Porous Iron-Carboxylate MetalâOrganic ... - ACS Publications

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Porous Iron-Carboxylate Metal−Organic Framework: A Novel Bioplatform with Sustained Antibacterial Efficacy and Nontoxicity Sha Lin,† Xiangmei Liu,† Lei Tan,† Zhenduo Cui,‡ Xianjin Yang,‡ Kelvin W. K. Yeung,§ Haobo Pan,∥ and Shuilin Wu*,†,‡ †

Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei Key Laboratory of Polymer Materials, School of Materials Science & Engineering, Hubei University, Wuhan 430062, China ‡ School of Materials Science & Engineering, Tianjin University, Tianjin 300072, China § Department of Orthopaedics & Traumatology, Li Ka Shing Faculty of Medicine, The University of Hong Kong 999077, Pokfulam, Hong Kong, China ∥ Center for Human Tissues and Organs Degeneration, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China S Supporting Information *

ABSTRACT: Sustained drug release plays a critical role in targeting the therapy of local diseases such as bacterial infections. In the present work, porous iron-carboxylate metal−organic framework [MOF-53(Fe)] nanoparticles (NPs) were designed to entrap the vancomycin (Van) drugs. This system exhibited excellent chemical stability under acidic conditions (pH 7.4, 6.5, and 5.5) and much higher drug-loading capability because of the high porosity and large surface area of MOF NPs. The results showed that the drug-loading ratio of Van could reach 20 wt % and that the antibacterial ratio of the MOF-53(Fe)/Van system against Staphylococcus aureus could reach up to 90%. In addition, this MOF-53(Fe)/Van system exhibited excellent biocompatibility because of its chemical stability and sustained release of iron ions. Hence, these porous MOF NPs are a promising bioplatform not only for local therapy of bacterial infections but also for other biomedical therapies for tissue regeneration. KEYWORDS: MOF, drug delivery, antibacterial, biocompatibility, osteogenic differentiation

1. INTRODUCTION

which combine the hybrid inorganic−organic framework, have been exhibiting great potential for biomedical applications9,10 because of their unique mesoporous structures such as high porosity,11 tunable particle size,12 and modifiable chemical surface,13,14 as well as chemical compositions.15 The most important feature is that the interconnected channel of MOFs makes this material a promising carrier platform for drug delivery systems.16,17 Some recent research has revealed that carboxylic acid iron MOFs [MOF-53(Fe)] constructed from iron ions and

Generally, surgical procedures are liable to suffer from bacterial infections, especially for artificial bone implants.1,2 The bacterial infection associated with the implants has also become a universal phenomenon;3 especially, Staphylococcus aureus (S. aureus) is a common pathogen causing wound infection. Therefore, it is urgent to develop novel antibacterial systems to avoid the risk of bacterial infections. Some antibacterial agentloading strategies have been proposed for targeting drug delivery system, including the inorganic nanocarriers4,5 and organic polymer nanocarriers.6,7 However, the poor stability, the uncontrolled release behaviors, and the poor biocompatibility of these materials have limited their extensive applications.8 Recently, metal−organic frameworks (MOFs), © XXXX American Chemical Society

