Antibacterial Countermeasures via MOF â Supported Sustained

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Biological and Medical Applications of Materials and Interfaces

Antibacterial Countermeasures via MOF Supported Sustained Therapeutic Release Dorina F Sava Gallis, Kimberly S. Butler, Jacob Ongudi Agola, Charles Pearce, and Amber McBride ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21698 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 27, 2019

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ACS Applied Materials & Interfaces

Antibacterial Countermeasures via MOF − Supported Sustained Therapeutic Release Dorina F. Sava Gallis,†* Kimberly S. Butler₸, Jacob O. Agola,‡ Charles J. Pearce,† Amber A. McBride₸ † Nanoscale Sciences Department, Sandia National Laboratories, Albuquerque, NM 87185, USA. ‡ Center for Micro-Engineered Materials, Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM 87131, USA. ₸ Nanobiology Department, Sandia National Laboratories, Albuquerque, NM 87185, USA. ABSTRACT: Long-term antimicrobial therapies are necessary to treat infections caused by virulent intracellular pathogens, including biothreat agents. Current treatment plans include injectable therapeutics given multiple times daily over a period for up to 8 weeks. Here we present a metal-organic framework (MOF), zeolitic imidazolate framework-8 (ZIF-8), as a robust platform to support the sustained release of ceftazidime, an important antimicrobial agent for many critical bacterial infections. Detailed materials characterization confirms the successful encapsulation of ceftazidime within the ZIF-8 matrix, indicating sustained drug release for up to a week. The antibacterial properties of ceftazidime@ZIF-8 particles were confirmed against E. coli, chosen here as a representative Gram-negative bacteria infection model in a proof-of-concept study. Further, we showed that this material system is compatible with macrophage and lung epithelial cell lines, relevant targets for antibacterial therapy for pulmonary and intracellular infections. A promising methodology to enhance the treatment of intracellular infections is to deliver the antibiotic cargo intracellularly. Importantly, this is the first study to unequivocally demonstrate direct MOF particle internalization using confocal microscopy via 3D reconstructions of z-stacks, taking advantage of the intrinsic emission properties of ZIF-8. This is an important development as it circumvents the need to use any staining dyes and addresses current methodology limitations concerning false impression of cargo uptake in the event of the carrier particle breakdown within biological media. Keywords: metal-organic frameworks, drug delivery, therapeutics, ZIF-8, photoluminescence, confocal microscopy.

1.

INTRODUCTION

Pathogens that can survive and replicate intracellularly, including many biothreat agents, are particularly difficult to treat as they can hide within the intracellular compartments, thereby evading the immune system. Burkholderia pseudomallei is one such virulent bacteria that causes an infection called melioidosis. This disease is prevalent in Southeast Asia and Northern Australia and its resistance to conventional antibiotics makes it difficult to treat effectively.1, 2 Ceftazidime is a third-generation, broad spectrum cephalosporin and is the main therapeutic for melioidosis. It is considered a critically important antimicrobial agent by the World Health Organization, who has identified ceftazidime as a member of an antimicrobial class which is the sole, or one of the limited available therapies for serious human bacterial infections.3, 4 In addition, this drug is a therapeutic choice for bacterial meningitis and Salmonella infection in children,4, 5 has broad spectrum activity for Gram-negative bacterial organisms,6 and is a vital drug in combination with avibactam for multi-drug resistant infections due to Enterobacteriaceae,

Escherichia coli, and pneumonia caused by a variety of organisms including Pseudomonas aeruginosa.4-7 Treatment of melioidosis is extremely difficult and often involves long-term antimicrobial therapies. The acute therapeutic phase lasts 10 days to 8 weeks and involves intravenous antimicrobials, while the eradication phase focuses on oral antimicrobials for 90 -180 days.8, 9 The acute phase therapy most commonly involves intravenous ceftazidime given every 8 hours.8, 9 Given the need to inject ceftazidime on regular intervals within a single 24 hrs period, the development of a delivery platform that would allow sustained or slower, time-dependent release could greatly enhance treatment while maintaining the efficacy of ceftazidime. In recent years, metal-organic frameworks (MOFs) have emerged as unique material platforms for a variety of biorelated applications, as facilitated by: (i) biocompatibility and precise particle size control; (ii) the ability to load a variety of cargos; (iii) facile surface chemistry that allows conjugation of various functionalities. MOFs have been shown to act as controlled-release antimicrobial agents through several mechanisms including: (i)

