Spatiotemporal Control of Supramolecular Self-Assembly and

Mar 2, 2017 - ... College of Life Sciences, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tian...
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Spatiotemporal Control of Supramolecular Self-Assembly and Function Jie Zhan,†,§ Yanbin Cai,† Shenglu Ji,† Shuangshuang He,§ Yi Cao,*,‡ Dan Ding,† Ling Wang,§ and Zhimou Yang*,†,§ †

State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, People’s Republic of China ‡ College of Physics, Nanjing University, Nanjing 210093, People’s Republic of China § College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Design, Nankai University, Tianjin 300071, People’s Republic of China S Supporting Information *

ABSTRACT: The enzyme-triggered self-assembly of peptides has flourished in controlling the self-assembly kinetics and producing nanostructures that are typically inaccessible by conventional self-assembly pathways. However, the diffusion and nanoscale chemical gradient of self-assembling peptides generated by the enzyme also significantly affect the outcome of selfassembly, which has not been reported yet. In this work, we demonstrated for the first time a spatiotemporal control of enzymetriggered peptide self-assembly. By simply adjusting the temperature, we could change both the catalytic activity of the enzyme of phosphatase and their aggregation states. The strategy kinetically controls the production rate of self-assembling peptides and spatially controls their distribution in the system, leading to the formation of nanoparticles at 37 °C and nanofibers at 4 °C. The nanofibers showed ∼10 times higher cellular uptake by 3T3 cells than the nanoparticles, thanks to their higher stability and more ordered structures. Using such spatiotemporal control, we could prepare optimized nanoprobes with low background fluorescence, rapid and high cellular uptake, and high sensitivity. We postulate that this strategy would be very useful in general for preparing self-assembled nanomaterials with controllable morphology and function. KEYWORDS: self-assembly, peptide, enzyme, kinetics, diffusion, molecular probe

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which have shown great promise in healthcare.22−29 A selfassembly process can be considered as a reaction navigating through a multidimensional free energy landscape. Recent studies have demonstrated that the reaction pathway to prepare supramolecular materials is crucial to the outcome of morphology and, more importantly, the function of selfassembled materials.30 However, most of these studies mainly focused on controlling the self-assembling pathways by varying the reaction kinetics at the time domain.31−33 Yet, self-assembly

INTRODUCTION

Self-assembly has been demonstrated as a powerful strategy to prepare supramolecular materials from individual molecules.1−7 Similar to the ubiquitous supramolecular structures in nature (e.g., cell membrane, tubulin microtube, etc.), individual molecules as building blocks can self-assemble into functional materials beyond molecular scale.8−14 Among the building blocks used to prepare supramolecular materials, peptides and peptide analogues have emerged as very promising ones to produce biofunctional materials due to their ease of design and synthesis, biocompatibility and bioactivity.15−21 Assisted by selfassembly, they can form many kinds of nanostructures including nanofibers, nanotubes, nanoparticles, and nanosheets, © XXXX American Chemical Society

Received: January 16, 2017 Accepted: March 2, 2017 Published: March 2, 2017 A

DOI: 10.1021/acsami.7b00784 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Scheme 1. Enzyme-Triggered Supramolecular Self-Assembly Could Be Spatiotemporally Controlled by Both Kinetics and Diffusiona

a (A) Conceptually illustration of self-assembly under spatiotemporal control. (B) Realizing spatiotemporal control in an enzyme-triggered peptide self-assembly system through temperature switch. Enzyme clusters quickly catalyzes the de-phosphorylation reaction (high kinetics) at high temperatures such as 37 °C, leading to a high local concentration of self-assembling peptides at nanoscale and the formation of nanoparticles. Individual enzyme molecule slowly catalyzes the de-phosphorylation reaction (low kinetics) at low temperatures such as 4 °C, or its Brownian movement is significant at high temperatures such as 37 °C. At either condition, self-assembling peptides are evenly distributed in the system, leading to the formation of nanofibers and gels.