Received: April 5, 2017 Accepted: May 19, 2017

A

DOI: 10.1021/acsami.7b04810 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces terephthalic acid are nontoxic and biocompatible.15 Owing to the breathing effect,18,19 the skeleton of this mesoporous material is very flexible and can reversibly adapt its pore size through drug adsorption.20,21 In addition, MOF-53(Fe) exhibits a good chemical stability in acidic solution (pH 7.4, 6.5, and 5.5)22,23 despite the trace amounts of degradation released from the MOF-53(Fe). The degradation products containing iron and small molecules are endogenous with low toxicity.24 Thus, drug-loading MOF-53(Fe) can be expected to control the release rate of drugs effectively. Furthermore, the trace amount of dissociative iron ions can be favorable for the human body.25−27 Among various strategies used for antibacterial therapy, organic antibacterial drugs are often employed to prevent bacterial infections because these drugs exhibit potent antibacterial efficiencies, even at low drug concentrations, such as vancomycin (Van). Van is one of the glycopeptide macromolecule antibiotics, mainly against Gram-positive bacteria such as S. aureus.28 Van presents the form of vancomycin hydrochloride in aqueous solutions; therefore, its surface is enriched with a large amount of positive charge, which enables it to be easily adsorbed on the negatively charged metal organic skeleton to form a strong electrostatic interaction. The drug delivery system of Van-loaded MOFs shows a controlled drug release and good antibacterial effect on S. aureus. To further confirm that the channel of porous MOFs can also load small-molecule drugs, ibuprofen (Ibu) was chosen as a typical drug model of small molecule in this work.29 Ibu is one of the common anti-inflammatory medications and has no antibacterial ability. In this work, Van not only has a strong antibacterial ability but also shows nontoxicity toward osteoblasts. As far as we know, MOFs have not been reported to be a carrier of antibacterial agents for antimicrobial applications. Meanwhile, MOFs have a good biocompatibility and even promote cell osteogenesis and differentiation. Herein, we utilized MOF-53(Fe) as the carrier platform of drugs to enhance the antibacterial efficacy. In this work, drugs were encapsulated into MOFs through a postsynthesis method. Briefly, MOF-53(Fe) was synthesized through the solvothermal method, and then, the drugs that included Van and Ibu acted as guest molecules and were encapsulated into MOF-53(Fe) nanoparticles (NPs) by physical absorption. The corresponding drug-loaded MOFs are marked as MOF-53(Fe)@Van and MOF-53(Fe)@Ibu. The measured Van-loading capacity could reach up to 20 wt %. It has been reported that bacterial infection will induce a local acidic environment;30 we demonstrated that MOF-53(Fe) had a controlled drug release behavior with a high antibacterial efficiency of 99.3%, especially under an acidic condition (pH = 5.5). In addition, the degradation products of Fe3+ are helpful to the human body. Meanwhile, Van had a slower and controllable release in an acidic environment, which is favorable for a more lasting antibacterial efficacy. In vitro tests disclosed that this drug delivery system of MOF-53(Fe)@Van showed a good chemical stability with noncytotoxicity.

the precursor solution was stirred until the metal source was dissolved completely. Simultaneously, 0.166 g (1 mmol) of terephthalic acid in 15 mL of DMF was added into the FeCl3·6H2O solution. After the mixture was stirred for 20 min, it was transferred into a steel autoclave and further heated for 72 h at 150 °C. After cooling to room temperature, the products were collected by centrifuging and washed three times with DMF. The as-synthesized particles were then dried overnight at 150 °C under a vacuum and preserved in a drying oven for further use. 2.2. Drug Encapsulation. The encapsulation of drugs, including Van and Ibu, was performed by a simple impregnation method. For Van encapsulation, Van was dispersed into 20 mL of deionized (DI) water with a concentration of 25 mg/mL, and then, 100 mg of MOF53(Fe) was added into the Van solution under stirring for 30 min. This mixture was placed in the vacuum environment for 1 day, and the mixtures of MOF-53(Fe)@Van were collected by filtering, washing with DI water, and then drying at room temperature. The crystalline structures of both MOF-53(Fe) and MOF-53(Fe)@Van powders were recorded using X-ray diffraction (XRD), and the loading amount of the drugs was quantified using an ultraviolet−visible (UV−vis) spectrophotometer (UV-3600, Japan). In addition, the encapsulation process of Ibu is described in detail in section S1 of the Supporting Information. 2.3. Characterization. The morphology and size of MOF NPs were visualized using a scanning electron microscope (SEM; JSM6510LV, Japan). The crystallinity and the purity of the particles were determined by XRD (D8A25, Bruker, Germany) using Cu Kα radiation (λ = 1.54051 and 1.54433 Å) over the 2θ range of 3°−30°. A transmission electron microscope (TEM; Tecnai G20, FEI, USA) was used to observe the ultrastructure of the particles. A Fourier transform infrared (FT-IR) spectroscopy detector (Nicolet IS10, USA) was chosen to identify Van and Ibu in the drug-loaded MOF-53(Fe). The specific surface area was measured from the results of nitrogen isotherms at 77 K (Quantachrome, QDS-MP-30) using the Brunauer− Emmett−Teller (BET) theory after the dehydration of the sample at 150 °C for 12 h under vacuum. The pore volume and pore size were calculated according to density functional theory (DFT) applied to the adsorption curve. X-ray photoelectron spectroscopy (XPS; Thermo Fisher Scientific 250Xi) measurements were taken to visualize the XPS spectra of the Fe 2p. Zeta potential (Zeta; Malvern, UK) was measured to detect the positive and negative charges of the sample solution. Dynamic light scattering (DLS; Malvern, UK) was utilized to detect the particle size distribution. 2.4. In Vitro Release Studies. A typical Van release was carried out by dispersing 25 mg of drug-loaded powders into a dialysis bag with 5 mL of phosphate buffer solution (PBS), then immersing the bag in 25 mL of PBS at different pH levels (pH = 7.4, 6.5, and 5.5) at 37 °C in a tube, and sampling out 5 mL of released liquid at set intervals while a fresh PBS solution of 5 mL was added back to the tube. The concentration of the released drug was measured from UV−vis absorbance at 280 nm. According to the released curve of Van, the released equilibrium concentration of Van can be used as the amount of drug loading (m1); therefore, the payload (wt %) = m1/m2, where m2 is the amount of drug-loaded particles. The Ibu loading was performed in a similar way. The release behavior of iron ions from MOF-53(Fe)@Van was analyzed by suspending 5 mg of MOF-53(Fe)@Van NPs into 50 mL of PBS at pH 7.4, 6.5, and 5.5. The releasing supernatant (3 mL) was collected at various intervals and centrifuged, and then, the concentration of iron ions was detected using inductively coupled plasma atomic emission spectrometry (Optimal 8000, PerkinElmer, US). 2.5. Antibacterial Property Test. S. aureus was used to evaluate the antibacterial activity of Van-loaded MOF-53(Fe) (MOF-53@Van) using agar-plating method and was cultured in the Luria−Bertani (LB) culture medium. In this study, 200 μL of mixtures of diluted bacterial suspension (107 CFU/mL) was incubated with LB culture medium containing PBS (control), free Van, MOF-53, MOF-53 + Van (nonencapsulated), and MOF-53@Van (12.5, 25, 50, 100, and 200 μg/mL). The payload of Van-loaded MOF-53(Fe) was 20%, and then,