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the release of antimicrobial metal ions, such as Ag+ and Co2+, from the framework;10-14 (ii) release of antimicrobial linkers from the framework;15 (iii) entrapping an antimicrobial agent within the framework for later release,16-19 or (iv) combining these intricate modalities.20 While therapy based on metal ion release shows great promise for topical applications, ions such as Ag+ and Co2+ can be toxic to human cells limiting their applicability for injection or inhalation.21-23 For delivery of intravenous or inhaled drugs, such as ceftazidime, MOFs created from more biocompatible metals, such as zinc,24 can be utilized to avoid metal ion based toxicity. Although MOFs are highly porous materials, the encapsulation of large cargos via post-synthetic methodologies is restrictive. Only a limited number of candidates possess appropriate prerequisites (large pore apertures and sizes).25-29 Recently, great progress has been made in extending the library of suitable MOFs for such large cargos, which are now reliably introduced via encapsulation.30-33 To be noted, in this case the cargo release mechanism involves the degradation of the MOF carrier. In particular, zeolitic imidazole frameworks (ZIFs) have been shown to be ideal hosts to allow this synthetic route, where the MOFs grows around the large biomolecule, while preserving its functionality. Additionally, ZIFs have been successfully utilized to encapsulate and release other antimicrobial agents including ciprofloxacin,18 gentamicin,19 and vancomycin,17 demonstrating their potential as carrier agents for antimicrobial therapy. In this study, we utilized the encapsulation strategy to load ceftazidime in ZIF-8 (Figure 1), as a potential therapeutic agent for systemic infections or for localized therapy, such as inhalation therapy for Burkholderia pseudomallei.34 A combination of experimental techniques including elemental analyses, high resolution microscopy for elemental mapping of individual nanocrystals, nitrogen gas sorption and thermogravimetric analyses was used to probe and confirm the ceftazidime drug loading within the ZIF-8 matrix. To confirm retention of therapeutic activity, the antibacterial effects of the ceftazidime loaded ZIF-8 were assessed against E. coli, a Gramnegative bacterium. Toxicity to mammalian cells was assessed in a human lung epithelial cell line (A549), as lung is a potential site for targeted ceftazidime delivery. Cell viability was also studied on mouse macrophage cell lines (RAW 264.7), which are target cells for antibacterial therapy for intracellular infection and are involved in the clearance of injected nanomaterials. Finally, the ability of the particles to be internalized into cells was directly evaluated on as-made materials using confocal microscopy via 3D reconstructions of z-stacks.

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(a) (a)

(b)

Figure 1. Ball-and-stick representation of (a) sodalite cage in ZIF-8 (11.6 Å); (b) ceftazidime (18 Å). Atom color scheme: S = yellow; C=grey; O= red; H= white; Zn= green.

2. EXPERIMENTAL SECTION All reactant materials were purchased from commercially available sources and used without further purification. 2.1. Materials synthesis. Synthesis of pristine ZIF-8: This synthesis has been adapted from a previous literature report.35 The reaction mixture containing Zn(CH3CO2) · 2H2O (0.200g, 0.911 mmol) and H2O (0.8 mL of pH = 8) was placed in a 20-mL scintillation vial. A total of additional 4 mL of H2O were added to this mixture. To this solution, a mixture of 2-methylimidazole (2.00 g, 0.0245 mol) in H2O (8mL) was added dropwise while stirring at room temperature over 24 hours. The product was centrifuged for 5 minutes at 6000 rpm and washed 3X with 10 mL of EtOH/H2O solution (1:1). Synthesis of ceftazidime@ZIF-8 (100 mg), compound 1: The reaction mixture containing ceftazidime hydrate (0.100 g, 0.183 mmol) and water (H2O, 4 mL) was added to a mixture containing Zn(CH3CO2) · 2H2O (0.200g, 0.911 mmol) and H2O (0.8 mL of pH = 8). To this solution, a mixture of 2-methylimidazole (2.00 g, 24.360 mmol) in H2O (8mL) was added dropwise while stirring at room temperature over 24 hours. The product was centrifuged for 5 minutes at 6000 rpm and washed 3X with 10 mL of EtOH/H2O solution (1:1). Synthesis of ceftazidime@ZIF-8 (400 mg), compound 2: The above synthesis was extended for the 400 mg sample, increasing the amount of ceftazidime accordingly (0.400 g, 0.732 mmol). To be noted, this sample was stirred over 3 days. 2.2. Powder X-ray Diffraction (PXRD). Measurements were collected over the 2θ range 2 − 30° and a step size of 0.02° in 2θ on a Bruker AXS D2 Phaser diffractometer, CuKα radiation (λ = 1.54178 Å). A standard least-squares (LSQ) peak refinement that was fit within the Jade 9.7 software package (Materials Data, Inc., Livermore, CA) was employed. The LSQ fit procedure allows for refining a zero-shift and this was performed during the LSQ


