Glucose-Responsive Metal-Organic-Framework Nanoparticles Act as

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Glucose-Responsive Metal-Organic-Framework Nanoparticles Act as “Smart” Sense-and-Treat Carriers Wei-Hai Chen, Guo-Feng Luo, Margarita Vázquez-González, Rémi Cazelles, Yang Sung Sohn, Rachel Nechushtai, Yossi Mandel, and Itamar Willner ACS Nano, Just Accepted Manuscript • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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Glucose-Responsive Metal-Organic-Framework Nanoparticles Act as “Smart” Sense-and-Treat Carriers Wei-Hai Chen,1 Guo-Feng Luo,1 Margarita Vázquez-González,1 Rémi Cazelles,1 Yang Sung Sohn,2 Rachel Nechushtai,2 Yossi Mandel,3 and Itamar Willner1,*

1

Institute of Chemistry, Center for Nanoscience and Nanotechnology, The Hebrew University of

Jerusalem, Jerusalem 91904, Israel 2

Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

3

School of Optometry and Vision Science, Faculty of Life Sciences, Bar-Ilan University, RamatGan, 5290002, Israel

* To whom correspondence should be addressed. Tel: 972-2-6585272. Fax: 972-2-6527715. E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT: Zeolitic Zn2+-imidazolate crosslinked framework nanoparticles, ZIF-8 NMOFs, are used as “smart” glucose-responsive carriers for the controlled release of drugs. The ZIF-8 NMOFs are loaded with the respective drug and glucose oxidase (GOx), and the GOx-mediated aerobic oxidation of glucose yields gluconic acid and H2O2. The acidification of the NMOFs microenvironment leads to the degradation of the nanoparticles and the release of the loaded drugs. In one sense-and-treat system GOx and insulin are loaded in the NMOFs. In the presence of glucose, the nanoparticles are unlocked, resulting in the release of insulin. The release of insulin is controlled by the concentration of glucose. In the second sense-and-treat system the NMOFs are loaded with the anti-vascular endothelial growth factor aptamer (VEGF aptamer) and GOx. In the presence of glucose, the ZIF-8 NMOFs are degraded, leading to the release of the VEGF aptamer that acts as a potential inhibitor of the angiogenetic regeneration of blood vessels by VEGF. As calcination of the VEGF-generated blood vessels leads to blindness of diabetic patients, the functional NMOFs might act as a “smart” materials for the treatment of macular diseases. The potential cytotoxicity of the NMOFs originated from the GOx-generated H2O2 is resolved by the co-immobilization of the H2O2-scavanger catalase in the NMOFs.

KEYWORDS: aptamer; insulin; VEGF; nanomedicine; diabetes; macular diseases


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Metal-organic frameworks (MOFs) represent a broad class of porous materials that attract substantial research interest in recent years.1-3 Different applications of MOFs include their use as porous matrices for the storage of gases,4,5 carriers of particles for catalysis,6,7 functionalization of MOFs with metal-ion/ligand complexes for catalysis,8,9 the use of MOFs for sensing,10-12 and their application for improving fuel cells performance.13-15 Special efforts are directed to the use of MOFs as drug delivery vehicles and controlled release systems.16-19 Different internal and external triggers have been used to release drugs entrapped in the MOFs matrices, and these include pH,20,21 temperature,22 light,23 reactive oxygen species (ROS),24,25 and chemical agents.26,27 Additional efforts are focus on the miniaturization of the MOFs into nanoparticle configurations, NMOFs. Besides the higher surface area and increased loading capacities per unit weight of nanoparticles, their enhanced suspendability may improve intravenous invasive treatment with minimal clotting or arterial deposition phenomena. In this context, nucleic acid-modified metal-organic framework nanoparticles provide promising stimuli-responsive platform for drug delivery and controlled release. Different nucleic acid structures were used to gate the controlled release of drug-loaded NMOFs, and different triggers such as pH,28 aptamer-ligand complexes29,30 or DNAzymes31 were used to unlock the NMOFs and release the drugs. One interesting biomaterial-NMOFs hybrid system includes the encapsulation of the biomaterials in the NMOFs structure within the process of formation of the NMOFs (one-pot synthesis). In these systems, the biomaterials are coated by nanodomains of the NMOFs, resulting in crosslinked NMOF-biomaterial matrices. Different enzymes were encapsulated in NMOFs matrices and their use as enzyme carriers for biocatalytic transformations32,33 or sensors34,35 were reported.