2. EXPERIMENTAL PROCEDURES 2.1. Synthesis of MOF-53(Fe). The iron-based MOF-53(Fe) was synthesized from a mixture of iron chloride (Alfa, 99%), terephthalic acid (Alfa, 97%), and N,N′-dimethylformamide (DMF, Alfa, 99%) with a molar ratio of 1:1:380 in a Teflon-lined steel autoclave. First, 0.27 g (1 mmol) of FeCl3·6H2O was dissolved in 15 mL of DMF, and B


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Scheme 1. Schematic Representation of MOF-53(Fe) Structure and the Process of MOF Packing Drug Molecules for Killing Bacteria

Figure 1. Surface topography and structural analysis of MOF-53(Fe): (a) SEM, (b) TEM, (c) XRD, and (d) Fe 2p XPS spectra of the MOF-53(Fe) after heated at 100 °C in vacuum. the concentration of free Van varied at 2.5, 5, 10, 20, and 40 μg/mL, correspondingly. The concentration of MOF-53 varied at 10, 20, 40, 80, and 160 μg/mL. The groups of free Van, MOF-53(Fe) + Van, and MOF-53(Fe)@Van have the same Van concentration, whereas the groups of MOF-53(Fe), MOF-53(Fe) + Van, and MOF-53(Fe)@Van have the same concentration of MOF-53(Fe). Each group of samples was prepared in triplicate, and the sample solution was placed in each well of a 96-well plate. Then, the samples were cultured in an incubator at 37 °C in 5% CO2 for 24 h. After that, the diluted bacterial solution of 20 μL was then coated on the culture dish covered with LB agar, and the samples were incubated at 37 °C for 24 h. At this point, we photographed the bacterial colony on the plates and counted the number of colonies. Meanwhile, the antibacterial rate was used to assess the antibacterial activity via the following equation