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refinement process for each material to accommodate any shift artifacts, and thereby correct the data. 2.5. Thermogravimetric analyses (TGA). Measurements were conducted on a SDTQ600 TA instrument. The samples were heated to 800°C at a 5 °C /min heating rate, under continuous nitrogen flow. 2.7. Scanning Electron Microscopy (SEM) - Energy Dispersive Spectroscopy (EDS). SEM analyses were captured on a FEI NovaNano SEM 230, at various accelerating voltages between 1 and 20 kV. EDS analyses were collected on an EDAX Genesis Apex 2 with an Apollo SDD detector. 2.8. Scanning Transmission Electron Microscopy (STEM) Energy Dispersive Spectroscopy (EDS). An image-aberrationcorrected FEI TitanTM G2 80-200 STEM with a Cs probe corrector and ChemiSTEM technology (X-FEGTM and SuperXTM EDS with four windowless silicon drift detectors) operated at 200 kV was used in this study. The EDS spectral imaging was acquired as a series of frames where the same region was scanned multiple times. An electron probe of size less than 0.13 nm, convergence angle of 18.1 mrad, and current of ~75 pA was used for data acquisition. High-angle annular dark-field (HAADF) images were recorded under similar optical conditions using an annular detector with a collection range of 60-160 mrad. 2.9. Sample activation and gas adsorption measurements. Prior to measuring the gas adsorption isotherms, the samples were immersed in 15 mL of methanol for 3 days, with the solvent replenished every 24 hrs. Following this treatment, all samples were activated under vacuum on a Micromeritics ASAP 2020 surface area and porosity analyzer, at 100° C for 12 hrs. Nitrogen gas adsorption isotherms were measured at 77 K using nitrogen of ultra-high purity (99.999%, obtained from Matheson Tri-Gas). 2.10. Photoluminescence (PL) measurements. The PL emission and excitation spectra of pristine and ceftazidime-loaded powder samples in 4-inch-long Pyrex NMR tubes were collected using a Horiba Jobin-Yvon Fluorolog-3 double-grating/doublegrating Fluorescence Spectrophotometer in front-face mode. Excitation spectra were collected by monitoring at the peak of the emission, and scanning over UV-visible wavelengths (320-550 nm). 2.11. Ceftazidime release studies. Release studies were performed on pristine ZIF-8 (control) and the highest loaded sample, ceftazidime@ZIF-8, 400 mg and were carried out using a New Brunswick Innova 44 incubator shaker. UV-Vis analyses were performed on a Bio-Tek SynergyTM H4 Hybrid microplate reader operated by the Gen5TM software on Thermo Scientific Nunc 96Well UV-Microplates. In a typical experiment, 20 mg of solid sample were dispersed into 40 mL of 1x Dulbecco’s Phosphate Buffer Saline (1x PBS, Thermo-Fischer Scientific, no calcium, no magnesium) and 0.01x PBS buffer at pH 7.4 and pH 5.0, respectively. The mixture was kept at 37°C under shaking at 150 rpm. 1 mL of release medium was sampled at regular time point intervals over 7 days. The aliquot was centrifuged at 4000 rpm for 2 minutes and 200 µL of the supernatant was analyzed via UV-Vis spectrophotometry. The remaining sample was returned to the original release system. The concentration of released ceftazidime, Cr, was determined by monitoring the absorbance intensity of the emission peak at 240 nm of the ceftazidime@ZIF-8, 400 mg sample; to eliminate any interferences associated with the MOF itself, the emission of pristine ZIF-8 was subtracted from that of the drug loaded sample. The final concentration was determined using a calibration curve of known concentration of free ceftazidime in PBS. The final ceftazidime release percentage was calculated according to the formula, percent released (%) = Cr/Ct, where Cr is