controls the self-assembly process. Thus, significant difference in morphology and especially the cellular uptake behavior of enzymatically formed nanomaterials were observed. We also demonstrated that this strategy could be applied for the production of molecular probes with much better performance to detect analytes inside cells.

often involves the redistribution of the building blocks at the spatial domain and thus is also diffusion-controlled. Therefore, it should be possible to control the reaction pathway in both the time and space domain simultaneously (Scheme 1A).34 Among methods to induce the self-assembly of peptides, enzymatic triggeration holds advantages in that it shares similarity to those applied to generate supramolecular structures in biological systems.35,36 Enzyme-triggered formation of nanostructures and hydrogels has therefore been widely applied for cancer cells inhibition,37,38 biomineralization,39,40 detections,41−44 and drug delivery.45,46 It would be natural to expect the heterogeneity and complexity of enzymetriggered molecular self-assembly, given that the perplexing array of time and space scale associated with the process. As such, both kinetics (temporal control) and diffusion (spatial control) will significantly affect the self-assembly of peptides (Scheme 1A). Several recent studies have shown that the kinetic control (temporal control)37,47 or spatial control48,49 is very important to the outcome of peptide self-assembly. However, it remains as a challenge to achieving both spatial and temporal controls (spatiotemporal control) of peptide selfassembly because it is very difficult to generate nanoscale chemical gradients in the presence of significant Brownian movements of both enzyme and peptide molecules at a high reaction kinetics. Here we report a simple strategy to achieve the spatiotemporal control of peptide self-assembly. As shown in Scheme 1B, our strategy utilizes enzyme clusters as the catalyst to reduce its Brownian motion and successfully generate a nanoscale chemical gradient of self-assembling peptide at high kinetics of enzymatic reaction. When enzyme clusters of n individual enzymes are formed, the distance between individual clusters, r, increases by n1/3 and the local concentration of enzyme-modified peptides increases by n*exp(Ea/T1 − Ea/T2), where Ea is the activation energy in the Arrhenius equation and T2 and T1 are the temperatures in which the enzyme form clusters and isolated enzymes, respectively. Clearly, the formation of enzyme clusters alters not only the spatial distribution of the enzymatically produced self-assembling peptides but also their local concentrations. This generates local concentration gradient of the peptides and

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RESULTS AND DISCUSSION Self-Assembly Behavior Control. We started with a peptide derivative of NBD-GDFDFpDY because it could be converted to NBD-GDFDFDY with excellent self-assembling property by the enzyme of phosphatase (Figure 1A), and the Dpeptides would have better stability in biological systems.50 This compound could form clear solution in phosphate buffer saline (PBS, pH = 7.4) solution at the concentration of 1 mg/ mL. We then added the enzyme of phosphatase (1 U/mL) to trigger its transformation. Interestingly, a hydrogel could be obtained at 4 °C whereas a clear solution remained at 37 °C (Figure 1B,C, respectively). The hydrogel and solution were stable for more than 1 month at room temperature (20−25 °C) without obvious changes in appearance. The results in Figure 1D show that more than 98% of NBD-GDFDFpDY had been converted to NBD-GDFDFDY in both the gel and solution by the enzyme in 10 h. However, the kinetics of such conversion at different temperature was dramatically different. The time needed to finish the conversion was less than 1 h at 37 °C, whereas was about 10 h at 4 °C. The transmission electron microscopy (TEM) images revealed nanofibers with the diameter of about 15−20 nm in the gel (Figure 1F) and nanoparticles with the diameter of about 100−150 nm in the solution (Figure 1G). We incubated nanofibers in the gel at 37 °C for 3 days. The gel would not change to a solution, and we still observed nanofibers in the sample (Figure S5A). We also incubated nanoparticles in the solution at 4 °C for 3 days. The solution did not convert to a gel and we observed nanoparticles in the sample (Figure S5B). These observations clearly indicated that they were not reversible. We then monitored the fluorescent property of nanofibers and nanoparticles, as shown in Figure 1E, the nanofiber solution exhibited a higher fluorescence intensity (about 6300 AU at 1 mg/mL) than the B