2.6. Morphological Studies. SEM could observe the morphology of S. aureus. The samples were put into 48-well plates with bacterial suspension. After an incubation period of 24 h, the bacteria were fixed with a 2.5% glutaraldehyde solution for 4 h, dehydrated sequentially in the ethanol solution with different concentrations (30, 50, 70, 90, and 100%) for 15 min, and then preserved in the cold closet for SEM observation. 2.7. Cell Culture. The MC3T3-E1 cells from the calvarial cells of mice (Tongji Hospital, Wuhan) were cultured with the samples of the solution to evaluate the cytocompatibility. They were cultured with αMEM (HyClone) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin solution (HyClone), followed by incubation in a humidified atmosphere of 5% CO2 at 37 °C. The culture medium was refreshed every 3 days. 2.8. Cytotoxicity Assay. The cytotoxicity of drug-loaded MOF53(Fe) was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of cellular activity on the preosteoblast MC3T3 cell of a mouse. Before the MTT assay, the MC3T3 cell was maintained in Dulbecco’s modified eagle medium (DMEM), then was seeded in 96-well plates, and cultured for 24 h.

Antibacterial ratio (%) = (number of CFUs in control group − number of CFUs in experimental group) /(number of CFUs in control group) × 100% C


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ACS Applied Materials & Interfaces After the cell attached to the wall, the solutions with different concentrations of pure MOF, MOF-53(Fe)@Van, MOF-53(Fe) + Van, free Van, and the same concentration of iron ions released from the MOF-53@Van NPs were added into the well and continued to be cultured for 24 h. After that, the MTT solution with a concentration of 5 μg/mL was added to each well and incubated for 4 h at 37 °C. The medium was then discarded, a dimethyl sulfoxide (DMSO) solution of 200 μL was added to each well, and the plate was shaken for 10 min; after that, the supernatant fluid was fetched out to test the optical density (OD) value, and a microplate reader was used to read the OD at 490 nm. The results were expressed as the percentage of cell viability. All measurements were taken in triplicate. 2.9. Fluorescence Morphology. The cell fluorescence was detected by cell staining. Typically, the MC3T3-E1 cells were first seeded in a 12-well plate for 24 h for cell attachment, and then, the samples were added to each well for a further incubation of 24 h. After being washed three times in PBS, the cells were fixed with 4% paraformaldehyde solution at room temperature for 10 min and then rinsed with PBS thoroughly. The cells were stained with fluorescein isothiocyanate (FITC; YiSen, Shanghai) at room temperature for 30 min in a dark place, rinsed with PBS, and then further stained with 4′,6-diamidino-2-phenylindole (DAPI; YiSen, Shanghai). The cytoskeletal actin (green fluorescence) stained by FITC and the cell nuclei (blue fluorescence) stained by DAPI were observed using the inverted fluorescence microscope (IFM, Olympus, IX73). The cells were washed with PBS, stained by propidium iodide (PI, dead cells, red fluorescence) for 30 min, rinsed with PBS, then stained by acridine orange (AO, live cells, green fluorescence), and finally observed via IFM.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of MOF-53(Fe). The synthesis of MOFs and subsequent encapsulation with Van and Ibu are schematically presented in Scheme 1. MOF-53(Fe) NPs were built from the self-assembly node of ferric ion clusters and ligands of the terephthalic acid. These threedimensional porous solids were octahedral with corner-sharing chains of iron ion clusters connected through terephthalate linkers to define diamond-shaped one-dimensional (1D) channels. Because of the myriad pores, drug molecules can be physically absorbed. The positively charged Van would be absorbed by the negatively charged MOF-53, and the Ibu acted as a small molecule would enter into the hole of the MOF-53. The SEM images of the synthesized materials, shown in Figure 1a, display that the MOF-53(Fe) NPs have an octahedral structure with an average diameter of 500 nm, and these NPs exhibit a homogeneous size distribution, similar to the reported results.31 The TEM examination further confirmed the unique structure of the 1D channel and neat hexagon projection, shown in Figure 1b. The XRD pattern shown in Figure 1c displays a high intensity of sharp diffraction for MOF-53(Fe).32 The chemical valence of Fe in the porous solid was identified using XPS, as shown in Figure 1d, in which the Fe 2p1/2 and Fe 2p3/2 peaks of MOF-53(Fe) are located at 725.2 and 711.6 eV, respectively, and their separation (Δ = 2p1/2 − 2p3/2) is 13.6 eV. These characteristics are consistent with those of Fe2O3.33,34 3.2. Drug Encapsulation. The textural properties of MOF53(Fe) and MOF-53(Fe)@Van assessed from the N 2 adsorption isotherm at −196 °C are presented in Figure 2, and the calculated values are listed in Table 1. The BET surface area and the pore volume for the MOF-53(Fe) are 83.76 m2 g−1 and 0.08 cm3 g−1, respectively, and the BET value of 12.3 m2 g−1 is higher than the reported value in the literature.35 After drug encapsulation, the corresponding values of surface area and the pore volume for the MOF-53(Fe)@Van were 10.338

Figure 2. (a) N2 adsorption isotherms at −196 °C and (b) DFT pore size distributions of the MOF-53(Fe) and MOF-53(Fe)@Van.