the concentration of released Ceftazidime and Ct is the total concentration of loaded ceftazidime. The release results after 7 days were normalized to 100%. 2.12. Assay of the antibacterial effect of ceftazidime@ZIF-8 particles. The antibacterial effects of ZIF-8, ceftazidime@ZIF-8 and free ceftazidime control were tested against model E. coli K12 T7 based on the optical density (OD) measurements. The widely recommended Mueller-Hinton (MH) bacterial growth medium,36 commonly recommended for antibiotic susceptibility assays, was used for all the tests. While stock concentrations (10 mg/mL) of both ZIF-8 and ceftazidime@ZIF-8 particles were prepared in dimethyl sulfoxide (DMSO), the free ceftazidime stock solution was prepared in molecular biology grade ultrapure water. Where applicable, semi-stock serial concentrations of the free ceftazidime were prepared before use. To initiate the assay, serial concentrations of ZIF-8 and ceftazidime@ZIF-8 particles were first prepared in MH medium (pH 5.0) for at least 24 hours while shaking at 200 rpm to enhance release of the loaded ceftazidime. E. coli K12 T7 was cultured overnight at 37°C in 40 mL LuriaBertani (LB) medium in 250 mL Pyrex flasks shaking at 250 rpm. A volume of 1 mL of this overnight E. coli K12 T7 culture was then withdrawn from, centrifuged (5000 g for 3 min), washed twice with 1X PBS and then suspended in the MH medium (pH 5.0). The optical density (OD) of this suspension was then obtained at 600 nm using Nanodrop (Thermo Fisher Scientific, Waltham, MA), before diluting this new stock to OD = 0.015 in MH medium (pH 5.0). In order to set up the 96-well microtiter plate assay, ZIF-8 and ceftazidime@ZIF-8 pre-incubated in MH medium (pH 5.0) was first transferred to the wells followed by 5 L of the diluted E. coli K12 T7 stock (OD= 0.015) to yield appropriate final concentrations of ZIF-8 and ceftazidime@ZIF-8 particles. The free ceftazidime was also tested in the MH medium (pH 5.0) in the same 96-well plate at final concentrations close to the MIC of ceftazidime reported in the literature for E. coli bacteria.37 The OD measurements were obtained on the microtiter plate reader (Biotek, Winooski, VT, USA) set at 37°C and shaking at 250 rpm. Experimental controls included blank MH medium and E. coli K12 T7 not exposed to ZIF-8 or ceftazidime. All measurements were acquired in triplicates. 2.13. Cell viability assessment. A549 (ATCC) were maintained in F-12K + 10% fetal bovine serum, FBS (by volume) and RAW 264.7 cells (ATCC) were maintained in DMEM + 10% FBS (by volume). For cell viability assessment, 1,000 cells were plated per well in 100 µL media in 96 well plates and allowed to adhere overnight. Fresh media containing ZIF-8, ZIF-8 loaded with ceftazidime or free ceftazidime at varied concentration (0 - 500 µg/mL) were then prepared. Cell exposure was performed by removing media from the adherent cells, and then replacing it with freshly prepared media containing MOF-based or free drug samples. Cells were incubated with to MOF-based or free drug for 24 or 48 hours at standard cell culture conditions (37ºC and 5% CO2). After exposure, cell viability was assessed using CellTiterGlo 2.0 Assay kit utilizing the standard protocol (Promega) with luminescence measured by a BioTek Synergy Neo2 microplate reader. The cell viability was calculated as a percentage of mock treated sample. Cell viability measurements were done in quadruplicate and graphed as the average and standard deviation. 2.14. Cellular uptake experiments. RAW 264.7 cells (ATCC) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) + 10% FBS (by volume). #1.5 coverslips were sterilized with ethanol and then placed on the bottom of the 6 well culture plates. RAW 264.7 cells were plated at 2 x 105 cells per well onto the coverslips in 6 well culture plates and allowed to adhere overnight. Plated cells were then exposed to pristine ZIF-8 or ceftazidime loaded ZIF-8 at 50 µg/mL in 2 mL culture media per well for 24 hrs. After 24 hrs, media was removed and the cells were rinsed 3


ACS Applied Materials & Interfaces times with 1mL of 1x PBS. Cells were then exposed to Cell Mask Orange Plasma Membrane Stain diluted at 1.5 µL of stock per 1 mL of media and used at 1 mL of media per well for 10 min. Media was removed and the cells were rinsed 3 times with 1 mL of 1x PBS. Cells were then fixed 4% paraformaldehyde in 1x PBS at 1 mL per well for 10 minutes. Cells were then rinsed 2 times with 1 mL of 1x PBS. The coverslips were then removed from the 6 well plate and mounted on slides using Prolong Gold mounting media. Mounting media was allowed to set overnight and then the edges of the coverslips were sealed to the microscope slide with clear nail polish. 2.15. Confocal microscopy. ZIF-8 samples were deposited on microscope cover glass by spin coating to create a thin film for microscopic analysis. Approximately 50 µg of ZIF-8 powder was dispersed in 50 µL of a 1% poly(vinyl alcohol) solution (PVOH). A #1.5 thickness circular microscope cover glass was mounted on the spin coater (Chemat Technology Spin Coater, KW-4A) and vacuum was applied. The MOF-PVOH solution was pipetted evenly on the cover glass and then allowed to spin at a low speed of 1000 rpm under vacuum for 60 seconds. The spin coated cover glass was mounted on a microscope slide. Edges of the cover glass were sealed to the microscope slide with clear nail polish. The sealed slides were imaged on a Leica SP8 confocal microscope with a 63x oil objective. The microscope was focused on the ZIF8 particles utilizing bright field mode. After focusing, a lambda scan was utilized to determine the fluorescence characteristics of the ZIF-8 particles in response to excitation with the 405 nm laser using a HyD detector. The detection bandwidth was set to 20 nm and the step size was set to 10 nm. In both cases the laser power was set to 5%. Data was collected from 410 - 760 nm with the 405 nm laser and 500-760 nm for the 488 nm laser. Lambda scan data was exported from the Leica software to Excel for graphical display. Using the lambda scan data to set the acquisition settings for the Leica scope, bright field and fluorescent images of the spun coated ZIF-8 particles was obtained. Images were obtained with the 405 nm laser at 1% power with collection window of 440 - 490 nm and the 488 nm laser at 1% power with the collection window from 550 - 600 nm. For visualization, the image from excitation with the 405 nm laser was false colored blue and the image from excitation with the 488 nm laser was false colored green. The sealed slides were imaged on a Leica SP8 confocal microscope with a 63x oil objective to create Z-stacks for a 3D image reconstruction. Sequential excitation was performed using the 405 nm laser at 1 % power for the ZIF-8 and the 552 nm laser at 0.1 % power for Cell Mask Orange. Sequential detection parameters were Hybrid Detector collecting from 420 - 475 nm for ZIF-8 and PMT detector collecting from 570 - 615 nm for the Cell Mask Orange. All images were deconvolved with Huygens Essential version 17.04 (Scientific Volume Imaging, The Netherlands, http://svi.nl), using the CMLE algorithm, with SNR 20 and 40 iterations for both channels. Images are shown as 3D reconstructions of z-stacks.