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An important subclass of hybrid NMOFs that can be loaded with nanoparticles or biomacromolecules includes zeolitic imidazolate frameworks (ZIFs) that reveal thermal and hydrothermal stabilities.36 ZIFs were used as metal nanoparticle carriers,37,38 drug carriers,39,40 enzyme encapsulation matrices41 and gas separation materials.42 Specifically, ZIF-8 is a biocompatible NMOF consisting of Zn2+ ions and 2-methylimidazolate ligand as bridging units.43,44 The ZIF-8 is stable under neutral physiological conditions but it is degraded under acidic conditions. This feature of ZIF-8 was used to apply the doxorubicin-loaded ZIF-8 as a pHresponsive carrier for the controlled release of the anti-cancer drug doxorubicin in cancer cells, exhibiting acidic microenvironments.45 The development of “smart” glucose-responsive carriers attracts substantial research efforts for the treatment of diabetic patients. Specifically, the development of insulin-loaded carriers that respond reversibly to glucose concentrations is of interest as a means of autonomous treatment of diabetic patients.46-49 For example, microgels loaded with glucose oxidase and insulin were used as functional glucose-responsive matrices for the release of insulin. The glucose oxidasecatalyzed oxidation of glucose yields gluconic acid that protonated the microgel matrices. This process led to the swelling of the carrier and the release of insulin.50 By an alternative approach, boronic acid modified gels were used as a glucose triggered self-regulated matrix for the release of insulin. The binding of glucose to the boronic acid functionalities yield charged boronate ester residues that resulted in the swelling of the hydrogel and release of insulin.51 Similarly, glucose accumulated in the eyes of the diabetic patients leads to macular diseases that, ultimately, leads to blindness originated from the calcination of blood vessels and their angiogenetic regeneration by the vascular endothelial growth factor (VEGF) protein. The concept of “aptamer-based therapies” whereby the binding of aptamers to proteins inhibit the activities of harmful proteins


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attracts recent research efforts.52 Accordingly, the enhanced VEGF-induced generation of blood vessels in cancer cells tissues, and the high levels of glucose in cancer cells, due to enhanced metabolism, may provide opportunities to design glucose-responsive NMOFs that release the anti-VEGF aptamer as inhibitor of VEGF. In the present study we apply ZIF-8 NMOFs as glucose-responsive carriers for the release of insulin or of the VEGF aptamer that inhibits angiogenesis via binding to the VEGF protein. We incorporate insulin or the VEGF aptamer and the enzyme glucose oxidase as biocatalyst into the ZIF-8 NMOFs. The GOx-catalyzed aerobic oxidation of glucose leads to the formation of gluconic acid. The local acidification of the NMOFs is then anticipated to degrade the ZIF-8, leading to the release of the loads. As the acidity in the NMOFs is controlled by the concentration of glucose, gated nanoparticle devices for the controlled release of the drugs may be envisaged.

RESULTS AND DISCUSSION In the first step, insulin-loaded ZIF-8 NMOFs were synthesized by mixing 2methylimidazole and Zn2+ in the presence of FITC-modified insulin (the fluorescence label was introduced to probe subsequently the release of insulin from the NMOFs), Figure S1. The resulting NMOFs revealed rhombic dodecahedral structures, ca. 300-350 nm, Figure S2(A), and the loading with FITC-labeled insulin corresponded to 76.2 µg/mg NMOFs. The insulin-loaded ZIF-8 NMOFs are pH-sensitive. Figure S2(B) shows the scanning electron microscopy (SEM) image of the NMOFs treated at pH = 5 for a time-interval of 30 minutes. The particles are corroded and reveal broken faces. It should be noted that after a time-interval of two hours the ZIF-8 nanoparticles are highly corroded and appear as aggregates in the SEM image, Figure S3,


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and the nanoparticles completely dissolve after a time-interval of two days at pH = 5. Figure S2(C) shows the time-dependent release of the FITC-modified insulin at different pH values. While at pH = 7.4 the insulin-loaded NMOFs are locked and no insulin is released, at pH = 6.0 and 5.0 unlocking of the NMOFs is observed. The release of insulin is faster as the pH is more acidic, suggesting that the release of insulin could be controlled by the active balancing of the pH at the NMOFs. The regulation of the pH at the NMOFs was achieved by the co-immobilization of glucose oxidase, GOx, and insulin in the NMOFs, and the use of glucose as activator for controlling the local pH at the NMOFs, Figure 1(A). The GOx and insulin were integrated in the NMOFs by the in situ synthesis of the ZIF-8 NMOFs in the presence of the two proteins. In the presence of glucose, the aerobic oxidation of the NMOFs proceeds, leading to the formation of gluconic acid. The local acidification of the NMOFs surface leads to the degradation of the NMOFs and to the release of insulin. The loading of GOx and insulin in the NMOFs was evaluated and corresponds to 15.6 µg/mg and 52.3 µg/mg, respectively, (see experimental section and supporting material). The crystallinity of the NMOFs with the entrapped GOx and insulin and analogues NMOFs without loading were examined by X-ray diffraction (XRD) measurements, Figure S4. The crystal structure of the NMOFs with encapsulated GOx and insulin overlap the XRD spectra of the NMOFs lacking the proteins and the reported spectra that correspond to the space group I43m. The similar crystallinity of the ZIF-8 NMOFs in the absence of the protein loads and in the presence of GOx/insulin is attributed to the formation of ZIF-8 nanocrystals surrounded by the respective proteins. This phenomenon was previously reported for other proteins entrapped in the ZIF-8 matrix.34,41 The confocal microscopy images of the ZIF-8 NMOFs loaded with the FITCmodified insulin (green) and the coumarin-modified GOx (blue) are shown in Figure 1(B), panel


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I and panel II, respectively. The bright field confocal microscopy image and merged image of the NMOFs are displayed in Figure 1(B), panel III and panel IV. The overlaped turquoise color is consistant with the internal integration of the two proteins in the NMOFs. Figure 1(C) I depicts the SEM image of the NMOFs loaded with GOx and insulin. Rhombic dodecahedral nanoparticles, ca. 300-350 nm, are generated by this procedure. Figure 1(C) II shows the SEM image of the NMOFs after treatment with glucose, 50 mM, for one hour, while image III shows the control image where the NMOFs were treated with the buffer solution, in the absence of glucose. Clearly, in the presence of glucose the NMOFs are degraded. Further control experiments revealed that the corrosion of the NMOFs occurred only in the presence of glucose and other saccharide, e.g., galactose, mannose or sucrose did not affect the NMOFs. These results imply that the corrosion of the NMOFs is specific to the GOx/glucose couple, consistent with the local pH changes occurring upon the aerobic biocatalyzed oxidation of glucose. Note that the high porosity of the NMOFs enable the permeation of the low-molecular-weight glucose into the NMOFs, thus allowing the GOx-catalyzed aerobic oxidation of glucose. Figure 1(D) and (E) depict the GOx/glucose-stimulated insulin release features from the NMOFs. Figure 1(D) shows the fluorescence spectra of the released FITC-labeled insulin after treatment with different concentrations of glucose for a fixed time interval of 60 minutes. As the concentration of glucose increases, the release of the insulin is enhanced, consistent with the accelerated, pH-stimulated degradation of the NMOFs. Figure 1(E) shows the time-dependent fluorescence changes upon the treatment of the FITC-labeled insulin/GOx-loaded NMOFs with variable concentration of glucose. As the glucose concentrations increase, the rates of release of insulin are enhanced. It should be noted that while the release of the FITC-labeled insulin from the ZIF-8 NMOFs, at pH = 5, is complete after ca. one hour, the GOx-stimulated release of the FITC-labeled insulin (as


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well as of the Cy3-labeled VEGF, vide infra) proceed, even after a time-interval of two hours. This suggests that the GOx-catalyzed aerobic oxidation of glucose yields higher pH values (ca. 6.0-6.5) in the NMOFs containers. Figure S5 shows the pH changes induced by the GOxmediated oxidation of variable concentrations of glucose. As the concentration of glucose increases the pH of the solution drops, consistent with the formation of gluconic acid. Control experiments revealed that no release of the FITC-modified insulin from NMOFs that lack the GOx occurs in the presence of added glucose, Figure S6. Also, the release of insulin from the insulin/GOx loaded NMOFs is specific to the glucose trigger, and other saccharides do not affect the release of insulin, Figure 1(F). These control experiments indicate that the oxidation of glucose by GOx provides the biocatalytic trigger for the release of insulin. The observation that the release of insulin from the NMOFs is regulated by the concentration of glucose suggests that by the optimization of the system, a “smart” controlled delivery system of insulin for diabetic treatment could be developed. To support the potential application of the GOx-loaded ZIF-8 NMOFs as “smart” nanoparticles for the controlled release of insulin, we examined the switchable release of insulin from the NMOFs at high and low concentrations of glucose encounter with diabetic patients, Figure 2. In this experiment, the FITC-labeled insulin/GOx loaded NMOFs were subjected to the elevated concentration of glucose (15 mM, 270 mg/dL) and lower concentrations of glucose (5 mM, 90 mg/dL). Evidently, at high concentrations of glucose, the effective release of insulin is observed from the corroded domains of the NMOFs. Lowering the glucose concentration decreases the release of insulin from the NMOFs due to the inhibition of the degradation of the NMOFs. By the cyclic increase and decrease of the glucose concentration, the switchable ON/OFF release of insulin is observed.


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A further aspect to consider rests on the evaluation of the cytotoxicity of the GOx-loaded NMOFs. To address this issue, MCF-10A epithelial breast cells were subjected to empty ZIF-8 NMOFs and insulin/GOx-loaded ZIF-8 NMOFs, Figure 3(A). No cytotoxic effect of the NMOFs on the MCF-10A cells could be detected within a time interval of ten days. In addition, the GOxstimulated degradation of the ZIF-8 NMOFs, originated from the aerobic GOx-catalyzed oxidation of glucose, and the accompanying pH changes introduce a further issue that needs to be addressed. The GOx-catalyzed oxidation of glucose yields gluconic acid and H2O2. The latter product might act as an undesired ROS generating promoter. Accordingly, we decide to integrate insulin/GOx/catalase into NMOFs carriers. In these nanoparticles, the co-immobilized catalase catalyzes the decomposition of H2O2 that is generated by the aerobic oxidation of glucose. Figure 3(B), curve (a), shows the time-dependent formation of H2O2 in the bulk solution (the generated H2O2 acts as substrate for horseradish peroxidase that catalyzes the oxidation of Amplex-Red to the fluorescent Resorufin product) upon the treatment of the insulin/GOx-loaded NMOFs in the presence of glucose 50 mM. Treatment of the insulin/GOx/catalase-loaded ZIF-8 NMOFs with glucose under similar conditions resulted in a very low concentration of H2O2 in the bulk solution (a low fluorescence intensity of Resorufin was detected), Figure 3(B), curve (b), implying that the catalase, indeed, degraded the accompanying H2O2 product. The incorporation of catalase in the insulin/GOx NMOFs did not affect the release of the insulin load, as it is evident from Figure 3(C). In the next step, the anti-vascular endothelial growth factor, VEGF aptamer, and GOx were immobilized in the ZIF-8 NMOFs as a potential “smart” drug carrier for macular diseases. The development of aptamer-based therapies attracts recent research efforts. Specifically, elevated concentrations of glucose in the eyes of diabetic patients result in blindness due to the clotting of


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blood vessels, a process that is accelerated by the VEGF-induced angiogenesis and formation of new blood vessels. Accordingly, substantial research efforts are directed toward the development of VEGF inhibition mechanisms. The availability of an anti-VEGF aptamer and the glucosestimulated dissolution of the GOx-loaded ZIF-8 NMOFs suggest that the incorporation of GOx and the VEGF aptamer in the ZIF-8 NMOFs could yield a “smart carrier” for the controlled release of the VEGF aptamer, and the inhibition of angiogenesis in macular diabetic-related diseases. Figure 4(A) schematically depicts the synthesis of GOx/Cy3-labeled VEGF aptamerloaded ZIF-8 NMOFs and outlines the release mechanism of the aptamer. The in situ formation of the ZIF-8 NMOFs using 2-methylimidazole and Zn2+, in the presence of the Cy3-fluorophorelabeled VEGF (Cy3-VEGF) aptamer and GOx, yielded the VEGF aptamer/GOx functionalized NMOFs. The aptamer was functionalized with the Cy3 fluorophore to allow quantitative evaluation of the aptamer loading, and to provide a means to probe the kinetics of the release of the aptamer. The loading of the NMOFs with GOx and Cy3-VEGF aptamer corresponded to 17.8 µg/mg and 50.2 µg/mg, respectively (for experimental details see experimental section). The loading of the ZIF-8 NMOFs with the VEGF aptamer and GOx was confirmed by confocal microscopy imaging. The ZIF-8 NMOFs loaded with the Cy3-modified VEGF aptamer (red) and the coumarin-functionalized GOx (blue) are shown in Figure 4(B), panel I and panel II. The bright field confocal microscopy image and merged image of the NMOFs are displayed in Figure 4(B), panel III and panel IV. The overlaped pink color is consistant with the integration of the aptamer and GOx in the NMOFs. The mechanism of release of the VEGF aptamer, in the presence of glucose, involves the GOx-mediated biocatalyzed oxidation of glucose, and the pHinduced dissolution of the NMOFs by the biocatalytically-generated gluconic acid. The corrosion and separation of the NMOFs under acidic conditions lead to the release of the aptamer. Figure


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S7 shows the SEM images of the resulting GOx/Cy3-VEGF aptamer loaded NMOFs. In addition, Figure S8 shows the release features of NMOFs loaded with Cy3-VEGF aptamer (lacking GOx) at different pH values. Evidently, at pH = 7.4 the VEGF aptamer is not released from the NMOFs. Subjecting the aptamer-loaded NMOFs to pH = 6.0 and pH = 5.0 results in the release of the aptamer, and the aptamer release is enhanced as the pH is lower. Also, no release of the VEGF aptamer was detected upon treatment of the aptamer-loaded NMOFs (that lack GOx) with glucose, implying that glucose itself (in the absence of GOx) does not affect the release of the aptamer, Figure S9. These control experiments demonstrate that the release of the VEGF aptamer from the NMOFs proceeds under acidic conditions and suggest that the GOx/Cy3-VEGF aptamer-loaded NMOFs could be triggered by glucose to release the aptamer through the local acidification of the NMOFs and their pH-induced degradation. Figure 4(C) shows the fluorescence spectra of the released Cy3-VEGF aptamer upon subjecting the GOx/Cy3-VEGF aptamer-loaded NMOFs to variable concentrations of glucose for a fixed timeinterval of 60 minutes. Figure 4(D) shows the time-dependent release of the Cy3-VEGF aptamer at different concentrations of glucose. Evidently, the release of the Cy3-VEGF aptamer is faster as the concentration of glucose is higher, consistent with the increased local acidity at the NMOFs as the concentration of glucose increases. That is, as the concentrations of glucose are higher, the local acidification of the NMOFs, via the GOx-catalyzed oxidation of glucose and the formation of gluconic acid increases, resulting in the enhanced release of the VEGF aptamer. Figure 4(E) shows the selectivity of the glucose-induced release of Cy3-VEGF aptamer from the NMOFs. Clearly, other saccharides do not affect the release of the aptamer. These results suggest that the GOx/Cy3-VEGF aptamer-loaded NMOFs may, indeed, act as “smart” materials for the glucose-triggered inhibition of VEGF-induced angiogenesis in macular diseases. The switchable,


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glucose-stimulated release of the fluorescent VEGF aptamer is demonstrated in Figure 4(F). At glucose concentration (15 mM) expected to cause macular damages, effective release of the VEGF aptamer is observed whereas lower concentrations of glucose (5 mM) decrease significantly the release of the aptamer load.

CONCLUSIONS In conclusion, the present study has introduced glucose oxidase (GOx)-loaded ZIF-8 NMOFs as versatile glucose-responsive nano-composites that may act as autonomous, senseand-treat vehicles for controlling diabetes or macular diseases. The pH-stimulated degradation of the ZIF-8 NMOFs was extended by applying GOx-loaded NMOFs as biocatalytic assemblies catalyzing the aerobic oxidation of glucose to gluconic acid and the dissolution of the NMOFs by the local acidification of the nanoparticles. The study demonstrated the synthesis of glucoseresponsive insulin/GOx- and VEGF aptamer/GOx-loaded ZIF-8 NMOFs and highlighted the glucose-triggered release of insulin or VEGF aptamer at physiological concentrations, relevant for controlling diabetics or macular diseases. While the results represent potential applications in future nanomedicine, further experiments optimizing the doses of the released drugs, and particularly, the application of the NMOFs in biologically-relevant environments, e.g., animals, are essential.

MATERIALS AND METHODS Materials: Zinc acetate, 2-methylimidazole, insulin, glucose oxidase (GOx), catalase, horseradish peroxidase (HRP), 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS), fluorescein isothiocyanate (FITC), 7-hydroxycoumarin-3-carboxylic acid N-succinimidyl ester,


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glucose, galactose, sucrose and β-lactose were purchased from Sigma-Aldrich. Amplex-Red was purchased from Life Technologies Corporation. Ultrapure water was obtained by a NANOpure Diamond

instrument

(Barnstead

International,

Dubuque,

IA,

USA).

5'-Cy3-

TGTGGGGGTGGACGGGCCGGGTAGA-3' (Cy3-VEGF) aptamer was purchased from Integrated DNA Technologies Inc. (Coralville, IA). FITC-modified insulin was prepared according to the previously reported method.53 Similarly, the coumarin-labeled GOx was also synthesized. Encapsulation of FITC-modified insulin in metal-organic framework nanoparticles: Briefly, 1 mL of 2-methylimidazole (1.4 M) containing 500 µg FITC-modified insulin was stirred gently at room temperature. Afterward, 1 mL of zinc acetate (20 mM) was added quickly and the mixture was stirred overnight. Then the obtained opaque solution was centrifuged and washed by deionized water for several times. The encapsulation efficiency of FITC-modified insulin in NMOFs was determined by fluorescent spectrophotometry using a calibration curve of FITCmodified insulin. Encapsulation of FITC-modified insulin and GOx in metal-organic framework nanoparticles: Briefly, 1 mL of 2-methylimidazole (1.4 M) containing 500 µg FITC-modified insulin and 200 µg GOx were stirred gently at room temperature. Afterward, 1 mL of zinc acetate (20 mM) was added quickly and the mixture was stirred overnight. Then the obtained opaque solution was centrifuged and washed by deionized water for several times. The loadings of insulin and GOx in the NMOFs were evaluated by labeling insulin with FITC and GOx with coumarin. The ZIF-8 NMOFs were prepared in the presence of known concentrations of the FITC-modified insulin and coumarin-labeled GOx. After the ZIF-8 synthesis with entrapped FITC-labeled insulin/coumarin-labeled GOx was completed, the NMOFs were separated, washed, and the


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residual fluorescence intensities of FITC-labeled insulin and coumarin-labeled GOx were evaluated. Using appropriate calibration curves relating to the fluorescence intensities of different concentrations FITC-labeled insulin or coumarin-labeled GOx, the total contents of the entrapped proteins in the NMOFs were evaluated, Figure S10 and Figure S11. It should be noted that the activity of GOx in the NMOFs is unaffected during the process of entrapment of GOx in the NMOFs. We evaluated the activity of the GOx-modified ZIF-8 NMOFs using the GOx/HRP catalyzed Amplex-Red to Resorufin assay. Knowing the content of GOx entrapped in the ZIF-8 NMOFs and using an appropriate calibration curve relating to the fluorescence intensities of Resorufin generated by different concentrations of GOx (fixed concentration of HRP), we estimated the activity of entrapped GOx in the NMOFs after the synthesis to the identical content of native GOx. We find that the activity of entrapped GOx was unchanged, as compared to the native enzyme, see Figure S12. Encapsulation of FITC-modified insulin, GOx and catalase in metal-organic framework nanoparticles: Briefly, 1 mL of 2-methylimidazole (1.4 M) containing 500 µg FITC-modified insulin, 200 µg GOx and 200 µg catalase were stirred gently at room temperature. Afterward, 1 mL of zinc acetate (20 mM) was added quickly and the mixture was stirred overnight. The obtained opaque solution was centrifuged and washed by deionized water for several times to obtain the NMOFs loaded with FITC-modified insulin, GOx and catalase. Encapsulation of Cy3-VEGF aptamer in metal-organic framework nanoparticles: Briefly, 1 mL of 2-methylimidazole (1.4 M) containing 500 µg Cy3-VEGF aptamer (60 nmol) was stirred gently at room temperature. Afterward, 1 mL of zinc acetate (20 mM) was added quickly and the mixture was stirred overnight. Then the obtained opaque solution was centrifuged and washed by deionized water for several times. The loading efficiency of Cy3-VEGF aptamer in NMOFs was


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evaluated by fluorescent spectrophotometry using a calibration curve of Cy3-VEGF aptamer, see Figure S13. Encapsulation of Cy3-VEGF aptamer and GOx in metal-organic framework nanoparticles: Briefly, 1 mL of 2-methylimidazole (1.4 M) containing 500 µg Cy3-VEGF aptamer (60 nmol) and 200 µg GOx were stirred gently at room temperature. Afterward, 1 mL of zinc acetate (20 mM) was added quickly and the mixture was stirred overnight. Then the obtained opaque solution was centrifuged and washed by deionized water for several times. The loading efficiency of Cy3-VEGF aptamer in NMOFs was determined by fluorescent spectrophotometry using a calibration curve of Cy3-VEGF aptamer. To evaluate the loading efficiency of GOx, we used the coumarin-labeled GOx to synthesize the NMOFs. And the encapsulation efficiency of of GOx was determined using appropiate calibration curve of coumarin-labeled GOx. Evaluation of the glucose sensing ability of FITC-modified insulin/GOx-loaded ZIF-8 NMOFs and monitoring the pH changes by a pH-sensitive dye of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS): The fluorescence intensities of HPTS (514-nm emission; 406-nm (I406) and 460-nm (I460) excitation) are strongly dependent on the degree of ionization of the 8hydroxyl group (pKa = 7.2) and hence on the pH value of the medium. The fluorescence intensities at I406 and I460 increase linearly with increasing HPTS concentration. As displayed in Figure S5, the titration curve of HPTS (0.5 µM) at different pH values was measured. To determine the glucose-sensing ability of FITC-modified insulin/GOx-loaded ZIF-8 NMOFs, HPTS (0.5 µM) was added together with FITC-modified insulin/GOx-loaded ZIF-8 NMOFs and glucose to act as the pH probe for measuring the pH variation in the bulk solution, induced by GOx-triggered glucose oxidation and the generated gluconic acid.


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pH-induced or glucose-triggered release of FITC-modified insulin from the NMOFs: Experiments were performed using the solution of protein-loaded NMOFs, concentration corresponding to 1 mg/mL for the FITC-modified insulin-loaded NMOFs and FITC-modified insulin- and GOx-loaded NMOFs. The NMOFs solutions (1 mg/mL) were treated with different pH buffers or variable concentrations of glucose. After incubation, the respective sample solutions were centrifuged at 6000 rpm for 10 min to precipitate the residual NMOFs, and the fluorescence of the released loads in the supernatant solution was measured by a Cary Eclipse Fluorescence Spectrophotometer (Varian Inc.). pH-induced or glucose-triggered release of Cy3-VEGF aptamer from the NMOFs: Experiments were performed using the solution of protein-loaded NMOFs, concentration corresponding to 1 mg/mL for the Cy3-VEGF aptamer-loaded NMOFs and Cy3-VEGF aptamerand GOx-loaded NMOFs. The NMOFs solutions (1 mg/mL) were treated with different pH buffers or variable concentrations of glucose. After incubation, the respective sample solutions were centrifuged at 6000 rpm for 10 min to precipitate the residual NMOFs, and the fluorescence of the released loads in the supernatant solution was measured by a Cary Eclipse Fluorescence Spectrophotometer (Varian Inc.). Cell viability experiments: Cell viability was assayed after incubation of the ZIF-8 NMOFs with or insulin/GOx-loaded ZIF-8 NMOFs in MCF-10A cells planted at a density of 1.8 × 105 cells per well in 24-well plates (1.9 cm2 growth area). After seeding of the cells overnight, cells were incubated with the ZIF-8 NMOFs (100 µg/mL) or insulin/GOx-loaded ZIF-8 NMOFs (100 µg/mL) for 6 h. The cells without any treatment were used as the control. Following intensive washing, the cells were further incubated for 1 day, 3 days, 5 days or 10 days with growth medium and the cell viability was determined with the fluorescent redox probe, Alamar Blue.


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The fluorescence of Alamar Blue was recorded on a plate-reader (Tecan Safire) after 1 h of incubation at 37 °C (λex = 560 nm; λem = 590 nm).

ASSOCIATED CONTENT The authors declare no competing financial interest. Supporting Information. Scheme of the synthesis of FITC-modified insulin-loaded NMOF, SEM image of FITC-modified insulin-loaded NMOFs and SEM image of FITC-modified insulin-loaded NMOFs after treatment at pH = 5.0, the time-dependent release of insulin from FITC-modified insulin-loaded NMOFs at different pH values, powder X-ray diffraction (PXRD) pattern of different NMOFs, pH changes induced by FITC-modified insulin/GOx-loaded ZIF-8 NMOFs, SEM image of Cy3-VEGF aptamer/GOx-loaded ZIF-8 NMOFs, the VEGF aptamer release properties from Cy3-VEGF aptamer-loaded ZIF-8 NMOFs at different pH values, and the results of other control experiments are provided. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Corresponding Author: [email protected] ACKNOWLEDGMENT This research is supported by the Israel Science Foundation. REFERENCES 1. Wang, Z.; Cohen, S. M. Postsynthetic Modification of Metal-Organic Frameworks. Chem. Soc. Rev. 2009, 38, 1315-1329.


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Figure 1. (A) Schematic synthesis of the insulin/GOx-loaded ZIF-8 NMOFs and the pH-induced degradation of the NMOFs through the GOx-catalysed oxidation of glucose. (B) Confocal microscopy images of the FITC-modified insulin- and coumarin-functionalized GOx-loaded ZIF-8 NMOFs (I and II, respectively) and the bright field and merged image of the loaded NMOFs (III and IV, respectively). (C) SEM images corresponding to: (I) The insulin/GOxloaded NMOFs. (II) The NMOFs after treatment with glucose (50 mM) for one hour. (III) Subjecting the NMOFs to a buffer solution pH = 7.4 for a time interval of two days. (D) Fluorescence spectra corresponding to the released FITC-labeled insulin upon subjecting the insulin/GOx-loaded NMOFs to different concentrations of glucose for a fixed time-interval of one hour: (a) 0 mM, (b) 1 mM, (c) 5 mM, (d) 10 mM, (e) 50 mM. (E) Time-dependent fluorescence changes upon releasing the FITC-labeled insulin from the insulin/GOx-loaded NMOFs in the presence of variable concentrations of glucose: (a) 0 mM, (b) 5 mM, (c) 10 mM, (d) 20 mM, (e) 50 mM. (F) Selectivity study demonstrating the selective glucose-induced release of insulin from the NMOFs. Figure shows the fluorescence spectra of the released FITC-labeled insulin form the NMOFs, after a time-interval of one hour, in the presence of: (a) add glucose, 50


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mM, (b) added galactose, 50 mM, (c) add β-lactose, 50 mM, (d) added sucrose, 50 mM, (e) NMOFs subjected to pure buffer solution.


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Figure 2. Switchable time-dependent release of insulin from the insulin/GOx-loaded ZIF-8 NMOFs in the presence of high concentration of glucose (15 mM, blue) and low concentration of glucose (5 mM, yellow).


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Figure 3. (A) Cytotoxicity of the unloaded ZIF-8 NMOFs (red) and insulin/GOx-loaded ZIF-8 NMOFs (green) toward epithelial MCF-10A cells. (B) Time-dependent fluorescence changes generated by the HRP catalyzed oxidation of Amplex-Red to Resorufin by the H2O2 generated from the insulin/GOx-loaded NMOFs (a) and the insulin/GOx/Catalase-loaded NMOFs (b) in the presence of glucose, 50 mM. The HRP is added as an auxiliary catalyst that probes the H2O2generated by the GOx-loaded NMOFs. (C) Time-dependent release of the FITC-labeled insulin from the insulin/GOx-loaded NMOFs (a) and from the insulin/GOx/Catalase-loaded NMOFs (b), in the presence of glucose, 50 mM.


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Figure 4. (A) The synthesis of the GOx/Cy3-labeled VEGF aptamer-loaded ZIF-8 NMOFs and the schematic glucose-driven release of the VEGF aptamer from the NMOFs due to the corrosion of the NMOFs by the local acid conditions generated by the GOx-catalyzed aerobic oxidation of glucose to gluconic acid. (B) Confocal microscopy images of the Cy3-modified VEGF aptamerand coumarin-functionalized GOx-loaded ZIF-8 NMOFs (I and II, respectively) and the bright field and merged image of the loaded NMOFs (III and IV, respectively). (C) Fluorescence spectra corresponding to the Cy3-labeled VEGF aptamer release from the GOx/Cy3-VEGF aptamer-loaded NMOFs in the presence of variable concentrations of glucose: (a) 0 mM, (b) 1 mM, (c) 5 mM, (d) 10 mM, (e) 50 mM. The particles were exposed to the respective concentrations of glucose for a time-interval of one hour. (D) Time-dependent fluorescence changes of the Cy3-labeled VEGF aptamer upon subjecting the GOx/Cy3-labeled VEGF aptamer-loaded ZIF-8 NMOFs to variable concentrations of glucose: (a) 0 mM, (b) 10 mM, (c) 50 mM. (E) The selective release of the Cy3-labeled VEGF aptamer in the presence of glucose. Fluorescence spectra of the released Cy3-labeled VEGF aptamer from the GOx/Cy3-labeled


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VEGF aptamer-loaded NMOFs after a time-interval of one hour in the presence of 50 mM glucose (a) and in the absence of glucose (b). The curves (c), (d) and (e) correspond to the fluorescence spectra of the released Cy3-labeled VEGF aptamer upon treating the NMOFs with galactose (50 mM), sucrose (50 mM) and β-lactose (50 mM), respectively. (F) Time-dependent fluorescence changes upon the switchable release of the Cy3-labeled VEGF aptamer from the GOx/VEGF-loaded ZIF-8 NMOFs in the presence of high concentration of glucose (15 mM, blue) and low glucose concentration (5 mM, yellow).


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Table of Contents Graphic


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Glucose-Responsive Metal-Organic-Framework Nanoparticles Act as

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