Table 1. Physicochemical Properties of MOF-53(Fe) and MOF-53(Fe)@Van MOF-53(Fe) MOF-53(Fe)@Van a

SBETa (m2 g−1)

Vpb (cm3 g−1)

pore width (nm)

83.76 10.338

0.08 0.03

1.688 1.614

BET surface area. bTotal pore volume.

m2 g−1 and 0.033 cm3 g−1, indicating a remarkable decrease compared with the parent MOF-53(Fe) and suggesting that a certain portion of the mesoporous cages of the MOF-53(Fe) were occupied by the drug molecules. Figure 2b shows the DFT pore size distribution curves of the MOF-53(Fe) and MOF-53(Fe)@Van. The corresponding pore size was about 1.688 and 1.614 nm, also indicating the presence of the mesoporous cages in these samples. In the further FT-IR analysis shown in Figure 3a, the characteristic peak of MOF53(Fe) at 800 cm−1 was assigned to δOOP (Ar C−H), indicating the existence of the phenyl substituent on the benzene ring. In addition, the characteristic peak of Van at 3430 cm−1 was derived from the N−H stretching vibrations, and the carbonyl absorption band was located at 1653 cm−1. After loading with MOF-53(Fe), the characteristic peak of Van still existed, suggesting the successful loading of Van in MOFs. As a small molecule, Ibu was also successfully loaded into the MOFs (Figure S1). According to the measurement of zeta potentials D


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Figure 3. (a) FT-IR spectra of starting MOF-53, MOF-53@Van, and free Van. (b) Zeta potentials of the MOF-53(Fe)@Van, MOF-53(Fe), and Van. Data represent mean ± standard deviation (SD) (n = 3, **P < 0.01).

shown in Figure 3b, the zeta potential of the aqueous solution of Van was tested as having positive potential, whereas the MOF-53(Fe) aqueous solution exhibited a negative electric potential. After drug loading, the MOF-53(Fe)@Van displayed a higher negative potential, indicating that a large number of Van molecules were adsorbed in the holes of MOF-53(Fe) through electrostatic interaction. In addition, the absorption of the drug did not change the structure of the MOF;36 similarly, the MOF-53(Fe) loaded with Ibu also did not alter the structure of MOF-53(Fe), which was proven by the XRD patterns shown in Figure S2. 3.3. Detection of Drug and Ion Concentration. Figure 4a,b shows the cumulative release percentage and concentration of Van from MOF-53(Fe)@Van with 20 wt % Van loading in PBS (pH = 7.4) within 72 h. The payload of Van loading in the flexible mesoporous MOF-53 may be exceptionally high (20 wt %). As shown in Figure 4a, the percentage of the released Van is 52% in the initial 2 h and reaches up to a maximum value of 97% when it came to an equilibrium concentration after immersion for 36 h. The corresponding drug concentrations are 92.6 and 175 μg/mL (Figure 4b). This released percentage was higher than that of the system of polymer NPs (79.6%),37 and the maximal released concentration was also higher than that with liposomes (62 μg/mL).38 In the earlier stage of 12 h, there was an obvious burst release ascribed to the adsorption of Van

Figure 4. (a) Released percentage of Van from MOF-53(Fe)@Van (0.8 mg/mL) in PBS under pH 7.4 at 37 °C. (b) Accumulated released concentration of Van from MOF-53(Fe)@Van (0.8 mg/mL) in PBS under pH 7.4 at 37 °C. (c) Accumulated released amount of Van drug release from MOF-53(Fe)@Van (0.8 mg/mL) in different PBS solutions (pH 7.4, 6.5, and 5.5). (d) Accumulated released amount of Fe3+ from MOF-53(Fe)@Van (100 μg/mL) in different PBS solutions (pH 7.4, 6.5, and 5.5).