3. RESULTS AND DISCUSSION In order to assess any changes in the ZIF-8 structure as function of ceftazidime loading, powder X-ray diffraction (XRD) studies were conducted on samples with two different guest loadings, Figure 2.

Ceftazidime@ZIF-8, 400 mg

Intensity, (a.u.)

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Ceftazidime@ZIF-8, 100 mg

Pristine ZIF-8

5

10

15

20

25

30

0

2-theta/

Figure 2. Powder X-ray diffraction patterns of the pristine ZIF-8 (blue) compared against ceftazidime loaded samples, 100 mg (black), and 400 mg (red).

It was confirmed that the crystalline structure of ZIF-8 remains largely unaffected by the presence of the ceftazidime guest molecules. Lattice parameter refinements, Table 1, identified a general trend for unit cell contraction with higher drug loading. In particular, when considering the pristine vs. the 400 mg sample, the cell contraction is ~ 0.04 Å, which is significant to indicate a change in the lattice via guest-framework interactions. Sample Pristine Ceftazidime@ZIF-8, 100 mg Ceftazidime@ZIF-8, 400 mg

a-axis (Å) 17.013 16.997

error (Å) 0.002 0.002

Cell volume (Å3) 4924.5 4910.8

16.974

0.006

4890.8

Table 1. Refined lattice parameters for pristine ZIF-8, Ceftazidime@ZIF-8, 100 mg and Ceftazidime@ZIF-8, 400 mg samples. High resolution STEM-EDS elemental mapping of individual nanocrystals was used to probe the presence of ceftazidime in ZIF-8, Figure 3. There is a well-defined presence of the S signal (atom found only in ceftazidime) at the nanoparticle level, highlighting a homogeneous distribution within the crystalline lattice. Moreover, a similar observation is noted when characterizing the bulk sample, at much lower magnification, via SEM-EDS elemental mapping, Figure S1. Elemental analyses conducted by Galbraith laboratories confirmed that the amount of ceftazidime loaded in the highest sample was ~ 10.8%. Calculated: C= 41.99%, H= 4.28%, N= 23.35; O= 3.66%; S= 1.04%; Zn= 25.65%. Found: C= 43.81%, H= 5.22%, N= 21.85; O= 3.00 %; S= 0.91%; Zn= 25.20%. To confirm guest encapsulation within the pores of ZIF-8, nitrogen gas sorption isotherms were measured at 77K, on pristine ZIF-8 and ceftazidime@ZIF-8 samples, Figure 3. The BET surface area for pristine ZIF-8 was 1993 m2/g, on par with calculated values and previous results.38 In the case of ceftazidime@ZIF-8, 100 mg sample, the BET surface area was


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1509 m2/g, while for ceftazidime@ZIF-8, 400 mg sample it was calculated to be 1127 m2/g, corresponding to a decrease of ~40% of the total available surface area in pristine ZIF-8.

Figure 3. TEM-EDS elemental mapping representative ceftazidime@ZIF-8 nanoparticles.

of

individual

Interestingly, with the maximum guest encapsulation, a subtle change in the pore environment is noticeable, as evidenced by the slight hysteresis upon desorption, Figure 4 (red trace). This behavior is also captured in the pore size distribution, which indicates a distinct change for this sample, with a slight shift to larger pore sizes, Figure S2.

