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Sep 15, 2017 - indicates standard deviation; * indicates p < 0.05 with Student's t test). ..... LDH-Co-PEG nanosheets have a high level of biosafety f...
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A Novel Type of Aqueous Dispersible Ultrathin-Layered Double Hydroxide Nanosheets for in Vivo Bioimaging and Drug Delivery Li Yan,▽,† Mengjiao Zhou,‡ Xiujuan Zhang,*,‡ Longbiao Huang,† Wei Chen,† Vellaisamy A. L. Roy,† Wenjun Zhang,*,† and Xianfeng Chen*,∥ ▽

Antibiotics Research and Re-evaluation Key Laboratory of Sichuan Province, Sichuan Industrial Institute of Antibiotics (SIIA), Chengdu University, Chengdu, Sichuan, P.R. China ∥ School of Engineering, Institute for Bioengineering, School of Engineering, The University of Edinburgh, King’s Buildings, Mayfield Road, Edinburgh EH9 3JL, United Kingdom † Center of Super-Diamond and Advanced Films (COSDAF) and Department of Materials Science and Engineering, City University of Hong Kong, Hong Kong SAR, P.R. China ‡ Institute of Functional Nano & Soft Materials (FUNSOM) and Collaborative Innovation Center of Suzhou Nano Science and Technology, Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou, Jiangsu, P.R. China S Supporting Information *

ABSTRACT: Layered double hydroxide (LDH) nanoparticles have been widely used for various biomedical applications. However, because of the difficulty of surface functionalization of LDH nanoparticles, the systemic administration of these nanomaterials for in vivo therapy remains a bottleneck. In this work, we develop a novel type of aqueous dispersible two-dimensional ultrathin LDH nanosheets with a size of about 50 nm and a thickness of about 1.4 to 4 nm. We are able to covalently attach positively charged rhodamine B fluorescent molecules to the nanosheets, and the nanohybrid retains strong fluorescence in liquid and even dry powder form. Therefore, it is available for bioimaging. Beyond this, it is convenient to modify the nanosheets with neutral poly(ethylene glycol) (PEG), so the nanohybrid is suitable for drug delivery through systemic administration. Indeed, in the test of using these nanostructures for delivery of a negatively charged anticancer drug, methotrexate (MTX), in a mouse model, dramatically improved therapeutic efficacy is achieved, indicated by the effective inhibition of tumor growth. Furthermore, our systematic in vivo safety investigation including measuring body weight, determining biodistribution in major organs, hematology analysis, blood biochemical assay, and hematoxylin and eosin stain demonstrates that the new material is biocompatible. Overall, this work represents a major development in the path of modifying functional LDH nanomaterials for clinical applications. KEYWORDS: layered double hydroxides, nanosheets, nanomaterials, anticancer therapy, drug delivery molecular weight heparin.14 Also, LDH nanoparticles can greatly increase siRNA delivery efficiency to mammalian cells and cortical neurons.18,19 Despite promising developments, the study of using LDH nanoparticles without involving formation of composites with other materials for biological and medical applications has been predominantly with in vitro cell lines for intracellular drug/gene delivery. So far, only a very small number of studies have been performed in mouse models, and the investigations were mainly confined to improving vaccine efficacy and the delivery of LDH administered through intradermal or intratumor injection. For example, it has been reported that LDH nanomaterials are able to act as an immune adjuvants to boost the immune responses

1. INTRODUCTION Layered double hydroxide (LDH) is an anionic clay, and the formula can be generally expressed as [M2+1−xM3+x(OH)2][An−]x/n·zH2O, where M2+ = Mg2+, Fe2+, or Co2+, M3+ = Al3+, Fe3+, or Gd3+, and nonframework anions An− = Cl−, CO32−, NO3−, etc.1−3 An− can be readily replaced by negatively charged molecules through either ion-exchange (molecules replace the anions after LDH preparation) or coprecipitation (adding molecules during LDH preparation). LDH nanoparticles have been broadly used in various biomedical applications,4−7 including bioimaging,8,9 antimicrobials,10 delivery carriers of drugs,11−17 genes18,19 and vaccines,20,21 protein separation,22 and photodynamic therapy.23 For example, drugs, genes, and biomolecules show greatly improved intracellular delivery efficacy when delivered by LDH nanoparticles.24−26 It has been reported that low molecular weight heparin loaded with LDH nanoparticles shows efficacy better than that of free low © XXXX American Chemical Society