onto the surface of MOF-53(Fe), which can be released into the PBS easily, and that was different from the Ibu-loading MOF delivery system (Figure S3) with a more sustained release E


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Figure 5. (a) Representative images of viable S. aureus grown on different samples after 24 h of culture are shown, and (b) colonies of viable S. aureus on different samples were grown for 24 h and counted. Data represent mean ± SD (n = 3, **P < 0.01 vs the control group). (c) Antibacterial activity of MOF-53 and MOF-53@Van with different concentrations against S. aureus via the agar plating method.

confirmed that the concentration of iron ions from the degradation of MOF was extremely low and that the actual degradation degree was 0.75% in pH 7.4 and 0.17% in pH 5.5. Therefore, the degradation can be ignored with respect to the as-prepared MOFs. According to the release profiles of iron ions shown in Figure 4d, the released amount of Fe3+ was increased with the increase in pH value. It could be inferred that the surface of MOFs exhibited negative charges, as presented in Figure 3b, and the surface charge would become more negative as the pH value increased.39,40 The accumulated negative charges would aggravate the degradation of the MOF-53(Fe), which demonstrated that the MOF-53(Fe) NPs had better stability under acidic conditions compared with that under neutral conditions. This result was in agreement with the released amount of Van in different pH values, as shown in Figure 4c. It can be speculated that the release rate of Van would be accelerated with the partial degradation of MOF-53(Fe). In other words, because MOF-53(Fe) NPs partly degraded under neutral conditions, the released amount of Van could have a bigger burst effect. Although MOF-53(Fe) NPs had a smaller degree of degradation under the acidic conditions, the release of Van was relatively slower. This result would contribute to the treatment of bacterial infections under acidic conditions. In addition, the iron ions with tolerable concentration are biocompatible, and the small molecules of the degradation product are absorbed by the body. In addition, this drug delivery system of iron-carboxylate MOFs had good stability in different solutions [DMEM, PBS, and simulated body fluid (SBF)], as shown in Figure S5, and the average diameter in

Figure 6. SEM morphology of S. aureus seeded on various samples after incubation at 37 °C for 24 h. The scale bar is 1 μm.

of Ibu. It was also partly associated with the slight dissolution of MOF-53(Fe) under the neutral condition. In addition, the accumulated release profiles of Van from MOF-53(Fe)@Van (0.8 mg/mL) (Figure 4c) and Fe3+ from MOF-53@Van NPs (100 μg/mL) (Figure 4d) were investigated in a series of PBS of pH 7.4, 6.5, and 5.5 within 168 h. According to the release profile shown in Figure 4, the accumulated released amount of Fe3+ showed the degradation of the MOF-53(Fe)@Van NPs under different pH conditions. To further evaluate the degradation of MOF-53(Fe) NPs, the MOF-53@Van NPs were fully dissolved in 4 mol/L concentrated hydrochloric acid. As shown in Figure S4, the content of free iron ions completely decomposed from 1 mg/mL MOF-53@Van NPs in hydrochloric acid was 196.24 μg/mL. However, the released amounts of iron ions from 1 mg/mL MOF-53@Van NPs in pH 7.4, 6.5, and 5.5 were 1.47, 1.16, and 0.33 μg/mL, respectively, which F


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Figure 7. MTT assay of MC3T3-E1 cell cultured with (a) MOF-53, MOF-53@Van, MOF-53 + Van, and free Van for 24 h and (b) different concentrations of iron ions; the iron ions of 0.24 μg/mL is equivalent to the content of iron ions produced in MOF-53@Van (200).

DMEM and SBF was 671.6 and 932.6 nm, respectively, which indicated that the Van-loaded MOF-53 NPs had a good dispersion without aggregations in the solution. As shown in Figure S6, the concentration of the iron ions was extremely low in these three solvents, which further confirmed the good stability of MOF-53@Van. 3.4. Antibacterial Activity. The antibacterial activities of Van-loading MOF-53(Fe) were analyzed via the spread plate method (Figure 5a).41 The number of viable bacterial colonyforming units (cfus) was counted, as shown in Figure 5b. The bacterial colony of the control group was dense with corresponding cfus of 296, indicating that the pure bacteria could grow well on the LB agar plate. The bacterial viability of the group MOF-53(Fe) with concentrations of 20, 40, 80, and 160 μg/mL was gradually reduced compared with that of the control group, and the corresponding number of cfus was 252, 232, 223, and 211, respectively. The group MOF-53@Van had better antibacterial activity, the number of cfus decreased significantly, and the corresponding number of cfus of MOF53@Van with concentrations of 25, 50, 100, and 200 μg/mL was 201, 142, 70, and 2, respectively, which verified that MOF53@Van with higher concentrations had better antibacterial

Figure 8. Fluorescent images of cells (scale bars = 50 μm) after culturing with various samples for 24 h with F-actin stained with FITC (green) and nucleus stained with DAPI (blue).

properties. Meanwhile, the antibacterial ratio using the agarplating method is shown in Figure 5c, the group MOF-53 NPs had a lower antimicrobial effect, and it could be inferred that the antimicrobial efficacy of MOF-53(Fe) was attributed to the antibacterial ability of iron ions.42 As shown in Figure S8, the antibacterial activity increased with the increase in the amount of iron ions, 0.24 μg/mL iron ions were equivalent to the concentration of iron ions degraded in MOF-53 (160), and the antibacterial rate could reach up to 12%. As shown in Figure 5c, it could be found more clearly that the antibacterial ratio of G


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shapes and incomplete membranes. Similarly, the free Van showed a great bacterial destruction, which may be attributed to Van inhibiting the synthesis of the cell wall structure of S. aureus44 and the death of S. aureus resulted from the destruction of the membrane integrity. 3.5. Cytotoxicity. The cell viabilities of MOF-53(Fe), MOF-53(Fe)@Van, MOF-53(Fe) + Van, and free Van were evaluated by MTT assays45,46 after being incubated for 24 h. As shown in Figure 7a, all samples displayed increasing cell viability with the increasing concentration and that they had a dose-dependent effect. When the concentration increased to 200 μg/mL, the cellular activity had an obvious increase. It has been inferred that a high concentration of iron ion could contribute to the cell viability.47 To verify the contribution of iron ions to the cell proliferation, the cell viability of free iron ions separated out from the same concentrations of the MOF53@Van was measured, as shown in Figure 7b, and they exhibited a high cellular viability. As to the group of free Van that exhibited no significant inhibition to cell proliferation, it showed a very low cytotoxicity when they were below the concentration of 200 μg/mL and did not apparently affect the cell proliferation.48 To further confirm whether the free iron ions could promote cell osteogenesis and cell differentiation,49 alkaline phosphatase (ALP) activity of different concentrations of iron ions at 1, 3, and 7 days was measured, which is shown in Figure S10. After incubation for 1 day, osteoblasts showed a good cellular activity, and the cellular activity increased with the increase in the concentration of iron ions. As the culture time increased to 3 days, ALP activity reduced to a lower level, and there was no obvious osteogenic differentiation in the early stage. After 7 days of culturing, ALP activity increased significantly, indicating good osteogenic differentiation ability. To investigate the influence of MOF-53(Fe) acting on the cell attachment, fluorescence morphology was observed through F-actin/nucleus cell staining. Figure 8 shows the fluorescence morphologies of MC3T3-E1 after culturing for 24 h. The cells on all samples displayed a polygonal and spreading morphology, the antennas of osteoblasts stretched well, and the number of nucleus changed little, suggesting the good biocompatibility of all samples. Figure 9 shows the visualized fluorescent dead/live staining images of MC3T3-E1 after acting with the sample that had cultured for 24 h. Live cells were stained green by AO, and dead cells were stained red by PI. The results demonstrated that the cells had a higher survival rate on MOF-based materials, especially for MOF-53 (160) and MOF53@Van (200).

Figure 9. Fluorescent images of cells (scale bars = 50 μm) stained with AO (live cells, green fluorescence) and PI (dead cells, red fluorescence) after cells cultured with various samples for 24 h.

MOF-53@Van could reach up to 99.3%, which demonstrated a great antibacterial efficacy. At the same time, the antibacterial activity of the groups MOF-53, MOF-53@Van, MOF-53 + Van, and free Van against S. aureus using OD method is shown in Figure S7. The group MOF-53 had a little bit of antibacterial activity, and the results were consistent with the results shown in Figure S8, whereas the MOF-53@Van had an increasing antibacterial ratio with the increasing concentration of drug-loaded MOF-53(Fe) NPs. After MOF-53@Van was treated with S. aureus at a concentration of 50 μg/mL, it could reach up to an antibacterial efficiency of 91.7%, which was attributed to the increasing concentration of Van from the drug-loaded MOF-53(Fe) NPs. However, when the concentration of the particles continued to increase to 200 μg/mL, the antibacterial ratio of MOF-53@Van dropped slightly because of the scattering effect caused by the remaining MOF-53(Fe) NPs that were mixed with bacterial suspension, resulting in the shielding effect on the OD measurement of bacteria. Consequently, the measured value reduced through this method. This was the limitation of OD method. It could be confirmed that the drug-loaded particles with higher concentrations (100 and 200 μg/mL) also had high antibacterial properties, in the area marked A in Figure S9. As shown in Figure S7, the antibacterial ratios of MOF53(Fe) + Van against S. aureus could reach up to more than 96% at low concentrations [MOF-53(Fe) + Van (20 + 5) and MOF-53(Fe) + Van (10 + 2.5)], whereas the group MOF-53@ Van at the same concentration had a lower antibacterial effect. It could be explained that MOF-53(Fe)@Van had a controlled drug release; the amount of the Van was gradually increased in the bacterial solution during the antibacterial test. The nonencapsulated Van + MOF had the same antibacterial activity with the free Van, which could be ascribed to the free state of drugs in the solution at the initial stage. Although the shielding effect caused by the remaining NP residues also existed in the group MOF-53 + Van, the antibacterial activity of this group increased with the increasing concentration, which can be found in the areas marked B in Figure S9. The free Van had a strong antibacterial effect, even at low concentration, which can be seen in the areas marked C in Figure S9. Regarding the morphology and membrane integrity of S. aureus, which were observed using SEM and are shown in Figure 6, the typical morphology was a spherical shape, and the surface was smooth on the control group.43 Bacteria on the group MOF-53@Van(25) appeared with little membrane corrugation. When the concentration of MOF-53@Van increased to 200 μg/mL, the bacteria corrugated with distorted

4. CONCLUSIONS In this study, we have successfully fabricated a drug delivery system of MOF-53(Fe)@Van, in which the MOF-53(Fe) NPs acted as the carrier platform and encapsulated antibacterial drug of Van. This system had an efficient drug-loading capacity of 20 wt %. The degradation degree of MOF-53(Fe) was 0.75% in PBS at pH 7.4 and 0.17% at pH 5.5, disclosing the good stability of this system. In addition, the as-prepared MOF53(Fe) showed a slower release of Van at pH 5.5. During or after surgical implantation, considering the fact that there was the inflammatory response caused by the implant-associated bacterial infection, inducing a local acidic environment around the implants, the as-prepared Van-loading MOF-53(Fe) in this study could exhibit a lasting antibacterial effect and a high antibacterial efficiency of 99.3% without cytotoxicity. AdditionH


Research Article


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ally, the iron ions from the degradation of MOF-53(Fe) had been proven to be biocompatible. Meanwhile, the proliferation and osteogenetic differentiation of MC3T3 cells could also be promoted by Fe3+ in this system.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b04810. Experimental procedures of incorporation of Ibu, release of Ibu, the particle size distribution of MOF-53@Van NPs, antibacterial property test, and osteogenic differentiation; FT-IR spectra of MOF-53 powders, Ibu-loaded MOF-53, and free Ibu; XRD patterns of MOF-53, MOF53@Van, and MOF-53@Ibu; Ibu release behavior; in vitro concentration detection of Fe3+ from the MOF53@Van NPs (1 mg/mL) degraded completely; DLS of MOF-53@Van NPs; the concentration detection of Fe3+ produced from the MOF-53@Van NPs (1 mg/mL) scattered in DMEM, PBS, and SBF; in vitro antibacterial activity of MOF-53, MOF-53@Van, MOF-53 + Van, and free Van; in vitro antibacterial activity of iron ion solution; photograph of 96-well plate; and ALP activity of MC3T3-E1 cells cultured on iron ions (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or [email protected]. ORCID

Shuilin Wu: 0000-0002-1270-1870 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is jointly supported by the National Natural Science Foundation of China (nos. 51422102 and 81271715), the National Key Research and Development Program of China [no. 2016YFC1100600 (sub-project 2016YFC1100604)], and Shenzhen Peacock Program (1108110035863317).

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REFERENCES

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