600

Amount of N2 sorbed, cc/g

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500 400 300 200 100

Pristine ZIF-8 Ceftazidime@ZIF-8, 100 mg Ceftazidime@ZIF-8, 400 mg

0 0.0

0.2

0.4

0.6

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1.0

P/P0

are present in these samples. As expected, pristine ZIF-8 is thermally stable until close to ~ 500°C, losing only ~2.25% of its entire mass. In contrast, the two ceftazidime loaded samples proceed with a distinct gradual weight loss starting at ~ 200°C and reached 4.50% mass loss for the 100 mg sample and 13.15 % mass loss for 400 mg sample at 500°C. This finding confirms a drug loading of ~ 10.9% in the highest loaded sample, in very good agreement with that found via elemental analysis, 10.8%. Previous literature findings suggested that ZIF-8 is robust in biologically relevant media, preserving its crystalline structure at pH 7.4, while degrading at pH 5.0, making it suitable as a pHtriggered cargo release platform.35 To the best of our knowledge, a systematic evaluation of its stability in unaltered PBS or PBS of well-known concentrations is still lacking. In order to gather a comprehensive understanding of ZIF-8’s stability in PBS, we tested its robustness in 1x, 0.1x and 0.01x PBS at pH 7.4 and pH 5.0 after 1 day and 7 days of incubation at 37°C, respectively, Figures S4 and S5. Interestingly, under the studied conditions (5 mg of ZIF-8 in 10 mL of PBS), ZIF-8 is stable at pH 7.4 at 0.01x and 0.1x both after 1 and 7 days. However, the crystalline structure is clearly affected in 1x PBS, even after 1 day. A more pronounced degradation is noted after 7 days. These results differ from those of a related stability study, where ZIF-8 was found to be stable in PBS with 10% (v/v) fetal bovine serum (FBS) at pH 7.4 37°C over 7 days.34 ZIF-8 is stable pH 5.0 in 0.01x and 0.1x PBS after 1 day, and in 0.01x after 7 days. The structure appears to be affected at 0.1x after 7 days. Finally, a new crystalline phase is formed (Zn phosphate) when the pristine ZIF-8 was tested at pH 5.0 in 1x PBS, after both 1 and 7 days. Our finding highlights the importance of the concentration and composition of the buffer with respect to structural stability, and cargo release, over time. Selection of buffer for structural analysis and cargo release is also important for understanding behavior within mammalian systems, including humans. PBS at 1x is designed to match osmolarity and ionic strength of the fluids within mammalian systems and therefore closely represents the conditions encountered after injection of ceftazidime in the body. Ceftazidime@ZIF-8 release studies were designed according to these findings, for the highest and lowest PBS concentrations, 1x and 0.01x, respectively. As detailed in the experimental section, the concentration of released ceftazidime was determined by monitoring the absorbance intensity of the emission peak at 240 nm of the ceftazidime@ZIF-8, 400 mg sample; to eliminate any interferences associated with the MOF itself, the emission of pristine ZIF-8 was subtracted from that of the drug loaded sample. The final concentration was then determined using a calibration curve of known concentration of free ceftazidime in PBS, Figure S6. The drug release in 1x PBS, Figure 5, occurs primarily during the first day, with roughly 70% of the total amount released at pH 5.0 and 50% for pH 7.4, respectively.

Figure 4. Nitrogen gas sorption experiments on pristine and ceftazidime@ZIF-8 nanoparticles at two different concentrations. The adsorption range is depicted using closed symbols, while the desorption is shown with open symbols.

Additional information concerning changes in the samples upon ceftazidime encapsulation was provided by thermogravimetric analyses. These measurements further confirmed the presence of guest molecules within the ZIF-8 pores and not on the surface, Figure S3. That is, no weight loss is observed before 200°C, an indication that no surface species


ACS Applied Materials & Interfaces 100

1X PBS pH= 7.4 1X PBS pH= 5

80

% Release

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

60 40 20 0 0

1

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3

4

5

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7

Time (Days)

Figure 5. Ceftazidime release from ceftazidime@ZIF-8, 400 mg sample in 1x PBS as function of time and pH.

This result is consistent with the XRD stability studies that showed ZIF-8 starts degrading at both pH 7.4 and pH 5.0 after just 1 day in 1x PBS. Interestingly, the release kinetics are much slower after the first day and the release plateaus after ~ 5 days. This suggests a rate dependent sample degradation. In contrast, only a very small amount of ceftazidime is released in 0.01x PBS, at both pH 5.0 and pH 7.4, Figure S7. Once again, this result is consistent with the XRD stability data, Figures S4b and S5b, that indicate the crystalline structure of ZIF-8 is mainly preserved under those conditions, even after 7 days of incubation. After confirmation that ceftazidime was released from the ZIF-8, the antibacterial properties of ceftazidime@ZIF-8 and pristine ZIF-8 particles were tested. Ceftazidime is often used for its broad-spectrum activity against Gram-negative bacterial organisms, which are often more difficult to kill than Grampositive organisms, therefore Gram-negative E. coli was selected as a representative organism. Ceftazidime release studies (Figure 5) demonstrated accentuated antibiotic release at pH 5.0. While release at pH 7.4 is important for release over time in blood and other biofluids, release at pH 5 presents the opportunity for release intracellularly from endosomal compartments after cell uptake. Intracellular infection is a major therapeutic challenge as intracellular persistence provides a reservoir of infection that is associated with recurrence.39 Intracellular responsive delivery, such as response to the acidic pH in intracellular compartments like the endosome has previously demonstrated enhanced antibacterial efficacy against intracellular pathogens.40 As pH can affect the efficacy of antibiotics, prior to testing the antibacterial properties of ZIF-8 particles, the sensitivity of the E. coli strain to ceftazidime at pH 5.0 was confirmed (Figure S8). The antibacterial effect of free ceftazidime was observed at the concentration range consistent with the literature,37 attesting to the high susceptibility of the E. coli strain to ceftazidime. Following confirmation of bacterial susceptibility, the antibacterial activity of the ceftazidime@ZIF-8 and ZIF-8 alone were assessed. The samples were incubated for 24 hrs in bacterial growth medium to allow breakdown of the ZIF-8. Following incubation, the media was inoculated with E. coli and