Received: April 15, 2017 Accepted: September 15, 2017 Published: September 15, 2017 A

DOI: 10.1021/acsami.7b05294 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces of vaccines.20 We also previously showed that LDH nanoparticles can be used to mechanically strengthen dissolving polymer microneedles for improved transdermal vaccine delivery.27,28 Furthermore, LDH nanoparticles were recently used as a host for photosensitizers to increase photodynamic therapy efficiency. In the study, it was reported that zinc phthalocyanine (ZnPc)-loaded LDH nanoparticles showed excellent anticancer behavior in photodynamic therapy in vivo, but the administration was performed by intratumor injection.23 Studies show that proper surface modification of nanomedicines can improve their stability, reduce toxicity, extend half-life in the bloodstream, and diversify their functions and applications.29 For instance, poly(ethylene glycol) (PEG)modified graphene and MoS2 nanosheets possess greatly improved stability in buffer solution and alleviate toxicity for in vivo administration.30−32 Moreover, nanoparticles including silica, polymer, metals, and iron oxides can be surface-modified with targeting ligands, antibodies, polymers, and fluorescent molecules for targeted delivery, bioimaging, and disease diagnosis.33−37 Therefore, it is highly desirable to prepare surface-modified LDH nanoparticles for a range of biological and medical applications including bioimaging and drug delivery. Overall, LDH nanoparticles without formation of composites with other materials have been strictly limited to deliver negatively charged molecules, but an in vivo study through the blood system has not yet been performed.38−40 To solve these problems and develop surface-tailored LDH nanomaterials for potential clinical applications, in this report, we develop a new type of aqueous dispersible ultrathin LDH nanosheets and modify the surface with different functional molecules. First, we covalently bind positively charged rhodamine B molecules to the surface of LDH nanostructures. The incorporation of rhodamine B molecules allow the material to be used for bioimaging. Second, the LDH nanosheets are tailored with neutral PEG by covalent bonding. The PEG modification will increase the circulation time of the nanomaterial in blood and make it more suitable for in vivo drug delivery through systemic administration. These PEG surfacemodified nanostructures can be readily used to deliver negatively charged anticancer drug methotrexate (MTX), through which a greatly enhanced drug efficacy is achieved, indicated by the effective inhibition of tumor growth in a mouse model. Overall, such surface-modified LDH nanostructures can be conveniently used for theragnostics.

photoluminescence spectra were determined with a Varian Cary 50 UV−visible spectrophotometer and an Edinburgh Instrument FLS920P spectrometer, respectively. Powder X-ray diffraction (XRD) patterns were recorded on a Smartlab instrument using Cu Kα radiation. 2.2. Preparation of SDS-Expanded LDH (LDH-SDS) Nanoparticles. An 80 mL amount of 0.15 M NaOH and 4 mmol of SDS were mixed with 20 mL of solution containing 2.0 mmol of MgCl2 and 1.0 mmol of AlCl3 under vigorous stirring for 10 min. The solution was centrifuged and washed once with Milli-Q water. The resultant slurry was redispersed in 80 mL of Milli-Q water and stirred at 80 °C for the next 24 h, airtight and under N2 protection. The LDH-SDS nanoparticles were collected by centrifuge at 4000 rpm for 5 min and dried by freeze-drying. 2.3. Preparation of PEG-Conjugated LDH (LDH-Co-PEG) and Rhodamine B-Conjugated LDH (LDH-Co-RB) Nanosheets. (1) LDH-Co-RB nanosheets: To conjugate rhodamine B to APTES, 8 mg of rhodamine B was mixed with 500 μL of APTES in 1 mL of DMSO for 48 h in the presence of EDC, airtight and under N2 protection. Ten milliliters of methylene chloride was mixed with 100 mg of CTAB at ∼45 °C. Then the prepared rhodamine B-conjugated APTES and CTAB−methylene chloride solution were quickly placed into a 20 mL glass vial containing 50 mg of LDH-SDS nanoparticles and kept airtight and under N2 protection. Next the solution was ultrasonicated for 30 min at ∼45 °C and then stirred for 48 h at the same temperature. At the end, the nanoparticles were collected by centrifugation and washed with methylene chloride, ethanol, and Millipore water in a few separate steps. The as-prepared sample was subjected to strong sonication for the next 24−48 h to make it welldispersed in water before the in vitro test. The final product of LDHCo-RB contains 0.15 wt % rhodamine B. (2) LDH-Co-PEG nanoparticles: To conjugate PEG-carboxy to APTES, 2.5 mg of PEG-carboxy was mixed with 25 μL of APTES in 1 mL of DMSO for 48 h in the presence of EDC, airtight and under N2 protection. Ten milliliters of methylene chloride was mixed with 100 mg of CTAB at ∼45 °C. Then the prepared PEG-carboxy-conjugated APTES was conjugated to LDH-SDS nanoparticles using the same procedures as described above. The final product of LDH-Co-PEG contains 5 wt % PEG. 2.4. Laser Scanning Confocal Microscopy. A HeLa cell suspension was seeded to a sterile glass coverslip in a 35 mm tissue culture dish for 24 h. Then the cell culture medium was removed, and a fresh medium containing 50 g mL−1 of LDH-Co-RB nanosheets was added followed by postculture for 4 h. Finally, after being washed with PBS, the coverslip was used for confocal microscopy. Imaging was performed on a confocal microscope (Leica TCS SPE). 2.5. Cell Viability Measurement. HeLa or A549 cells were washed twice with PBS. HeLa cells suspended in DMEM (with 10% FBS, 1% penicillin/streptomycin) were plated into 96-well plates (100 μL of DMEM; 1500−3000 cells per well). The cells were incubated at 37 °C for 24 h before further treatment. Then another 100 μL of DMEM containing various concentrations of MTX, nanoparticles, and MTX-loaded nanoparticles was added to 96-well plates for an additional 48 and 72 h incubation. After certain periods of incubation, the original medium in each well was removed. Subsequently, 180 μL of DMEM (without FBS) and 20 μL of MTT stock solution (5 mg mL−1 in PBS) were added and incubated for 4 h. Then the medium containing MTT was completely removed, followed by adding 200 μL of DMSO to each well. Cell viabilities were determined by reading the absorbance of the plates at 540 nm using a BioTek Powerwave XS microplate reader. 2.6. In Vivo Drug Delivery for Cancer Therapy. Tumor-bearing BALB/C mice were developed by injection of 4T1 cells near the right flank (1 × 1064T1 cells in 60 μL of PBS). After 6 days, the mice were randomly divided into four groups. A 200 μL amount of MTX (0.5 mg mL−1), MTX loaded with traditional nonmodified LDH (LDH-MTX; 5 mg mL−1 LDH and 0.5 mg mL−1 MTX), MTX loaded with PEGmodified LDH (LDH-Co-PEG MTX; 5 mg mL−1 LDH-Co-PEG and 0.5 mg mL−1 MTX), and PBS solution were injected into the different groups of mice via the tail vein, respectively. The tumor size and body

2. EXPERIMENTAL SECTION 2.1. Chemicals and Characterization. NaOH and MgCl2 were purchased from International Laboratory USA (San Francisco, CA). Sodium dodecyl sulfate (SDS), N-(3-(dimethylamino)propyl)-Nethylcarbodiimide hydrochloride (EDC), methylene chloride, Ncetyl-N,N,N-trimethylammonium (CTAB), and MTX were ordered from Acros (Morris Plains, NJ). AlCl3, (3-aminopropyl)triethoxysilane (APTES), dimethyl sulfoxide (DMSO), and PEG-carboxy (O-(2carboxyethyl)poly(ethylene glycol), Mw = 5000) were from SigmaAldrich (St. Louis, MO). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and penicillin/streptomycin were obtained from Life Technologies (Camarillo, CA). The 96-well cell culture plates and cell culture dishes were obtained from Corning (Corning, NY). Zeta potential and size distribution were measured with a Zetasizer (Malvern). Transmission electron microscopy (TEM) images were taken on a Philips Technai 12. The UV−vis absorption and B

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ACS Applied Materials & Interfaces weight were monitored daily for 2 weeks. These experiments were performed following the guidelines issued by Soochow University. 2.7. Hematology and Blood Analysis. The blood samples were collected from the tumor bearing mice at 7 days after intravenous injection of different formulations (PBS, MTX, LDH-MTX, and LDHCo-PEG MTX). After blood collection, the heart, liver, spleen, lung, kidney, and tumor were also harvested. The tissues were fixed in a 10% formalin solution, embedded in paraffin, sectioned, and stained with different antibody−dyes. Subsequently, all of the samples were processed for hematoxylin and eosin (H&E) staining, hematology analysis, blood biochemical assay, and nanomaterial biodistribution measurement. These experiments were carried out in accordance with the guidelines issued by Chengdu University and Sichuan Province.

Figure 1a is a TEM image of as-prepared LDH-Co-RB nanosheets. It is apparent that the majority of LDH-Co-RB

3. RESULTS AND DISCUSSION 3.1. Materials Preparation and Characterizations. Figure S1 illustrates the covalent conjugation of positively charged rhodamine B molecules to LDH nanosheets. Briefly, rhodamine B is first bonded to APTES in the presence of EDC (Figure S1a). The product in this step is denoted as APTESRB. In parallel, LDH nanoparticles with large interlayer spacing were prepared by coprecipitating with addition of SDS in the reaction solution (Figure S1b). The product is indicated by LDH-SDS. SDS was added to expand the LDH gallery. Next, APTES-RB was reacted with LDH-SDS for 48 h in the presence of CTAB to synthesize surface-modified LDH nanosheets (LDH-Co-RB). In this step, the role of CTAB is to extract SDS molecules from LDH as shown in Figure S1b. Figure S2a presents the XRD patterns of SDS-expanded LDH nanosheets (LDH-SDS) and conventional LDH nanoparticles without using SDS during preparation. In the pattern of LDHSDS, the (003) peak shifts from 11.5° of conventional LDH to 3.4°, indicating that the interlayer spacing is expanded to 26 Å with the addition of SDS in the preparation (Figure S3). In comparison, the XRD pattern of LDH-Co-RB nanosheets does not display any obvious diffraction peaks. This indicates that these nanosheets do not possess the layered structure of traditional LDH nanostructures. The Fourier transform infrared (FTIR) spectra of LDH, LDH-SDS, and LDH-Co-RB nanomaterials are presented in Figure S2b. The spectrum of LDH-SDS displays three peaks at 2963, 2932, and 2842 cm−1, indicating C−H stretching modes. The disappearance of these three peaks in the spectrum of LDH-Co-RB suggests that SDS molecules are removed from the nanomaterials when CTAB is added. The broad peak at 1000−1200 cm−1 in the spectrum of LDH-Co-RB nanosheets can be ascribed to Si−O−Si asymmetric stretching vibration, which confirms the successful covalent attachment of APTES-RB to LDH nanoparticles.41 To further confirm the covalent conjugation between APTES and RB, and between APTES-RB and LDH, X-ray photoelectron spectroscopy (XPS) analysis was carried out. From the XPS spectra, it is clear that O, Si, N, Al, Mg, and C elements are present (Figure S4a). The spectrum of C 1s is resolved to four peaks at 288, 286.3, 286, and 284.6 eV, which are related to OC−N, C−O, C−N, and C−C bonds, respectively. In the N 1s spectrum, the three peaks at 399.3, 401.1, and 402.0 eV are assigned to NH2, C−N(−C)−C and HN−CO bonds, respectively. In the spectrum of O 1s, the existence of Si− O−Si (O−C), Si−O−Al/Mg and OC bonds is demonstrated by the peaks at 532.1, 531, and 531.6, respectively. Overall, the XPS spectra confirms the successful covalent conjugation of RB to LDH via Si−O−Mg/Al bonds and the conjugation of RB to APTES via peptide bonds.

Figure 1. (a) TEM image of LDH-Co-RB nanosheets. (b) AFM image of LDH-Co-RB nanosheets. (c, d) Zoom-in AFM image of LDH-CoRB nanosheets and the thickness analysis along the marked lines.

structures are thin nanosheets with a lateral size of about 50 nm. This is in line with the result obtained by dynamic light scattering (Figure S5). The size, morphology, and thickness of the LDH-Co-RB nanostructures were further characterized by atomic force microscopy (AFM) (Figure 1b−d). From the AFM results, the size (43 ± 13 nm, n = 50) of LDH-Co-RB nanosheets is very similar to that observed from TEM. The thickness of the majority of these nanomaterials is about 1.4 nm, but thicker nanosheets with a thickness of around 3−4 nm were also observed (Figure 1c,d, Figures S6 and S7). This demonstrates that these LDH-Co-RB nanosheets are ultrathin. Because the thickness of the parent brucite layer is about 4.9 Å, one can conclude that the majority of the nanomaterials probably contain a single layer of metal hydroxides with covalently attached APTES and rhodamine B molecules. The thickness is similar to that of 2-D nanostructures such as graphene and graphitic carbon nitride nanosheets. In contrast, the thickness of conventional LDH nanoparticles is on average about 15 nm.42 3.2. LDH-Co-RB Nanosheets for Bioimaging and Intracellular Drug Delivery. Figure 2a displays digital images of LDH-Co-RB nanosheet powder taken under daylight and UV light, with free rhodamine B dye powder as a reference for comparison. Attractively, LDH-Co-RB nanosheets are highly luminescent under UV light, while no luminescence can be observed from rhodamine B powder. This observation is confirmed by solid-state fluorescence spectra (Figure 2b), in which strong emission of LDH-Co-RB nanosheets can be measured while the fluorescence of rhodamine B dye is completely quenched in powder form. The high luminescence of LDH-Co-RB nanosheets in the dry state may be explained as follows: it is possible that covalently doping rhodamine B molecules to LDH nanoparticles helps keep spacing between rhodamine B molecules and thus effectively minimizes their quenching.8 C

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Figure 2. (a) Digital images LDH-Co-RB nanosheets and rhodamine B powder under daylight and UV illumination. (b) Solid-state emission spectra of rhodamine B powder and LDH-Co-RB nanosheets.

Figure 3. Absorption (a) and emission (b) spectra of LDH nanoparticles with covalently conjugated rhodamine B molecules (LDH-Co-RB) and a free dye. Laser scanning confocal microscopy images of HeLa cells after incubation with LDH-Co-RB for 4 h: (c) bright-field image, (d) fluorescent image, (e) merged image.

Figure 4. Viabilities of HeLa cells after 48 h (a) and 72 h (b) of incubation with LDH-Co-RB nanosheets (NS), free MTX anticancer drug (MTX), and the LDH nanosheets loaded with MTX (NS-MTX). The concentrations of different materials in group 1 are NS: 2 μg mL−1, MTX: 0.25 μg mL−1, NS-MTX: 2 μg mL−1 of NS, and 0.25 μg mL−1 of MTX. From group 1 to 4, the solutions were serially diluted five times (n = 3, error bar indicates standard deviation; * indicates p < 0.05 with Student’s t test).

After confirming the excellent solid photoluminescence property of the LDH-Co-RB nanosheets, we also studied its

application in liquid form for bioimaging (Figure 3). Compared with free rhodamine B dye, LDH-Co-RB nanosheets show very D

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conveniently released during the endocytosis process of the nanomedicine (Figure S10). For the in vivo study, we first studied the biodistribution of LDH nanoparticles by taking fluorescence images of various organs and tumor (Figure S11). For this purpose, we attached Cy5 dye to LDH-Co-PEG by reaction with residual amine groups on the surface of the LDH-Co-PEG nanosheets and then intravenously injected the material into mice. The results in Figure S10 indicate that, during the initial 6 h, LDH-Co-PEG nanosheets are first accumulated in kidney (45.6% of fluorescence intensity), and limited fluorescent signal is in the tumor (5.2% of fluorescence intensity). By 24 h, the fluorescence intensity of LDH-Co-PEG nanostructures in kidney drops to 33% and, in the meantime, more LDH-CoPEG nanosheets accumulate in the tumor (8.8% of fluorescent intensity). Different from many previous reports in which nanoparticles are found to be heavily delivered to liver, these LDH nanosheets are preferably transported to kidney. This will be very good for clearance of the nanostructures from patients and therefore minimize the concern for long-term toxicity of the nanomedicine in the human body. Further efforts will be needed to further modify the nanostructure to improve the accumulation of the nanomaterial in the tumor site. Nevertheless, we subsequently performed in vivo anticancer therapy to explore the function of this type of LDH nanosheets as a carrier for drug delivery in the mouse model. Figure 5a shows the in vivo cancer therapeutic efficiency of free MTX drug, MTX drug loaded in PEG-modified LDH nanosheets, and MTX incorporated in traditional LDH nanoparticles. In Figure 5a, it can be seen that the efficacy of MTX loaded in nonmodified LDH nanoparticles and free MTX is very similar (on day 14, n = 5, p > 0.1 for the tumor volume in the two groups with Student’s t test). In a revealing comparison, on day 14, MTX loaded in LDH-Co-PEG nanosheets exhibits inhibition of tumor growth significantly better than that of free MTX (p < 0.05 for the tumor volume in the two groups with Student’s t test) and MTX loaded in nonmodified LDH nanoparticles MTX (p < 0.05 for the tumor volume in the two groups with Student’s t test). Moreover, mouse weight loss (from day 1 to 6) was found in the groups treated with MTX (weight loss of 2.4 ± 1.9% by day 6, Mean ± SD) and MTX loaded in nonmodified LDH nanoparticles (weight loss of 2.0 ± 3.9% by day 6, Mean ± SD) (Figure S12). In comparison, the mouse weight constantly increases in the group treated with MTX-loaded LDH-Co-PEG nanosheets (weight gain of 5.5 ± 2.1% by day 6 and 12.0 ± 6.7% by day 14, mean ± SD) (Figure S12). The weight difference of mice in different groups indicates that the PEG-modified LDH nanosheets with MTX displays reduced side effects. In addition, H&E staining of tumor slices was also evaluated (Figure 5c). From the H&E staining, one can observe higher cancer cell damage area (red stained) in LDH-MTX- and LDH-Co-PEG MTX-treated groups, indicating improved anticancer efficiency. To further understand the mechanism of LDH-Co-PEG MTX inhibition of tumor growth, immunohistochemistry analysis of caspase-3, KI-67, and CD31 was conducted (Figure 6). Caspase-3 is the central regulator and executes apoptosis, which is known as programmed cell death. In Figure 6, caspase3 expression of the LDH-Co-PEG MTX (mean optical density (MOD) = 0.1705)-treated group was higher than that of MTX (MOD = 0.1612)- and LDH-MTX (MOD = 0.1689)-treated groups, indicating that LDH-Co-PEG MTX is able to induce more cell apoptosis than that of MTX and LDH-MTX. For

similar absorption and emission spectra, indicating that covalent conjugation does not affect the optical properties of rhodamine B dye (Figure 3a,b). Figure 3c−e shows that LDHCo-RB nanosheets can easily penetrate cell membranes after 4 h incubation, making these nanosheets very promising for not only bioimaging application but also intracellular drug delivery at the same time. To confirm that LDH-Co-RB nanosheets can also be used as a vector for intracellular delivery, we studied intracellular delivery of anticancer drug MTX with the help of LDH-Co-RB nanosheets (Figure 4 and Figure S8). As LDH-Co-RB nanosheets are positively charged (∼40 mV) (Figure S5), it is convenient to load negatively charged drug MTX by electrostatic attraction. For drug loading, 2 mg mL−1 of free anticancer drug MTX was mixed with 2 mg mL−1 of LDH-CoRB nanosheets for 12 h, and then the MTX-loaded nanosheets and free MTX were separated by centrifugation at a speed of 12 000 rpm. By measuring UV−vis absorbance of the MTX suspension at 370 nm, it was found that the loading capacity of LDH-Co-RB nanosheets is about 12.5 wt %. In other words, 1 g of the nanomaterials can load 0.125 g of MTX. Figure 4 presents the viabilities of HeLa cells after incubation with LDHCo-RB nanosheets, anticancer drug MTX (MTX), and LDHCo-RB nanosheets loaded with MTX for 48 h (a) and 72 h (b). For the first 48 h of treatment with free MTX drug, the viability of HeLa cells remains high (over 80%) even at a high MTX concentration of 0.25 μg mL−1, but at the same concentration, MTX loaded with LDH-Co-RB nanosheets can decrease the cell viability to below 60%. After 72 h of treatment with free MTX drug, the viability of HeLa cells drops to ∼49% at a concentration of 0.25 μg mL−1, while the viability is ∼31% in the group in which MTX is delivered with the aid of LDH-CoRB nanosheets. Obviously, LDH-Co-RB nanosheets represent a promising nanocarrier to deliver drug molecules into cells for improved therapeutic efficacy. 3.3. LDH-Co-PEG Nanosheets for in Vivo Drug Delivery for Cancer Therapy. Although many studies have demonstrated that LDH-based nanomaterials can effectively deliver a wide range of molecules (e.g., genes, anticancer drugs, and vaccines) for in vitro biomedical applications, very limited evidence shows that LDH nanoparticles can improve cancer therapy in vivo. The reason may be that it is difficult to modify the surface of LDH nanoparticles due to their lack of functional groups and that non-surface-modified LDH nanoparticles can hardly survive in the blood system, resulting in low circulation time and reduced delivery efficiency in vivo. The PEG molecule is widely used to improve the biocompatibility, stability, and circulation time of nanomaterials for in vivo applications, because of the “stealth effect” that suppresses nonspecific interaction and reduces blood clearance.43−46 Therefore, we modified the surface of our LDH nanostructures with PEGcarboxyl molecules (O-(2-carboxyethyl)polyethylene glycol, Mw = 5000) using the same strategy as that employed to attach rhodamine B (Figure S9) and test the application for in vivo anticancer therapy in a mouse model. PEG-modified LDH (LDH-Co-PEG) nanosheets still carry strong positive charge, with a surface zeta potential of ∼+32 mV, and MTX was loaded by electrostatic attraction. Before the in vivo tumor study, we investigated the release profile of MTX from LDH-PEG nanosheets. The results show that MTX displays a prolonged release curve at pH 7.4 in PBS solution. However, when the pH drops to 5.0, the pH value of late endosomes/lysosomes, there is a burst release. This indicates that the loaded drug can be E

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cells in the LDH-Co-PEG MTX-treated group was 46%, which was higher than that in the MTX (27%)- and LDH-MTX (32%)-treated groups. Therefore, one can conclude that LDHCo-PEG MTX works better than MTX and LDH-MTX to induce tumor cell apoptosis. KI-67 is a nuclear protein and believed to be required for maintaining cell proliferation. Therefore, higher KI-67 expression means that higher cancer cell proliferation occurs. In Figure 6, higher expressions of KI67 can be observed in control (MOD = 0.2642) and MTX (MOD = 0.2401)- and LDH-MTX (MOD = 0.2453)-treated groups, while the LDH-Co-PEG MTX-treated group has a low value (MOD = 0.2382). This indicates that LDH-Co-PEG can effectively inhibit cancer cell proliferation and tumor growth. CD31 protein is used to identify endothelial cells and can help estimate tumor angiogenesis, which is a vital process in tumor growth. In Figure 6, higher CD31 expression is shown in the control and MTX-treated groups but it is lower in the LDHMTX- and LDH-Co-PEG MTX-treated groups, revealing that the tumor growth rate is lowered in the LDH-MTX- and LDHCo-PEG MTX-treated groups compared with the control and MTX-treated group. Microvascular density (MVD) was also counted with the help of the CD31 marker to assess the neovasculature in the tumor, which is highly associated with tumor metastasis and prognosis. MVD counting was evaluated in the vascular hot spots (most vascularized area, in every slice five hot spots were selected), and the average MVD results are 14, 10, 8, and 7 in control, MTX, LDH-MTX, and LDH-CoPEG MTX groups, respectively. These MVD-CD31 results confirm that the tumors treated with LDH-MTX and LDH-CoPEG MTX have a lower tendency to grow. Overall, our experiments demonstrate that LDH-Co-PEG nanostructures are more suitable as a carrier for delivery of anticancer drugs by showing improved therapeutic efficacy of drugs and possibly alleviating side effects. 3.4. Biosafety of LDH-Co-PEG Nanosheets in Vivo. The biocompatibility of nanomaterials is of great importance for clinical applications; therefore, we next systematically investigated the potential influence of our LDH nanosheets. First, histopathological examinations (hematoxylin and eosin (H&E) stain) of the major organs, including heart, liver, spleen, lung and kidney, were conducted (Figure 7). From H&E staining images, we could not identify any obvious organ damage in the LDH-Co-PEG nanosheet-treated group compared with the control group of PBS-treated mouse. In addition, the liver and kidney functions (Figure 8) of the mice after LDH-Co-PEG nanosheets treatment were also evaluated by measuring the blood serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine (CRE). There is no obvious increase of ALT, AST, ALP, BUN, and CRE in the LDH-Co-PEG nanosheet-treated group compared with the control group, which indicates that LDH-Co-PEG nanosheets have minimal toxicity toward liver and kidney, which is in line with H&E results. In addition, there is also no significant change of the complete blood count data of red blood cells (RBC), white blood cells (WBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT) (Figures S14 and Figure S15). It is worth noting that the significantly high WBC level (compared with normal range) of all groups of mice may be caused by the tumor in the mice. Nevertheless, the LDH-Co-PEG MTX-treated mice possess

Figure 5. In vivo anticancer activities: (a) Tumor growth curves of 4T1 tumor-bearing BALB/C mice after intravenous injection with (1) PBS, (2) free MTX (MTX), (3) MTX loaded in traditional LDH nanoparticles (LDH-MTX), and (4) MTX loaded in PEG-modified ultrathin LDH nanosheets (LDH-Co-PEG MTX) (n = 5, error bar indicates standard deviation; the arrow indicates tail vein injection at day 1; * indicates p < 0.05 with Student’s t test). (b) The body weight evolution of 4T1 tumor-bearing mice at different times after intravenous injection of different materials (n = 5, error bar indicates standard deviation). (c) H&E staining images of tumors slices. The tumor-bearing mice were treated with MTX, LDH-MTX, and LDHCo-PEG MTX at day 1 and sacrificed at day 7. Higher cancer cell damage (red area indicates area of cell damage) was found in LDHMTX- and LDH-Co-PEG MTX-treated groups. Scale bar: 100 μm.

Figure 6. Immunohistochemistry images of tumor slices by using caspase 3, KI67, and CD31 antibody. The tumor-bearing mice were treated with PBS, MTX, LDH-MTX, and LDH-Co-PEG MTX at day 1 and sacrificed at day 7. Yellow-brown indicates positive; blue indicates negative. Scale bar: 20 μm.

quantity analysis, cell apoptosis was also determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Figure S13). The percentage of apoptotic F

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ACS Applied Materials & Interfaces

4. CONCLUSIONS In summary, we report a novel type of aqueous dispersible twodimensional ultrathin LDH nanosheets. We are able to surfacemodify the nanosheets with positively, negatively, and neutrally charged molecules to form multifunctional LDH nanohybrids for bioimaging and in vivo drug delivery for cancer therapy. The rhodamine B-modified LDH-Co-RB nanosheets prevent self-quenching of rhodamine B in powder form and display very strong photoluminescence. These LDH-Co-RB nanosheets can disperse well in the liquid state, penetrate the cell membrane for bioimaging, and increase intracellular delivery in vitro. PEGmodified LDH-Co-PEG nanosheets can significantly increase the therapy efficacy of anticancer drug MTX in the in vivo application in the mouse model compared with traditional LDH nanoparticles and free MTX drug. Also very importantly, through our systematic study of the safety profiles of the LDHCo-PEG nanosheets by carrying out body weight measurement, H&E staining, biodistribution in major organs, hematology analysis, and blood biochemical assay, it was found that this type of nanostructure is highly biocompatible.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 7. H&E staining images of major organs (heart, liver, spleen, lung, and kidney). The tumor-bearing mice were treated with PBS, MTX, LDH-MTX, and LDH-Co-PEG MTX at day 1 and sacrificed at day 7. Scale bar: 200 μm.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b05294. Schematic illustration of the preparation of LDH nanosheets (Figure S1), powder X-ray diffraction patterns (XRD) and Fourier transform infrared (FTIR) spectroscopy (Figures S2 and S3), XPS spectra (Figure S4), dynamic light scattering measurement (Figure S5), AFM image of LDH-Co-RB hybrid (Figures S6 and S7), viabilities of A549 cells (Figure S8), AFM image of LDHCo-PEG nanosheets (Figure S9), release profile of MTX

WBC levels much lower than those of the control group, which indicates the beneficial effect of the treatment. This again demonstrates that the LDH-Co-PEG nanosheets have very low toxicity in vivo. In general, all toxicity profiles suggest that LDH-Co-PEG nanosheets have a high level of biosafety for in vivo cancer therapy.

Figure 8. (a) Liver function (ALT, AST, and ALP) and (b) kidney function (BUN and CRE) of tumor-bearing mice. The mice were treated with (A) PBS as control, (B) MTX, (C) LDH-MTX, and (D) LDH-Co-PEG MTX at day 1 and sacrificed at day 7. The normal ranges of ALT, AST, ALP, BUN, and CRE are 33.0−99.0, 69.5−210.0, 40.0−190.0, 2.00−7.70, and 22.0−97.0, respectively. G

DOI: 10.1021/acsami.7b05294 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

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from LDH-Co-PEG (Figure S10), biodistribution of LDH-Co-PEG (Figure S11), body weight evolution of 4T1 tumor-bearing mice (Figure S12), TUNEL staining of apoptotic tumor cells (Figure S13), complete blood counts data: RBC, HGB, HCT, MCV, MCH, MCHC, PLT, and MPV (Figures S14 and S15) (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] ([email protected]). *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Vellaisamy A. L. Roy: 0000-0003-1432-9950 Wenjun Zhang: 0000-0002-4497-0688 Xianfeng Chen: 0000-0002-3189-2756 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (no. 51173124) and National Basic Research Program of China (nos. 2012CB932400, 2011CB808400), the School of Engineering of University of Edinburgh, the Royal Society Research Grant Scheme RG150564, and the Chengdu University (nos. 2081916010 and ARRLKF16-05).

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DOI: 10.1021/acsami.7b05294 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX