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Article
Versatile Synthesis and Fluorescent Labeling of
ZIF-90 Nanoparticles for Biomedical Applications
Christopher G Jones, Vitalie Stavila, Marissa A Conroy, Patrick
Feng, Brandon Slaughter, Carlee E. Ashley, and Mark D. Allendorf
ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11760 • Publication Date (Web): 07 Mar 2016
Downloaded from http://pubs.acs.org on March 11, 2016
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Page 1 of 23 ACS Applied Materials & Interfaces
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Versatile Synthesis and Fluorescent Labeling of
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6 ZIF-90 Nanoparticles for Biomedical Applications
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10 Christopher G. Jones, Vitalie Stavila,* Marissa A. Conroy, Patrick Feng, Brandon V. Slaughter, Carlee
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12 E. Ashley, Mark D. Allendorf*
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Sandia National Laboratories, Livermore, California, 94551 and Albuquerque, NM, 87123
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17 * E-mail: vnstavi@sandia.gov ; mdallen@sandia.gov
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23 ABSTRACT: We describe a versatile method for the synthesis and fluorescent labeling of ZIF-90
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nanoparticles (NPs). Gram-scale quantities of NPs can be produced under mild conditions,
26 circumventing the need for high temperatures and extended reaction periods required by existing
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28 procedures. Monitoring the reaction in situ using UV-Vis spectroscopy reveals that ZIF-90 NP
29 nucleation in solution starts within seconds. In addition to reporting a method to reproducibly form sub-
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31 100 nm ZIF-90 particles, we show that particles of various sizes can be produced, ranging from 30 nm
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to 1000 nm, by altering amine chemistry or reaction temperature. The presence of linker aldehyde
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34 groups on the NP surface allows for post-synthetic labeling with amine-functionalized fluorescent dyes,
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36 providing utility for imaging within biological systems. In vitro cell studies show that ZIF-90 NPs have
37 a high rate of cellular internalization, provide finite degradation periods of the order of several weeks,
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39 and are biocompatible with six different cell lines (> 90% viable when incubated with NPs for up to 7
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days). These features highlight the potential for use of ZIF-90 nanostructures in bioimaging and targeted
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42 drug delivery applications.
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45 KEYWORDS: ZIF-90, nanoparticle, surface functionalization, cellular uptake, bioimaging
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INTRODUCTION
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52 Nanomaterials are an increasingly important medical research tool due to their ability to
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54 facilitate the transport of therapeutic and diagnostic agents through barriers within biological
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56 environments. These properties are particularly advantageous for tumor targeting and preferential
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delivery of high toxicity anticancer drugs.1 A key motivation for the development of such materials is
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3 the nonspecific uptake of commonly used drugs, which often induces a cytotoxic effect on healthy
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5 tissues and organs throughout the body. To reduce systemic toxicity, it is highly desirable to
6 synthesize biocompatible nanomaterials within specified size ranges that will stabilize imaging or
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8 therapeutic agents until they reach their desired target.2 Nanoparticles (NPs) (particles in the 1 to 100
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10 nm range), interact on the same scale as naturally occurring biological molecules and systems and
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12 can therefore be designed to suit specific parameters and functions.3 Exploiting the preferential
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cellular uptake range with specifically designed particles can drastically improve the efficacy of
15 imagining particular biological sites within the body, as well as reduce the overall systemic toxicity
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17 of potent anticancer drugs.4
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20 The majority of nanocarriers for imaging and therapeutic applications are either purely
21 inorganic (quantum dots, silica, Fe3O4, Au NPs) or purely organic (liposomes, polymers,
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23 dendrimers) in nature. Recently, metal organic frameworks (MOFs) were introduced as promising
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25 nanoscale delivery agents for various biomedical applications.5-7 MOFs are porous crystalline
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27 materials comprised of metal ions or clusters interconnected through a network of organic bridging
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ligands, giving MOFs several distinct advantages over existing nanocarriers. First, the synthetic
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30 space for these materials is vast, since rational design principles can be applied to both the metal
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32 clusters and organic linkers, and the number of reported frameworks with nanoporosity is now in
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34 the thousands.8 Second, MOFs are compositionally and structurally tunable, allowing systematic
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variation of pore size, shape, and chemistry.8,9 Third, many MOFs are intrinsically biodegradable as
37 a result of relatively labile metal–ligand bonds.10 Finally, both the bulk and surface of MOFs can be
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39 altered using post-synthetic modification methods11,12 to, for example, impart a specific chemical
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41 functionality12 or control water stability and solubility.13 Prior research demonstrated the
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43 effectiveness of MOFs in a variety of different applications, including membrane separations,
44 catalysis, gas storage, and active layers in functional devices.14,15 These characteristics of MOFs
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46 provide the means to tailor the chemical and structural properties of delivery agents to maximize
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48 effectiveness for specific biological targets.6,10,16,17 Moreover, controllable methods of preparing
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50 nanoscale MOFs are beginning to emerge,6,18,19 an important development, since cellular uptake of
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individual NPs is strongly determined by their size, shape, and chemical composition.20
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54 Zeolitic imidazolate frameworks (ZIFs) are a large subclass of MOFs that have considerable
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56 potential in biomedicine.21-23 In addition to permanent porosity and adjustable pore dimensions and
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58 geometries, ZIFs can also display tunable chemical stability under biological conditions.24
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3 Remarkably, very recent work by Zheng et al.23 demonstrated that the anticancer drug curcumin can
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5 be loaded inside ZIF-8 pores using ship-in-a-bottle synthesis; the resulting curcumin@ZIF-8
6 particles are more stable and display higher cytotoxicity toward HeLa cells under acidic
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8 microenvironments compared to pure curcumin. Of particular interest to biomedical applications is
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10 ZIF-90 (Zn(II) imidazolate-2-carboxyaldehyde), as the aldehyde group on the bridging imidazolium
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12 ligand provides chemical handle for functionalizing both the pore interior and the external surface
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with, for example, imaging and/or therapeutic agents. A recent study by Li et al.25 demonstrated the
15 functionalization and stability of ZIF-90 crystals serving as fluorescent probes for H2S detection and
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17 thio-aminoacid recognition. Post-synthetically modified ZIF-90 not only showed a high selectivity
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19 towards biothiols (H2S and cysteine), but also exhibited exceptional biocompatibility when observed
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21 in vitro. This highlights the potential for using ZIF-90 as a contrast agent for optical imaging, as well
22 as for sensing and molecular recognition in biological applications.