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the bacterial growth curve was followed hourly (Figure S9). The pristine ZIF-8 showed significant inhibition of E. coli growth (Figure S9a) consistent with other studies demonstrating E. coli inhibition by pristine ZIF-8 at 24 hrs. 18,19 No difference in the antibacterial effect was noted between the ceftazidime@ZIF-8 and pristine ZIF-8 particles after 24 hrs incubation (Figure S9). Examination of two prior studies of antibiotic loaded ZIF-8 targeting E. coli has demonstrated variability in antibiotic response at 24 hrs with ZIF-8 constructs, with one study demonstrating an enhanced effect with ciprofloxacin loaded ZIF-8 and a second study showing no enhancement with gentamicin loaded ZIF-8 when compared to pristine ZIF-8.18,19 One possible explanation for this variability is the potential for media components, such as proteins, to affect the breakdown of the ZIF-8. This is supported by previous literature showing the extended stability of ZIF-8 in PBS stabilized with FBS, which has a high protein content.35 To determine if additional time is necessary to allow ZIF-8 breakdown and release of active ceftazidime, pristine ZIF-8 and ceftazidime@ZIF-8 were incubated for 72 hrs in media prior to addition of E. coli (Figure 6). After 72 hrs incubation, the ceftazidime@ZIF-8 showed very strong antibacterial properties resulting in complete inhibition of E. coli growth at 100 µg/mL and almost complete growth inhibition at 50 µg/mL. Interestingly, the antibacterial effect of the pristine ZIF-8 is no longer present with the longer incubation time, suggesting the breakdown process or reactive intermediates in the ZIF-8 breakdown may be responsible for the antimicrobial effect of

(a)

(b)

Figure 6. Assessment of the growth of E. coli exposed to (a) ceftazidime@ZIF-8 and (b) pristine ZIF-8 after pre-incubation in mildly acidic Mueller-Hinton (MH) medium at pH 5.0 for 72 hrs at 37°C.


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pristine ZIF-8. These results indicate that the antibacterial activity is dependent on the degradation kinetics of the ceftazidime@ZIF-8 particles. While the need for the particles to breakdown does present some limitations for use, it also provides some opportunities for these particles to be used in concert with free therapeutic, which has limited half-life within the body, as long-term release vehicle to provide delivery over a longer time scale without the need for reinjection. Future development of the MOF can also focus on alterations of the linkers to alter the time needed to breakdown the structure to provide different availability of ceftazidime.

low cytotoxicity to both cell types, with noticeable toxicity only occurring at high doses, 200 and 500 µg/mL, after 48 hrs of exposure. With exposure to ZIF-8 and ceftazidime@ZIF-8 in both the macrophage and lung epithelial cell lines, there was a dose dependent toxicity, which demonstrated increased toxicity at 48 hrs compared to 24 hrs only at the highest doses. The toxicity between the macrophage and the lung epithelial cells was very similar at lower doses and both cell lines showed greater than 75 % viability at 20 µg/mL at both 24 and 48 hrs. The observed toxicity is very similar to previously observed toxicity in other

Figure 7. Cellular viability of A549 human lung epithelial cells (a & b) and RAW 264.7 mouse macrophage cells (c & d) after 24 hrs. (a & c) and 48 hrs (b & d) of incubation with free ceftazidime, unloaded ZIF-8, and ceftazidime loaded ZIF-8.

Given the potential use of ZIF-8 as a for use as a therapeutic delivery platform, the ZIF-8 particles were assessed for toxicity to mammalian cells. A human lung epithelial cell line (A549) and a mouse macrophage cell line (RAW 264.7) were chosen as representative cell lines for cytotoxicity assessment. Lung is a potential site for targeted ceftazidime delivery and macrophages are involved in the clearance of injected nanomaterials, making them useful target cells for antibacterial therapy for intracellular infection, including Burkholderia pseudomallei. Cell viability was assessed after both 24 and 48 hrs of incubation with free ceftazidime, pristine ZIF-8 and ceftazidime@ZIF-8, 400 mg samples at concentrations ranging for 0.5-500 µg/mL, Figure 7. The free ceftazidime shows very

cell lines for pristine ZIF-8.19, 41 At the highest doses, 200 µg/mL and 500 µg/mL, greater toxicity was noted with the pristine vs. ceftazidime loaded ZIF-8 which is due to the contribution of the ceftazidime, which is non-toxic in both cell lines, as the samples were dosed by total compound weight. This significant impact on the toxicity due to the presence of the ceftazidime is expected as ceftazidime accounts for 10.8 % 10.9 % of the weight of ceftazidime loaded ZIF-8 as assessed by elemental analysis and TGA, respectively. Additionally, at these highest doses there is increased toxicity to the macrophage cell line compared to the lung epithelial cell line. Differential toxicity between cell types and specifically increased toxicity to



