127 nanocomposites exhibited lower cytotoxicity than free DOX. As mentioned above, free DOX is slightly more cytotoxic than DOX-loaded MoS 2 -GSH-CYS-PF127 toward HeLa cells. This is because of the low molecular weight of DOX enables its easy diffusion into the cell without the drug release process.  nanocomposites. Hence, we employed HeLa cells for the evaluation and a uorescence microscope for visualization. The concentration of free DOX and DOX in the MoS 2 carrier was xed to 5 mg mL À1 . Fig. 6A shows the uorescent microscopic images of HeLa cells incubated with DOX-loaded MoS 2 -GSH-CYS-PF127 nanocomposites. These cells displayed a faint red Paper DOX uorescence within the cell membrane or cytoplasm of cells at the beginning of incubation (i.e., 2-4 h). However, aer 6 h of incubating the MoS 2 carrier with HeLa cells, a strong red uorescence was observed in the nucleus of HeLa cells depicting DOX release from the carrier, followed by internalization into the nucleus. Drug-loaded MoS 2 -GSH-CYS-PF127 nanocomposites efficiently released the drug under the intracellular GSH-rich condition and the released drug subsequently diffused into the nuclei of HeLa cells. The internalization of free DOX by HeLa cells also resulted in a strong red color and this was even evident at 2 h (Fig. 6B). However, a higher number of dead cells was observed with free DOX than DOX-loaded MoS 2 nanocomposites. 63,64

Conclusion
In this study, we prepared a DOX-loaded PF127 self-assembled MoS 2 nanocomposite and demonstrated its ability to release the transported drug under reduced GSH conditions. To construct the GSH-sensitive MoS 2 nanocomposite, we introduced a disulde-containing CYS to the exfoliated MoS 2 prior to introducing PF127. By TEM imaging, the synthesized MoS 2 nanocomposite was found to have a spherical shape and size of $82.3 nm while the DOX-loaded MoS 2 nanocomposite had a size of 102 nm. In 5 mM GSH, the MoS 2 nanocomposite released 52% of the transported drug in 72 h. In addition, by performing an MTT assay, the biocompatibility of the nanocomposite was conrmed. The DOX-loaded nanocomposite was demonstrated to be highly toxic due to the release of DOX in the GSH-rich cancer cell environment. The DOX release behavior of the carrier was further supported by images obtained via uorescence microscopy. Compared to 2 h, aer 6 h of incubation, the DOX-loaded nanocomposite effectively released DOX which was internalized into the nucleus. Aer a 4 h incubation, DOX was only found in the cell membrane and cytoplasm.

Conflicts of interest
There are no conicts to declare.