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25 However, for MOFs in general and ZIFs in particular to serve as practical nanocarriers, new
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27 synthetic protocols for NP synthesis and surface functionalization need to be developed that provide
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greater control over NP properties, in particular size and shape. Most reported strategies for
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30 synthesizing ZIF powders yield particle sizes in the micron range.26 Several literature reports
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32 describe the synthesis of sub-micron ZIF-90 particles;27,28 however there are no established
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34 procedures for controlling particle size within a narrow range required for efficient intracellular
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delivery of therapeutics and imaging agents. Particles between 1 nm and 100 nm are the most
37 suitable for these applications, given their favorable diffusion characteristics across cellular
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39 membranes, but development of reliable synthetic routes for controlling NP growth in this nano-
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41 regime remains a significant challenge.20 Here, we describe a rapid and versatile approach for
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43 synthesizing ZIF-90 particles between 30 nm and 1000 nm in diameter using mild reaction
44 conditions at or near room temperature (RT). Although a RT synthesis of ZIF-90 was previously
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46 reported, this method produces particles greater than 80 nm.28 Our standard procedure for ZIF-90
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48 NP synthesis involves the reaction of DMF solutions of Zn(NO3)2 and 2-imidazolecarboxaldehyde
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50 (IcaH) in the presence of trioctylamine (vide infra). By this procedure we demonstrate reproducible
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synthesis of multi gram-scale batches of ZIF-90 NPs in 1 minute or less. In contrast, other methods
53 to synthesize sub-micron and sub-100 nm ZIF-90 NPs require extended reaction periods of up to 24
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55 hours.26,28 In addition to our standard synthetic procedure in DMF/trioctylamine, we examined the
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57 effect of the amine and temperature on particle growth and show that NPs with sizes ranging from
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3 30 nm to 1000 nm and narrow size distributions can be obtained. To assess the potential for ZIF-90
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5 to serve as a nanoscale delivery platform in biological systems, post-synthetic modification was
6 conducted on as-synthesized NPs to attach fluorescent dyes to the surface aldehyde group. Finally,
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8 using in vitro studies, the fluorescently labeled ZIF-90 NPs were tested to determine potential
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10 cellular uptake, particle degradation periods, as well as impact on overall cell viability.
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14 EXPERIMENTAL
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17 Materials and NP characterization. All synthesis materials and solvents were purchased from
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19 Sigma-Aldrich and Fisher Scientific. Alexa Fluor® 633 Hydrazide and Alexa Fluor® 647 Hydrazide
20 fluorescent dyes were purchased from Invitrogen. Powder X-ray diffraction (PXRD) patterns were
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22 collected using a PANalytical Empyrean X-ray diffractometer equipped with a PIXcel3D detector and
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24 operated at 44 kV and 40 mA using Cu Kα radiation. The patterns were collected in the 2θ range of 5
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26 to 50° with a step size of 0.026°. Scanning Electron Microscopy (SEM) was performed using a
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Hitachi S-4500 field-emission electron microscope. SEM slides were prepared by first suspending
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29 particles in ethanol and sonicating for 10 minutes. Using a pipet, 2-3 drops of the suspended particles
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31 were placed on the surface of a gold wafer and air dried to evaporate remaining solvent. Wafers were
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33 coated with a conductive layer using a Denton Vacuum Desk II equipped with a Au/Pd target.
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Porosimitry was conducted using a Quadrasorb SI prorosometer from Quantachrome Instrument.
36 Optical turbidity was measured using a Shimadzu UV-3600 Spectrophotometer at 360 nm. Elemental
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38 analysis measurements were performed by ALS Labs, Inc. Isotherms were measured on 55–80 mg
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40 samples using a Quantachrome Quadrasorb-SI (Kr/MP) porosimeter (Quantachrome Instruments,
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42 USA). BET analysis was conducted from N2 isotherm measurements collected at 77 K using a liquid
43 nitrogen bath. Steady-state and time-resolved photoluminescence (PL) spectra were obtained using a
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45 Horiba Jobin-Yvon Fluorolog 3-21 fluorometer. Absolute quantum yield measurements were
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47 collected using a QuantaPhi integrating sphere attachment in the range of 300-800 nm and corrected
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49 for the sphere reflectivity, transport optics, and photodetector spectral sensitivity.
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51 Synthetic procedure for growing ZIF-90 NPs. Our “standard” preparation method for ZIF-90
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53 NPs, which yields particle sizes of 60-90 nm, was performed by dissolving 223 mg (0.75 mmol) of
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55 Zn(NO3)2·6H2O in 50 mL DMF with stirring. In a separate vial, 200 mg (2.10 mmol) of IcaH (IcaH =
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57 imidazolate-2-carboxyaldehyde) was added to 100 mL of DMF and was heated at 50 °C with stirring
58 until fully dissolved and then cooled to RT. A third solution was prepared by adding 0.86 mL (1.96
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3 mmol) of trioctylamine (TOA) to 50 mL of DMF. At RT, Zn(NO3)2·6H2O was added to the IcaH
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5 solution, followed by addition of TOA while stirring. Upon addition of TOA, a white precipitate
6 formed immediately in the solution (Figure S1, Supp. Info). After one minute, the reaction was
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8 quenched by adding 100 mL of ethanol. Particles were then isolated by centrifugation at 8000 RPM
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10 for 15 minutes. They were then washed with EtOH and centrifuged again at 8000 RPM for 15
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12 minutes. This washing procedure was repeated five times. The washed material was kept in vacuum
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overnight to generate “dry” ZIF-90 NPs; the overall yield was between 74 and 79% (calculated based
15 on Zn(NO3)2).
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18 Effect of amine. ZIF-90 NPs were also synthesized by replacing TOA with either triethylamine
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20 (TEA), yielding 200-300 nm particles, or tributylamine (TBA), which yields 100-200 nm particles.
21 In each case the standard procedure was followed, with the exception of either 1.96 mmol of TEA or
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23 TBA added in place of TOA. All other reaction conditions remained the same (Table S1).
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26 Effect of temperature. ZIF-90 synthesis was conducted by systematically varying the reaction
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temperature. A low temperature synthesis of ZIF-90 NPs (30-50 nm) was conducted at 0 °C by
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29 following a similar procedure as described above. The metal salt, linker and surfactant solutions were
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31 placed in a freezer at 0 °C for one hour. Once solutions reached 0 °C, they were placed on a stir plate
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33 and quickly mixed. After reacting for one minute, the solution was quenched with 100 mL ethanol.
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Particles were then isolated and washed using EtOH as described above. For high-temperature
36 synthesis, which yielded 100 – 1000 nm particles, each reactant was placed inside an oven at 50 °C,
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38 75 °C, 100 °C, 125 °C, or 150 °C. Once all solutions reached their desired temperatures, they were
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40 placed on a stir plate and mixed. The reaction was stirred for one minute, after which the solution
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42 was placed in the oven at the previously set temperature for 60 minutes. After 60 minutes, the
43 reaction mixture was quenched with ethanol, and ZIF-90 particles were isolated by centrifugation as
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45 described above.
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48 Synthesis of ZIF-7. ZIF-7 microrods were synthesized by adding 117 mg (0.188 mmol) of
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Zn(NO3)2·6H2O, 120 mg (1.02 mmol) of benzimidazole, and 0.43 mL (0.983 mmol) of trioctylamine
51 to 150 mL of DMF. The solution was placed in the oven for 60 minutes at 150 °C. The reaction was
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53 quenched with ethanol, and the product was washed with ethanol as in the case of ZIF-90 NPs.
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56 Synthesis of ZIF-8. ZIF-8 NPs were synthesized by adding 235 mg (0.376 mmol) of
57 Zn(NO3)2·6H2O, 84 mg (1.02 mmol) 2-methylimidazole, and 0.43 ml (0.983 mmol) of trioctylamine
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3 to 150 mL of DMF. Solution was placed in the oven for 60 minutes at 150 °C. After 60 minutes,
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5 solution was removed from the oven and cooled to RT. The as-synthesized particles were washed
6 with ethanol and isolated through centrifugation as described above.
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9 Synthesis of TIF-2. TIF-2 NPs were synthesized by adding 235 mg (1.07 mmol) of
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11 Zn(CH3COO)2·2H2O, 140 mg (1.06 mmol) 5-methylbenzimidazole, 88 mg (1.29 mmol) of
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imidazole, 8 mL of 2-amino-1-butanol, 2 mL of trioctylamine and 7.2 mL of DMF in a steel
14 autoclave. The solution was placed in the oven for 48 hours at 150 °C and then cooled to RT.
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16 Particles were washed with EtOH, centrifuged and dried in air.
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19 Fluorescent labeling of ZIF-90 nanoparticles. ZIF-90 NPs were labeled by dissolving 1 mg
20 of either Alexa Fluor® Hydrazide 633 or 647 in 50 mL of methanol. ZIF-90 NPs synthesized using
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22 the standard procedure outlined above were then added to the dye solution. The solution was placed
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24 on an orbital shaker for 24 hours at RT. After 24 hours the particles were isolated by centrifugation
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26 at 8000 RPM for 15 minutes. Isolated particles were washed with ethanol and centrifuged for 15
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minutes. This wash cycle was repeated 6 times, at which point no further dye was observed in the
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29 ethanol. Isolated particles were left to air dry at RT for 24 hours. A schematic representing the
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31 chemical reaction occurring between the linker and dye is shown in Figure 1. Although the dye
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33 loading is difficult to quantify, the elemental analysis results (Table S2) on the dye-labeled ZIF-90
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NPs shows a slightly higher carbon content and a lower nitrogen content compared to as-
36 synthesized ZIF-90 NPs.
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54 Figure 1. Schematic illustration of synthesis and functionalization of ZIF-90 NPs with Alexa Fluor®
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56 Dyes.
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3 Cell Culture. All cells and growth media were purchased from American Type Culture
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5 Collection (Manassas, VA) and grown according to manufacturer’s instructions. Briefly, A549
6 (ATCC cat. no. CCL-185) and CHO-K1 (CCL-61) were grown in F-12K medium with 10% (v/v)
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8 fetal bovine serum (FBS). HEK 293 (CRL-1573), HeLa (CCL-2), and HepG2 (HB-8065) were
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10 maintained in Eagle’s Minimum Essential Medium (EMEM) with 10% FBS. LNCaP (CRL-1740)
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12 was grown in RPMI-1640 medium with 10% FBS. All cells were maintained at 37°C in a humidified
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atmosphere (air supplemented with 5% CO2) and were passaged with 0.05% trypsin at a sub-
15 cultivation ratio of 1:3.
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18 Colloidal Stability of ZIF-90 NPs. To assess colloidal stability, ZIF-90 NPs were incubated in
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20 simulated body fluid (see Marques, et al.29 for the recipe) at a concentration of 25 mg/mL for up to
21 72 hours at 37 °C. At 0, 1, 6, 12, 24, 48, and 72 hours, 48 µL was removed, diluted in 2.4 ml of 1X
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23 D-PBS, transferred to 1 mL polystyrene cuvettes (Sarstedt; Nümbrecht, Germany) for Z-average
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25 measurements or to 1-mL folded capillary cells (Malvern; Worcestershire, United Kingdom) for zeta
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27 potential measurements, and analyzed using a Zetasizer Nano (Malvern; Worcestershire, UK).
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29 Dissolution Rates of ZIF-90 NPs in Simulated Body and Lysosomal Fluids. To determine
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31 dissolution rates, 1 mg of ZIF-90 NPs was incubated in a simulated body fluid or a simulated
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33 lysosomal fluid (see the recipe for ‘artifical lysosomal fluid’ from Marques, et al.29 for up to 8 weeks
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at 37°C. Total mass was then determined at 0, 3, 5, 7, 14, 21, 28, 42, and 56 days by filtering the
36 solution to capture ZIF-90 NPs and allowing them to dry for 3 days in the presence of a desiccant.
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39 Biocompatibility of ZIF-90 NPs. To assess the biocompatibility of ZIF-90 NPs, 1 x 106 A549,
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41 CHO-K1, HEK 293, HeLa, HepG2, or LNCaP cells were incubated with 0.1, 0.5, 1, 5, 10, 50, 100,
42 500, or 1000 µg of ZIF-90 NPs in 1 mL of serum-free growth medium for 1 hour at 37 °C; cells were
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44 then washed three times with 1X PBS to remove unbound ZIF-90 NPs and incubated in complete
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46 growth medium for 0, 1, 3, 5, 7, 14, 21, 28, or 42 days at 37 °C. To quantify the percentage of viable
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48 cells in each population, cells were stained with 5 µL/mL of SYTOX® Green Dead Cell Stain
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(Invitrogen Life Sciences; Carlsbad, CA) for 20 minutes at 37 °C and analyzed using a FACSCalibur
51 flow cytometer (Becton Dickinson; Franklin Lakes, NJ) equipped with BD CellQuestTM software,
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53 version 5.2.1. Samples were acquired with the fsc channel in linear mode and all other channels in
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55 log mode. Events were triggered based upon forward light scatter, and a gate was placed on the
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57 forward scatter-side scatter plot that excluded cellular debris. Samples were excited using the 488-
58 nm laser source, and emission intensity was collected in the FL1 channel (530/30). Mean
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3 fluorescence intensity (MFI) was determined using FlowJo Software, version 6.4 (Tree Star, Inc.;
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5 Ashland, OR). Cells with a MFI ≥ 100 times the MFI of unstained cells were considered dead. For
6 experiments that lasted more than 3 days, growth medium was replaced every 48-72 hours.
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9 Internalization of ZIF-90 NPs by CHO-K1 Cells. To promote cellular uptake of ZIF-90 NPs,
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11 Alexa Fluor 647-labeled ZIF-90 NPs were modified with an octarginine (R8) peptide, synthesized
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with a C-termine cysteine residue by New England Peptide (Gardner, MA), using the
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14 heterobifunctional crosslinker, Sulfo-SMCC (Pierce Protein Research Products; Thermo Fisher
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16 Scientific LSR; Rockford, IL). 1 x 106 CHO-K1 cells were then incubated with 0.1, 0.5, 1, 5, 10, 50,
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18 100, 500, or 1000 µg of Alexa Fluor 647 hydrazide, Alexa Fluor 647-labeled ZIF-90 NPs, or Alexa
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20 Fluor 647-labeled, R8-modified ZIF-90 NPs in 1 mL of serum-free growth medium for 1 hour at
21 37°C. Cells were washed three times with 1X PBS to remove unbound dye molecules or ZIF-90 NPs,
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23 analyzed using a FACSCalibur flow cytometer, treated with 0.05% trypsin for 10 minutes at RT to
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25 remove surface-bound dye molecules or ZIF-90 NPs, and analyzed again. Alexa Fluor® 647
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27 fluorescence was excited by the 633-nm laser and collected in the FL3 channel (670-nm long pass
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filter), and MFIs were determined using FlowJo Software.
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31 Intracellular Fate of ZIF-90 NPs in CHO-K1 Cells. To assess the intracellular fate of ZIF-90
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33 NPs, 1 x 106 CHO-K1 cells were incubated with 10 µg of Alexa Fluor 647-labeled, R8-modified
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ZIF-90 NPs in 1 mL of serum-free growth medium for 1 hour at 37°C. Cells were then washed three
36 times with 1X PBS, fixed with 3.7% formaldehyde for 15 minutes at RT, permeabilized with 0.2%
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38 Triton X-100 for 5 minutes at rt, and blocked with Image-iT® FX signal enhancer (Invitrogen Life
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40 Sciences; Carlsbad, CA) for 30 minutes at RT. The sample was then incubated with a mouse
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42 monoclonal antibody against lysosomal-associated membrane protein 1 (Abcam, Inc.; Cambridge,
43 MA), diluted 1:500 in 1X PBS with 1% BSA, for 1 hour at 37 °C and an Alexa Fluor® 488-labeled
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45 goat antibody against mouse IgG (Invitrogen Life Sciences; Carlsbad, CA), diluted 1:250 in 1X PBS
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47 with 1% BSA, for 90 minutes at 37 °C. The sample was washed three times with 1X PBS between
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49 each step, mounted with SlowFade® Gold containing DAPI (Invitrogen Life Sciences; Carlsbad,
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CA), and imaged with a laser scanning confocal microscope as described below.
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53 Persistence of ZIF-90 NPs within CHO-K1 Cells. To quantify the intracellular persistence of ZIF-
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55 90 NPs, 1 x 106 CHO-K1 cells were incubated with 10 µg of Alexa Fluor 647-labeled, R8-modified
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57 ZIF-90 NPs in 1 mL of serum-free growth medium for 1 hour at 37 °C. Cells were then washed
58 three times with 1X PBS, incubated in complete growth medium for 0, 3, 5, 7, 14, 21, 28, or 42
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3 days at 37 °C, and analyzed via a FACSCalibur flow cytometer as described above. To confirm
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5 flow cytometry data, 1 x 106 CHO-K1 cells were incubated with 10 µg of Alexa Fluor 647-labeled,
6 R8-modified ZIF-90 NPs in 1 mL of serum-free growth medium for 1 hour at 37 °C, washed three
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8 times with 1X PBS, and incubated in complete growth medium for 7 or 42 days at 37 °C. Cells were
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10 then fixed with 3.7% formaldehyde for 15 minutes at RT, mounted with SlowFade® Gold
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12 containing DAPI, and imaged with a laser scanning confocal microscope as described below. For
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experiments that lasted more than 3 days, growth medium was replaced every 48-72 hours.
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16 Laser Scanning Confocal Microscopy. Two and three-color images were acquired using a Zeiss
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18 LSM510 META (Carl Zeiss MicroImaging, Inc.; Thornwood, NY) operated in Channel mode of the
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20 LSM510 software; a 63X, 1.4-NA oil immersion objective was employed in all imaging. Typical
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laser power settings were: 30% transmission for the 405-nm diode laser, 5% transmission (60%
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23 output) for the 488-nm Argon laser, and 85% transmission for the 633-nm HeNe laser. Gain and
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25 offset were adjusted for each channel to avoid saturation and were typically maintained at 500-700
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27 and -0.1, respectively. 8-bit z-stacks with 1024 x 1024 resolution were acquired with a 0.7 to 0.9-
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µm optical slice. LSM510 software was used to overlay channels and to create 3D projections of z-
30 stack images.
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35 RESULTS AND DISCUSSION
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37 Nanoparticle synthesis. Previously reported room-temperature methods for ZIF-90 synthesis
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39 typically yielded particle sizes ranging from hundreds of nanometers27 to several microns.26 A
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41 notable exception is the work of Yang et al.,28 who reported particles within the 80 to 200 nm range.
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We found that reacting a Zn(II)-salt with the IcaH linker in DMF in the presence of a ternary amine
44 consistently yields ZIF-90 particles in the sub-100 nm range. A tertiary amine with large alkyl
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46 substituents can act as both a deprotinating agent and a surfactant, facilitating the nucleation and
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48 growth of NPs. Addition of trioctylamine (TOA) to the reaction mixture not only causes rapid
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50 nucleation of particles at RT, but also serves as a capping agent that inhibits particle growth. Once
51 added to a solution of Zn(NO3)2 and IcaH, the reaction occurs rapidly; formation of ZIF-90 NPs is
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53 observed in as little as 1 minute. Scanning electron microscopy (SEM) revealed that isolated NPs
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55 have an average diameter of 60-90 nm (Figure S2). Powder X-ray diffraction (PXRD) patterns match
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57 those previously reported and indicate highly crystalline materials.26
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3 To gain further insight into the growth kinetics of ZIF-90 NPs, turbidity measurements were
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5 conducted using UV-Vis absorption spectroscopy. Turbidity measurements are widely used to
6 monitor NP formation rates30 and NP interactions in solutions.31 UV-Vis analysis is also useful for
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8 quantifying the rate at which NPs nucleate, since solution turbidity depends on the volume fraction
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10 of suspended NPs in solution. In a study by Smith et al.32 the optical turbidity was measured in situ
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12 to characterize formation rates of covalent organic frameworks (COFs). Here, we follow a similar
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procedure, using 360-nm light, to observe ZIF-90 NP formation rates during standard room-
15 temperature synthesis with TOA. Time-lapse images demonstrating the changes in turbidity for a
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17 typical ZIF-90 reaction performed at RT over 60 seconds are shown in Figure 2A. The rapid rate at
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19 which this reaction occurs is evident, forming appreciable amounts of product within the first 10
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21 seconds of mixing. This is quantified by monitoring the reaction using a UV-Vis spectrometer over a
22 two-minute period (Figure 2B). An essentially constant growth rate is observed up to a reaction time
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24 of ~50-55 s, at which point the turbidity saturates. The negligible increase in slope after
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26 approximately 60 seconds suggests that most of the product forms during the first minute of reaction.
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3 Figure 2. (A) Time lapse images of ZIF-90 reaction solution over 60 s. (B) Optical turbidity
4 measurement for RT ZIF-90 NP formation using UV-Vis spectroscopy.
5
6
7
8 In the absence of a base, ZIF-90 does not form at RT due to the increased acidity of the IcaH
9
10 linker; Morris et al. employed TEA26 using vapor diffusion and Yang et al. used pyridine28 in DMF
11
12 to deprotonate IcaH, so that the MOF forms at RT under non-solvothermal conditions. However,
13
14 these two amines have different pKa values, so to clarify the relationship between NP size and
15
basicity, we performed the synthesis of ZIF-90 NP using TEA or TBA in place of TOA. PXRD
16
17 patterns confirmed the identity of ZIF-90 particles synthesized in the presence of TBE and TEA to
18
19 be identical to that of TOA (Figure S3). SEM images of synthesized particles reveal increasingly
20
21 larger particle sizes from TOA, TBA, and TEA, respectively (Figure S4 – S5). Particle sizes were
22
23
estimated through measurements using SEM imaging. Individual particles were counted and
24 measured to determine size distributions and average particle size. Interesting enough, porosimetry
25
26 measurements indicate that BET surface area slightly decreases as a function of MOF particle size
27
28 (Table S1, Supp. Info). Although particles tended to agglomerate after centrifugation, it was found
29
30 that they could be dispersed after thorough sonication. As noted initially, synthesis using TOA
31 produced particles in the range of 60 to 90 nm (Figure 3B). Synthesis using TBA yielded faceted
32
33 particles of 100 to 200 nm (Figure 3D), whereas the largest particles were produced using TEA,
34
35 yielding particles between 200 to 300 nm (Figure 3F). From these results, it is evident that amine
36
37 chemistry plays an important role in determining particle size. Although further studies are needed
38
39
to fully understand the NP growth mechanism, the capping effect demonstrated by these ternary
40 amines provides an effective method for facilitating RT reactions and for controlling particle growth
41
42 in the nano-regime.
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11 Figure 3. ZIF-90 NPs synthesized in TOA at (A) 0 °C, (C) 100 °C, and (E) 150 °C and particles
12
13 synthesized at RT using (B) trioctylamine, (D) tributylamine, (F) trimethylamine.
14
15
16
17 Given that solvothermal conditions can lead to micron-scale crystals,26 it is important to evaluate
18
19 the influence of reaction temperature on the growth mechanisms leading to sub-micron ZIF-90
20 particles. Starting from 0 °C, we increased the reaction temperature to 150 °C in 25 °C intervals at a
21
22 constant reaction time of 60 minutes. Interestingly, SEM images of isolated particles indicate that
23 particle size increases as a function of reaction temperature. A plot of average particle size as a
24
25 function of reaction temperature is shown in Figure 4, which shows that particle size is
26
approximately a linear function of temperature. Particles synthesized at 0 °C ranged between 30–50
27
28 nm (Figure 3A), the smallest reported to date for ZIF-90. In contrast, particles formed at 25 °C were
29
30
60–90 nm (Figure 3B) and particles formed at 100 °C were 300–400 nm (Figure 3C). Finally,
31 reactions performed at 150 °C produced rhombic dodecahedron microcrystals (Figure 3E and S6).
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53 Figure 4. Average particle size of ZIF-90 synthesized at temperatures between 0 °C and 150 °C.
54
Error bars represent the mean ± the standard deviation.
55
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3 The increasing particle size with temperature is rather unusual. Typically, NP formation follows a
4 nucleation and growth mechanism,33 which involves a short burst of nuclei followed by their
5
6 growth. The activation energy for nucleation is normally larger than that for subsequent particle
7
growth, which makes the nucleation rate more sensitive to changes in temperature than the growth
8
9 rate.33 Particle growth is controlled by diffusion and/or surface reactions and typically occurs via
10
11
Ostwald ripening34 or oriented attachment.35 In the first case, the growth of the larger particles is
12 due to dissolution of the smaller ones, whereas in the second mechanism they grow by merging of
13
14 smaller ones. We hypothesize that in the case of ZIF-90, growth is related to the degree of
15 stabilization of the particles by the TOA surfactant. Elevated temperatures increase the rate of
16
17 surfactant desorption from the MOF particle surface, enabling growth species such as MOF
18 secondary building units (SBUs) to interact and attach to the particle surface. This is supported by
19
20 the fact that shorter alkyl chains on the ternary amine lead to increasingly larger particle sizes from
21 TOA, TBA, and TEA, respectively (Figures 3 (B, D, F), S4, S5). In addition, higher temperatures
22
23 favor the formation of rhombic dodecahedron microcrystals of ZIF-90, which seems to be a
24 thermodynamically stable crystal shape for this system.
25
26
27 We also performed a limited number of experiments to assess whether other ZIF NPs can be
28 isolated using the reaction conditions established for ZF-90. However, our attempts to synthesize
29
30 ZIF-7, ZIF-8, and TIF-2 NPs at RT from Zn(II)-salts and the corresponding linkers in the presence
31 of a ternary amine were unsuccessful. Although the RT synthesis seems to be specific for ZIF-90,
32
33 the high-temperature (150 °C, DMF/TOA) reaction conditions allowed us to produce ZIF-7
34 microrods, sub-150 nm ZIF-8 NPs and submicron TIF-2 spherical particles (Figures S7, S8 and S9,
35
36 Supp. Info). The PXRD patterns confirm the identity and phase purity of the as-synthesized
37
materials. In contrast to ZIF-7, TIF-2 and ZIF-90, ZIF-8 forms small (<150 nm) particles from
38
39 DMF/TOA solutions at 150 °C, suggesting a relatively efficient surface binding of the surfactant to
40
41
limit the particle growth. Although other methods exist for the synthesis of ZIF-7 and ZIF-8
42 nanomosphologies,19 as far as we know, there are no other methods known to yield nano- or
43
44 submicron particles of TIF-2.
45
46 Fluorescent labeling and bioimaging. To show the functionalization capabilities of ZIF-90 and
47
48 to help measure diffusion characteristics across cellular membranes, NPs were covalently labeled
49
with fluorescent molecules via post-synthetic modification. For this we chose Alexa Fluor® 633
50
51 Hydrazide which has amine groups that can react with aldehyde groups on the MOF linkers. Alexa
52
53 Fluor® 647 Hydrazide was also used to post-synthetically label NPs to show functionalization is
54
55 possible using other far red flourescent dyes. The electrophylic carbon atoms of the aldehyde –CH=O
56
57
groups in ZIF-90 are nucleophilically attacked by the amine group –NH2 of the hydrizide moiety of
58 the dye to form an imine –CH=N– compound, also known as a Schiff base (Figure 1). The Alexa
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60
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3 Fluor® dyes are too large to enter the pores of the ZIF-90 NPs, so the labeling (performed by by
4
5 soaking NPs in a methanolic solution of the dye; Figure S10, Supp. Info) occurs entirely on the
6 surface. SEM images show no noticeable changes in particle shape or size upon post-synthetic
7
8 surface treatment. Similarly, PXRD patterns were identical to those of unlabeled ZIF-90 NPs
9
10 showing no change in MOF structure or crystallinity (Figure S11, Supp. Info). Steady-state
11
12 fluorescence measurements revealed that the respective Alexa Fluor 633- and Alexa Fluor 647-
13
14
labeled NPs possess red shifts of 8 nm and 34 nm when compared to the unbound dyes (Figure S12,
15 Supp. Info). Corresponding quantum yields of 11.3% and 18.1% for the dye labeled NPs compare to
16
17 solution-phase values of 70.6% and 33.0% for unbound Alexa Fluor 633 and Alexa Fluor 647,
18
19 respectively. These observations suggest changes in the dye electronic environment and/or structure
20
21 upon binding to the ZIF-90 NPs.
22
23 To assess the utility of fluorescently-labeled ZIF-90 NPs as bio-imaging agents, we employed
24
25 dynamic light scattering (DLS) to monitor their colloidal stability in complex biological fluids (see
26 Figure 5A). The hydrodynamic size of the RT-synthesized ZIF-90 NPs increased from ~70 nm to
27
28 only ~100 nm upon incubation in a simulated body fluid (SBF) for 72 hours at 37 °C. This indicates
29 that ZIF-90 NPs will not form large (>1 µm) and potentially toxic aggregates upon intravenous
30
31 injection. Under the same conditions, the zeta potential of ZIF-90 NPs decreased from 6.4 mV to –
32 2.1 mV, likely due to absorption of serum proteins onto the NP surface. In general, NPs with near-
33
34 neutral surface charges avoid non-specific uptake by phagocytic and non-phagocytic cells and are
35
thus less cytotoxic than NPs bearing a large negative or positive charge.36,37 At the same time, the
36
37 fluorescence measurements revealed no significant dye leakage out from the Alexa Fluor 633- and
38
39
Alexa Fluor 647-labeled ZIF-90 NPs incubated in SBF at 37 °C.
40
41 The chemical stability of ZIF-90 NPs was further by measuring their time-dependent dissolution
42 upon incubation in a SBF or a simulated lysosomal fluid (SLF) at 37 °C. As shown in Figure 5B,
43
44 ZIF-90 NPs degrade within 4 weeks under conditions that simulate intracellular environments (i.e.
45 the SLF) and within 6 weeks in conditions that simulate the blood (i.e. the SBF). These data indicate
46
47 that ZIF-90 NPs have sufficient chemical stability to enable long-term labeling and tracking of cells
48
49
in vitro and in vivo. The rather slow dissolution rates we observe are consistent with the generally
50 hydrophobic nature of ZIFs.38 The water adsorption isotherm of ZIF-90 was recently reported and
51
52 the material was found to be weakly hydrophobic, a result of competing hydrophilic aldehyde
53 groups and imidazole rings within the pores.39
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18 Figure 5. ZIF-90 nanoparticles are stable in simulated body and lysosomal fluids for up to 6 weeks. (A)
19 Mean hydrodynamic size and zeta potential of ZIF-90 NPs upon continuous incubation in a simulated
20
21 body fluid for 3 days at 37 ºC. (B) Dissolution of 1 mg of ZIF-90 NPs upon continuous incubation in a
22 simulated body fluid (SBF) or simulated lysosomal fluid (SLF) for 8 weeks at 37 ºC. Error bars
23
24 represent the mean ± the standard deviation for n=3.
25
26
27
28
29 To demonstrate the utility of fluorescently-labeled ZIF-90 NPs in long-term cell labeling and
30 tracking, an arginine-rich peptide (R8) that has been shown to induce micropinocytosis,40 was
31
32 conjugated to the surfaces of ZIF-90 NPs using the heterobifunctional crosslinker, Sulfo-SMCC.
33 Flow cytometry and laser scanning confocal microscopy were then used to quantify internalization,
34
35 intracellular fate, and persistence of ZIF-90 NPs within CHO-K1 cells as has been similarly done
36
for ZIF-8 and MIL-101.19,22 To assess the internalization efficiencies of ZIF-90 NPs before and after
37
38 modification with the R8 peptide, 1×106 CHO-K1 cells were incubated with increasing
39
40
concentrations of Alexa Fluor 647, ZIF-90 NPs, or R8-modified ZIF-90 NPs for 1 hour at 37°C, and
41 the mean fluorescence intensity (MFI) of each cell population was measured using flow cytometry;
42
43 cells were then treated with trypsin to remove surface-bound dye molecules or NPs, and their MFIs
44 were measured again (see Figure S13). The results demonstrate that Alexa Fluor 647 and ZIF-90
45
46 NPs bind to the surfaces of CHO-K1 cells, but are not internalized. In contrast, R8-modified ZIF-90
47 NPs were efficiently internalized by CHO-K1 cells and trafficked to lysosomes, as evidenced by the
48
49 high degree of colocalization between Alexa Fluor 647-labeled ZIF-90 NPs and an Alexa Fluor 488-
50 labeled antibody against lysosomal-associated membrane protein 1 (LAMP-1) in the confocal
51
52 fluorescence microscopy image depicted in Figure 6A-D. Despite being localized in the harsh
53 lysosomal environment, R8 modified ZIF-90 NPs were detectable in CHO-K1 cells for several
54
55 weeks. After incubation, localized NPs continued to show observable fluorescence at 2 weeks
56 (Figure 6E), while after 4 weeks (Figure 6F) the fluorescence was no longer detectable.
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24 Figure 6. Confocal fluorescence microscopy images of CHO-K1 cells 3 hours (A-B) and 1 week (C-D)
25
26 after being incubated with Alexa Fluor 647-labeled ZIF-90 NPs (red). Lysosomes were stained with an
27 Alexa Fluor 488-labeled antibody against LAMP-1 (green), cell nuclei were stained with DAPI (blue).
28
29 Panels (A) and (C) have the green channel removed. Confocal fluorescence microscopy images of
30 CHO-K1 cells 2 weeks (E) and 1 month (F) after being incubated with Alexa Fluor 647-labeled ZIF-90
31
32 NPs (red) for 1 hour at 37ºC and washed three times with 1X PBS. Cell nuclei were stained with DAPI
33
34 (blue). Scale bars = 20 µm.
35
36
37
38 Biocompatability. The biocompatibility of ZIF-90 NPs was evaluated by incubating various
39
40 immortalized cell lines with 0.1-1000 µg/mL of ZIF-90 NPs for 1 hour at 37°C or 10 µg/mL of ZIF-
41
90 NPs for 1-42 days at 37 °C. As demonstrated by Figure 7, ZIF-90 NPs had a minimal impact on
42
43 the viability of three of the six cell lines that were tested. Even at a ZIF-90 NP concentration of 1
44
45
mg/mL (~10X higher than concentrations tested for other Zn-containing nanomaterials41) or
46 incubation times of 6 weeks (~20X longer than time periods tested for other Zn-based nanomaterials
47
48 and MOFs),21,41 more than 80% of a Chinese hamster ovarian epithelial cell line (CHO-K1), a
49 human cervical epithelial cell line (HeLa), and a human lung carcinoma cell line (A549) remained
50
51 alive. ZIF-90 NPs had a greater impact on the viability of a human embryonic kidney epithelial cell
52 line (HEK 293), a human prostate carcinoma cell line (LNCaP), and a human liver carcinoma cell
53
54 line (HepG2), all of which we have found are especially sensitive to primary amine-containing
55 molecules (e.g. the cationic lipid-based transfection reagent, Lipofectamine 3000).42 It is important
56
57 to note, however, that all cell lines were > 90% viable when incubated with ≤ 10 µg/mL of ZIF-90
58 NPs for ≤ 7 days.
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18 Figure 7. (A) Percentage of 1x106 cells that remain viable after incubation with increasing
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20 concentrations of ZIF-90 NPs for 72 hours at 37ºC. (B) The percentage of 1x106 cells that remain viable
21 after incubation with 10 µg/mL of ZIF-90 NPs for up to 6 weeks at 37 ºC. Error bars represent the mean
22
23 ± the standard deviation for n = 3.
24
25
26
27
28
29 CONCLUSIONS
30
31 In summary, in the present study we demonstrate that ZIF-90 particles of various sizes and
32
33 shapes (including sub-100 nm NPs) can be synthesized under mild reaction conditions in a facile
34
35 and reproducible manner. The addition of a ternary amine, such as trioctylamine, not only enables
36
the room-temperature synthesis, but also provides a capping effect to limit the NP growth.
37
38 Increasing reaction temperature from 0 to 150 °C results in a systematic size increase from 30 nm to
39
40 about 1 µm. Such a finding of unconventional particle growth effect may offer a novel protocol for
41
42 MOF particle size modulation. Further adaptions of this procedure showed it is possible to isolate
43
44 nano- and micron-sized particles of other ZIFs, including ZIF-7, ZIF-8 and TIF-2. Covalent surface
45 functionalization of ZIF-90 NPs was achieved by imine condensation of the surface aldehyde
46
47 groups of the MOF linker by the hydrazide moiety of the Alexa Fluor® fluorescent dyes. When
48
49 modified with the R8 peptide, ZIF-90 NPs could effectively penetrate cellular membranes and
50
51 localize within cell lysosomes where they were detectable for up to 2 weeks. Upon incubation with
52
various cells, ZIF-90 NPs showed limited cytotoxic effects over a range of concentrations and
53
54 extended incubation periods, demonstrating a high degree of biocompatibility. The exceptional
55
56 stability of ZIF-90 in both the SBF and the SLF media, as well as their long-term intracellular
57
58 persistence, indicate that ZIF-90 may be well-suited for applications where a steady, sustained
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3 release of NP-stabilized cargo is important. The versatility provided by the surface functional group,
4
5 along with finite degradation periods and a high degree of biocompatibility, highlight the potential
6 of ZIF-90 nanostructures to serve as a possible instrument for further use in imaging, drug delivery,
7
8 and other biomedical applications.
9
10
11
12
13 ASSOCIATED CONTENT
14
15 Supporting Information
16
17 Experimental procedures and additional characterization details for ZIF-7, ZIF-8, TIF-2 and ZIF-90
18
particles synthesized under various reaction conditions. This material is available free of charge via
19
20 the Internet at http://pubs.acs.org.
21
22 AUTHOR INFORMATION
23
24
25 Corresponding Authors:
26
27 Vitalie Stavila, vnstavi@sandia.gov.
28
29
Mark D. Allendorf, mdallen@sandia.gov.
30
31 Present Addresses
32 Sandia National Laboratories, 7011 East Avenue, Livermore, CA, 94551-0969, USA
33
34
35 Notes
36 The authors declare no competing financial interests.
37
38
39 ACKNOWLEDGMENTS
40 This work was supported by the Sandia Laboratory Directed Research and Development Program.
41
42 Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia
43
44 Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department
45
46 of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000. The
47 authors thank Dr. Christopher A. Lino for helpful discussions.
48
49
50 ____________________________________________
51
52
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