DOI: 10.1021/acsami.7b00784 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 2. (A) CD spectra show an α-helix conformation of peptide in nanofibers formed at 4 °C and a random conformation of peptide in nanoparticles formed at 37 °C; (B) proposed mode of molecular selfassembly of the peptide at different temperatures, nanofibers show an enhanced cellular uptake by 3T3 cells than nanoparticles; (C) cellular uptake of nanomaterials at different time points and fluorescence confocal images of cells treated with 120 μM of (D) nanofibers and (E) nanoparticles for 2 h (scale bars in panels D and E represent 25 μm). Figure 1. (A) Chemical structures of NBD-GDFDFpDY and NBDGDFDFDY and schematic illustration of enzymatic conversion by phosphatase; optical images of samples formed by treating PBS solution of NBD-GDFDFpDY (1 mg/mL) with 1 U/mL of phosphatase for 12 h; (B) gel formed at 4 °C; (C) clear solution formed at 37 °C; (D) conversion rate of NBD-GDFDFpDY by the phosphatase at different temperatures determined by LC-MS; (E) fluorescence spectra of PBS solutions of 1 mg/mL of NBD-GDFDFpDY treated with 1 U/mL of phosphatase for 12 h at different temperatures; TEM images of (F) gel formed at 4 °C and (G) solution formed at 37 °C.

Different Behavior of Enzyme Molecule at Different Temperature. We hypothesized that the aggregation of enzymes to form clusters at elevated temperature accounted for such distinct self-assembly behaviors. To confirm this, we used dynamic light scattering (DLS) to study the aggregation of enzyme molecules at different temperatures. The results indicated that the phosphatase existed as individual molecules with a size of 5−7 nm at 4 °C, whereas it formed clusters with diameter of 35−50 nm at 37 °C at the concentration of 1 U/ mL (Figure S7) containing ∼30−60 enzymes. The individual enzyme molecule slowly catalyzed the dephosphorylation of NBD-GDFDFpDY in a homogeneous way at 4 °C. The formed NBD-GDFDFDY remained at low concentrations near the enzyme molecule and could diffuse easily from the enzyme. The homogeneously distributed NBD-GDFDFDY self-assembled with each other to form nanofibers (Figure 2B). The nanofibers entangled with each other to form a three-dimensional network of the gel. However, at higher temperature of 37 °C, the enzyme clusters rapidly produced high concentrations of NBDGDFDFDY, which instantly formed nanoparticles around the enzyme clusters before diffusing away (Figure 2B). To confirm further that the formation of enzyme clusters is the key to obtain the peptide nanoparticles, we used a much diluted concentration of enzyme (0.1 U/mL) to trigger the selfassembly of NBD-GDFDFpDY at 37 °C. Under this condition, the enzyme also exhibited as individual molecules because of its low concentration (Figure S8). It took about 10 h to finish the conversion from NBD-GDFDFpDY to NBD-GDFDFDY (Figure S9). Because of the lack of high concentration of selfassembling peptides near the enzyme, the solution turned into a gel formed by nanofibers (Figures S10 and S11). Recent pioneering work from Ulijn group had showed that the clusters of enzyme with difference sizes would lead to nanofibers with different diameters.32 Our results showed that enzyme molecule exhibited different behaviors at different temperatures, leading

nanoparticle solution at the same concentration (about 3500 AU). Because NBD was an environment-sensitive fluorophore, the higher emission from nanofibers suggested the more ordered molecular arrangement in nanofibers. These observations clearly demonstrated that temperature had dramatical effects on the kinetics of conversion, morphology of nanostructures, molecular arrangement, and macroscopic property of resulting samples. Molecular Arrangement. To explore the molecular arrangement of NBD-GDFDFDY in the resulting nanomaterials, we collected the circular dichroism (CD) spectra of the gel and the solution, respectively. The CD spectra in Figure 2A clearly demonstrated that NBD-GDFDFDY adopted helical conformation in the gel with a positive peak at 192 nm and negative peaks at 208 and 224 nm, whereas it exhibited disordered conformation in the solution with a major negative peak at about 200 nm and a minor one at 226 nm. The results clearly showed that the molecular arrangement of NBD-GDFDFDY was more ordered in nanofibers than that in nanoparticles. The difference of the self-assembled structures prepared at the two conditions was also confirmed by FT-IR spectra (Figure S6). These observations clearly indicated that enzymatic triggeration at lower temperature resulted in the formation of nanomaterials with more ordered molecular arrangement and lower energy (higher stability). C