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Figure 8. Macrophage uptake of ZIF-8 and ZIF-8@Ceftazidime after 24 hrs exposure. Cell membranes were visualized with Cell mask orange (red) and ZIF-8 are directly visualized by their inherent emission spectra (green). (a) 3D z-stack images showing the ZIF-8 and Ceftazidime@ ZIF-8 inside RAW 264.7 cells. Individual channels and the merged images are shown. Merged images show the particles internal to the membrane. Scale bar represents 5 µm. (b) 3D z-stack with cut away panels of RAW 264.7 cells to allow visualization of internal ZIF-8@Ceftazidime. Green, red and blue outlines show the x, y, and z planes.

macrophages has been observed with both ZIF-8 and other MOFs previously.41, 42 After evaluating toxicity to mammalian cells, the uptake and cell internalization of pristine and loaded nanoparticles was assessed in macrophage cells. Macrophages are the major component of Mononuclear Phagocyte System (MPS) responsible for clearance and biodistribution of injected nanomaterials through phagocytosis.43, 44 Additionally, macrophages are the most common cells for replication of intracellular bacterial pathogens of both public health and biodefense concerns including: Coxiella burnetii, Salmonella enterica, Mycobacterium tuberculosis, Francisella tularensis and the target of ceftazidime therapy, Burkholderia pseudomallei.45 The potential for intracellular ceftazidime release from ZIF-8 in acidic intracellular compartments shown by the increased response at pH 5 makes assessment of intracellular localization important for future development as a therapeutic for intracellular infections. Intracellular infections are particularly difficult to treat due the ability of the bacteria to hide within macrophages. Nanocarriers present the potential to deliver the antibiotic cargo intracellularly, which is a promising methodology to enhance the treatment of intracellular infections.46, 47 Prior ZIF-8 cell internalization studies have focused on imaging a fluorescent loaded cargo within the ZIF-8 for an indirect visualization of uptake.34, 41 The limitation of this methodology is that breakdown of ZIF-8 within biological media could release the cargo and the cargo could interact directly with the cell, leading to a false impression of ZIF-8 uptake. Interestingly, ZIF-8 possesses intrinsic emission properties, when excited at 365 and 488 nm, Figure S10.48 To examine the potential to directly visualize ZIF-8 by confocal microscopy, pristine ZIF-8 nanoparticles were coated onto a microscope slide and scanned for emission spectra using the 405 nm laser, Figure S11. Utilizing these spectra to guide the emission

collection windows, the ZIF-8 particles were directly visualized on the microscopy slides, Figure S12. Following identification of the native excitation and emission of pristine ZIF-8, the uptake of both pristine and ceftazidime loaded ZIF-8 after 24 hrs exposure was assessed utilizing RAW 264.7 macrophages and confocal microscopy, Figure 8. Examination of the merged 3D images in the last column of Figure 8a shows that the membrane, displayed in red, is laying on top of the ZIF-8 particles thereby completely obscuring the green signal from the ZIF-8 particles, which is clearly visible in the channel shown in the first column of Figure 8a. The complete concealment of the ZIF-8 particles by the membrane demonstrates that the ZIF-8 particles are internal to the cell membrane. To further confirm internalization, the 3D images were sliced in the x,y and z plane allowing clear visualization of the ZIF-8 particles within the macrophages, Figure 8b. This is the first example to unequivocally demonstrate cell internalization of ZIF-8 based on its intrinsic emission properties.

5. CONCLUSIONS In summary, here we presented a comprehensive evaluation of a MOF-based system as an effective slow-release therapeutic carrier for treating intracellular infections. Sustained release of ceftazidime, an efficient antibacterial agent, is noted over the course of a week. Furthermore, bacterial susceptibility of ceftazidime loaded ZIF-8 to E coli cultures is shown, demonstrating this construct as a versatile nanoparticle platform which can be adapted for in vitro antibiotic delivery and intracellular bacterial killing. Noteworthy, cell internalization of ZIF-8 is visualized directly, taking advantage of the intrinsic emission properties of


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this material. Together, the ability of the ZIF-8 particles to be effectively internalized and release cargo within cell lines of interest qualify this system as an effective countermeasure for pathogens difficult to treat, including many biothreat agents. Future work will assess the relevance of related materials systems to support encapsulation of protein and peptide based advanced therapeutics, amenable to selectively treat intracellular infections.

ASSOCIATED CONTENT Supporting Information. TGA, XRD, SEM-EDS and analyses. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected]

ACKNOWLEDGMENT The authors would like to acknowledge Grace Vincent and Matthew Sanchez (materials synthesis), Ping Lu (TEM-EDS), Lauren Rohwer (PL) and Mark Rodriguez (XRD). This work is supported by the Laboratory Directed Research Development Program at Sandia National Laboratories. Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia, LLC., a wholly owned subsidiary of Honeywell International, Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA-0003525. This paper describes objective technical results and analysis. Any subjective views or opinions that might be expressed in the paper do not necessarily represent the views of the U.S. Department of Energy or the United States Government.

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