DOI: 10.1021/acsami.7b00784 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces to different reaction−diffusion processes at nanoscale and resulting in different molecular arrangements of the peptides and morphologies of nanostructures. The different peptide conformations at different temperatures might also account for the different morphologies of resulting nanomaterials. The molecular vibration of the peptide was more significant at 37 °C than at 4 °C, leading to more ordered molecular selfassembly in the nanofibers formed at 4 °C. Cellular Uptake. We further explored the functional consequence of different self-assembled structures. Many pioneering works have shown that nanomaterials with different morphologies and surface properties would show significant difference in many behaviors such as cellular uptake,51 circulation time in blood,52 etc. We therefore measured the cellular uptake behavior of our nanofibers and nanoparticles. NIH 3T3 cells were treated with the same concentration of the nanofibers and nanoparticles (120 μM). The amount of NBDGDFDFDY in the cells were determined at different time points. The results in Figure 2C indicate that nanofibers showed a more rapid and much higher uptake by the cells than the nanoparticles. For instance, the intracellular concentration of NBD-GDFDFDY was 662, 1088, 1321, and 1127 μM at 0.5, 1, 2, and 4 h time point for nanofiber-treated cells. Whereas it was 17, 136, 188, and 308 μM at 0.5, 1, 2, and 4 h time point, respectively for nanoparticle-treated cells. The nanofibers therefore exhibited more than 10 times higher cellular uptake than the nanoparticles. The fluorescence confocal microscopic images also showed similar results (Figure 2D,E). Cells treated with nanofibers exhibited much stronger yellow fluorescence than those treated with the same concentration of nanoparticles at the same incubation time. The yellow fluorescence from NBD-moiety matched well with the red fluorescence from LysoTracker (Figure S12), suggesting that the uptake of the nanomaterials was through endocytosis. The peptide molecules in nanofibers formed at 4 °C exhibited a more ordered conformation than those in nanoparticles formed at 37 °C, and the nanostructures formed at lower temperature would be thermodynamically more stable, which ultimately led to much higher and faster cellular uptake of our nanofibers by 3T3 cells.33 Optimized Fluorescence Probes Generated with Spatiotemporal Control. We subsequently opted to test whether this novel strategy could be used to optimize the performance of molecular probes by spatiotemporally controlling the self-assembly pathway. Our recent studies showed that peptide self-assembly could be applied to prepare fluorescence molecular probes with better performance.53−55 Following this idea, we replaced the NBD by a quenched ratiometric fluorescence probe of 4-N3-1,8-naphthalimide (N3-NTI) to make N3-NTI-GDFDFpDY (Figure 3A). The N3-NTI group could be converted to NH2-NTI moiety with lower emission at 425 nm and higher emission at 550 nm by H2S, resulting in ratiometric fluorescence change. Similar to NBD-GDFDFpDY, N3-NTI-GDFDFpDY formed nanofibers with the diameter of about 10−20 nm and nanoparticles with the size of 70−120 nm by 1 U/mL of phosphatase at 4 and 37 °C, respectively (Figure 3B,C) due to the kinetics differences of enzymatic conversion (Figure S13). Compared with the emission pattern of N3-NTIGDFDFpDY in PBS solution, the formation of nanofibers and nanoparticles quenched the fluorescence peaks both at 425 and 550 nm (Figures 3D and S14). After H2S was added to convert totally N3-NTI-GDFDFDY to NH2-NTI-GDFDFDY, the signal fluorescence intensity at 550 nm from nanofiber solution

Figure 3. (A) Chemical structures of N3-NTI-GDFDFpDY and N3NTI-G DF DFD Y and schematic illustration of conversions by phosphatase and H2S; TEM image of (B) nanofiber solution formed at 4 °C and (C) nanoparticle solution formed at 37 °C; (D) emission spectra indicate that probe in nanofiber form exhibits lower background fluorescence and shows higher signal fluorescence than probe in nanoparticle form; (E) background F550/F425 ratio of nanofiber was smaller, whereas the signal F550/F425 ratio of nanofiber was bigger than that of nanoparticle, respectively.

(about 4500 AU) was about 2 times stronger than that from nanoparticle solution (about 2300 AU, Figure 3D). The F550/ F425 ratio was therefore changed from 0.5 to 54 and from 0.7 to 17 for nanofiber and nanoparticle solution, respectively. We also monitored the fluorescence change of nanofiber and nanoparticle solutions by adding different concentrations of H2S. We obtained the fluorescence spectra of both solutions with different concentrations of H2S at 0.5 h time point. The results in Figure 4A,B show that, as the concentration of H2S increased, the peak at 425 nm decreased and the peak at 550 nm gradually increased in intensity for both samples. However, with the same concentration of H2S, it was obvious that the nanofiber solution showed a lower fluorescence at 425 nm and a higher fluorescence at 550 nm than the nanoparticle solution. The F550/F425 ratio of nanofiber solution was therefore larger than that of nanoparticle solution with the same concentration of H2S (Figure 4C). For example, the ratio was 83 for nanofiber solution, whereas 25 for nanoparticle solution with 200 μM H2S. Upon irradiation by a UV lamp at 330 nm, we observed stronger yellowish green fluorescence from nanofiber solution than nanoparticle solution with 200 μM H2S (Figure 4D). The bigger fluorescence ratio suggested the higher sensitivity and better performance of nanofibers than nanoparticles in vitro. Imaging Application in Cells. Next, we tested the performance of both nanoprobes in cell experiments. NIH 3T3 cells were first treated with 120 μM nanofiber or nanoparticle for 2 h. Confocal fluorescence microscopy images were then obtained at an excitation wavelength of 405 nm for blue channel and 488 nm for yellow channel, respectively. The results in Figure 4E1,E2 show that cells treated with nanofibers and nanoparticles had nearly no intracellular fluorescence, D

DOI: 10.1021/acsami.7b00784 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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clearly indicated the better performance of the probe in the nanofiber form than that in the nanoparticle form. Although we were not able to optimize the function of the self-assembled structures a priori, the spatiotemporal control of the selfassembly pathway allowed us to access more functional structures that are otherwise inaccessible by conventional selfassembly methods. We thus envisioned that the performance of the probe in the nanofiber form may be further improved by screening more possible self-assembly pathways in both time and space domains.

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CONCLUSIONS Diffusion limit is a very important factor in biological systems, especially within cells, because of the crowding effect. It is therefore a great need to develop model systems to understand the importance of diffusion in the preparation of functional nanostructures. In this study, we have demonstrated that, by simply varying the temperature, the kinetics and reaction− diffusion process could be manipulated in enzyme-triggered molecular self-assembly, which ultimately led to different selfassembling behavior of peptides and functions of resulting nanomaterials. At the lower temperature, nanomaterials with more ordered molecular arrangement and lower energy would be obtained. Such nanomaterials showed faster and enhanced cellular uptake behaviors. The strategy in our study was used to prepare molecular probes with better performances. In this study, we only tested the cellular uptake difference by 3T3 cells between nanofibers and nanoparticles, the nanomaterials generated by our strategy might show differences in other properties such as circulation time in blood, cellular uptake by other cell lines, etc. We postulate that our study provided a useful strategy to manipulate spatiotemporally the property and function of supramolecular nanomaterials.

Figure 4. Emission spectra of (A) solution of nanofiber and (B) solution of nanoparticle treated with different concentration of H2S for 0.5 h (excitation wavelength = 340 nm) indicate that the signal fluorescence of nanofiber is bigger than that of nanoparticle at the same condition; (C) F550/F425 ratio of different solutions treated with different concentrations of H2S for 0.5 h indicated a bigger ratio of nanofiber than nanoparticle at the same condition; (D) optical images of different solutions irradiation by a hand-held UV lamp (330 nm channel) show a brighter fluorescence from nanofiber solution than that from nanoparticle solution upon the treatment of H2S; (E−G) confocal fluorescence images of 3T3 cells incubated with different samples (scale bars in panels E−G represent 25 μm) indicate a higher sensitivity and faster response to analyte of nanofiber than nanoparticle.

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EXPERIMENT SECTION

Chemicals and Materials. Fmoc-amino acids were obtained from GL Biochem (Shanghai, China). 2-Chlorotrityl chloride resin was obtained from Nankai Resin Co. Ltd. (Tianjin). Alkali phosphatase (30 U/μL) was obtained from Takara (D2250, Dalian, China) Bio. Inc. All the other starting materials were obtained from Alfa. Chemical reagents and solvents were used as received from commercial sources. General Methods. HR-MS was carried out with a VG ZAB-HS system (England). HPLC was conducted with a LUMTECH HPLC (Germany) system using a C18 RP column with MeOH (0.05% of TFA) and water (0.05% of TFA) as the eluents. LC-MS was conducted with a Shimadzu LCMS-20AD (Japan) system. The synthesized peptides were characterized by 1H NMR (Bruker, Switzerland) and high-resolution mass spectrometry (HR-MS, England). Circular dichroism spectroscopy data were obtained with a BioLogic (MOS-450) system. FT-IR spectra were tested by a Biorad FTS6000. Dynamic light scattering (DLS) was performed on a laser light scattering spectrometer (BI-200SM). The fluorescence spectrum was recorded on a BioTek Synergy 4 Hybrid Microplate Reader. TEM images were acquired on a Tecnai G2 F20 system, operating at 200 kV. Preparation of NBD-β-Alanine. The preparation of NBD-βalanine was according to our previous report.53 The brief process as shown in Scheme S1 (95.6% yield). Preparation of 4-N3-1,8-naphthalic-β-alanine. The preparation of NBD-β-alanine was according to our previous report.54 The brief process as shown in Scheme S2 (85.2% yield). Self-Assembly of NBD-GDFDFpDY and N3-NTI-GDFDFpDY. 1 mg of NBD-GDFDFpDY was dispersed in 0.9 mL of PBS buffer solution (pH = 7.4). Na2CO3 (1 M) was added to the above solution to adjust the final pH = 7.4, and PBS was then pipetted into the solution to make the volume of solution 1.0 mL. The solution was incubated at 4

suggesting the low background fluorescence signals of both samples. We then added different concentrations of H2S to the cells, which were then incubated for another 0.5 h. We observed stronger yellow fluorescence in cells treated with nanofibers than those treated with nanoparticles at the same concentration of H2S (Figures S15). For instance, when the concentration of H2S was 5 μM, we observed moderate yellow fluorescence in cells treated with nanofibers (Figure 4F1), whereas we observed very weak fluorescence in cell treated with nanoparticles (Figure 4F2). The stronger yellow fluorescence in cells treated with nanofibers than those treated with nanoparticles was due to the higher cellular uptake of nanofibers by cells (Figure S16). These observations suggested that the sensitivity of the probe in the nanofiber form was higher than that in the nanoparticle form. If we shortened the treatment time of cells with nanofibers or nanoparticles to be 0.5 h, we still observed strong yellow fluorescence in cells treated with nanofibers and H2S (50 μM, Figures 4G1 and S17), whereas we observed much weaker fluorescence in cells treated with nanoparticles and the same concentration of H2S (Figures 4G2 and S17). The shorter detection time and higher sensitivity E

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or 37 °C for 30 min. The phosphatase (1 U/mL) was then added to the above solution for 12 h at 4 or 37 °C. A similar procedure was used to trigger the self-assembly of N3-NTI-GDFDFpDY by phosphatase, except its concentration was 0.01 wt %. Determination of Conversion Ratio. 2 mL of the samples were used to determine the conversion ratio of peptides. After 1 U/mL of phosphatase was added to the sample, 100 μL of each sample was taken out at each time point and then 100 μL of methanol was added to terminate the reaction. The conversion ratio was then analyzed by the LC-MS. We detected the peaks of precursor and hydrogelator at each time point, and the conversion ratio was calculated by integrating the peaks area. The gradient of the mobile phases and the representative LC-MS traces are shown in Table S1 and Figure S18. Fluorescence Detection. 100 μL of nanoprobe solution at 4 and 37 °C was added in a 96-well plate. The fluorescence spectrum was acquired on a BioTek SynergyTM 4 Hybrid Microplate Reader (NBDGDFDFDY, λexc. = 470 nm; N3-NTI-GDFDFDY, λexc. = 340 nm). Cell Staining Experiments with both Our Probe and the LysoTracker Red. After incubation with 120 μM NBD-GDFDFDY at 37 °C for 0.5, 1, 2, 4 h, the NIH 3T3 cells were washed with PBS for three times and then incubated with LysoTracker red (1 μM) for 15 min for lysosome labeling and then washed with PBS for three times. Both groups of cells were fixed by using 4% of paraformaldehyde; we recorded the images by a laser scanning confocal microscopy (λexc. = 588 nm). Fluorescence Responses of the Nanoprobes to H2S. For the fluorescence responses of nanofiber and nanoparticles to H2S, the stock solution (0.1 mg/mL) of the probe was diluted with PBS buffer (pH 7.4). The exogenous source of H2S was prepared by sodium hydrogen sulfide (NaHS) with dissolving in pH 7.4 PBS buffer. The test solution was prepared by adding the sensor and NaHS solution together to the final concentration of the sensor of 0.01 mg/mL. The tested solution was stirred for 15 min, and then the fluorescence spectra were recorded. Laser Scanning Confocal Microscopy for Imaging in 3T3 Cells. NIH 3T3 cells were incubated in class bottom cell culture dish at a density of 40 000 cells per dish. After incubation for 24 h, the Dulbecco’s modified Eagle’s medium (DMEM) solution containing 120 μM of each compound was then added to the cells. The DMEM solution was then removed at 0.5, 1, 2, or 4 h, and cells were washed for three times with PBS. We recorded the images by a laser scanning confocal microscopy (NBD-GDFDFDY, λexc. = 488 nm; N3-NTIGDFDFDY, λexc. = 405 nm for blue channel and 488 nm for yellow channel). All images were taken by a laser scanning confocal microscopy instrument (Leica TSC SP8) at the same voltage. Determination of Cellular Uptake in 3T3 Cells.56 3T3 cells were incubated in 12-well plates for 24 h at a density of 1.2 × 106 cells. The DMEM solution containing 120 μM of each compound was added to the cells and the upper medium was removed at 0.5, 1, 2, or 4 h. Cells were washed for three times with PBS. After treatment with cell lysis solution (1 mL per well contained 200 μL DMSO) for 15 min, the solutions were centrifuged at 1570g for 10 min. The amount of compound in the upper solution was determined by a microplate reader (Bio-RAD iMarkTM, America). Intracellular concentration = (c × 1 mL)/(cell number × 1.7 × 10−9); c, calculated from the standard curve equation. An average diameter for NIH 3T3 was about 15 μm for a suspended cell (volume: 4/3πr3 = 1.7 × 10−9 cm3, r = 7.5 μm)

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Research Article

AUTHOR INFORMATION

Corresponding Authors

*Y. Cao. Email: [email protected]. *Z. Yang. Email: [email protected]. ORCID

Yi Cao: 0000-0003-1493-7868 Zhimou Yang: 0000-0003-2967-6920 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the International S&T Cooperation Program of China (2015DFA50310), National Natural Science Foundation of China (51373079 and 31370964) and Program for Changjiang Scholars and Innovative Research Team in University (IRT13023).

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b00784. Characterization of compounds; FT-IR spectra; dynamic light scattering; cellular uptake; emission spectra; confocal images; details in cell assays (PDF) F

DOI: 10.1021/acsami.7b00784 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.7b00784 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX