Article pubs.acs.org/ac
Environment-Sensitive Fluorescent Supramolecular Nanofibers for Imaging Applications Yanbin Cai,† Yang Shi,† Huaimin Wang,† Jingyu Wang,† 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), ‡College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, P. R. China S Supporting Information *
ABSTRACT: The combination of an environment-sensitive fluorophore, 4-nitro-2,1,3-benzoxadiazole (NBD), and peptides have yielded supramolecular nanofibers with enhanced cellular uptake, brighter fluorescence, and significant fluorescence responses to external stimuli. We had designed and synthesized NBD-FFYEEGGH that can form supramolecular nanofibers and emit brighter than its counterpart of NBDEEGGH without the self-assembling property. The nanofibers of NBD-FFYEEGGH could specifically bind to Cu2+, leading to the formation of fluorescence quenched elongated nanofibers. This fluorescence quenching property was enhanced in self-assembling nanofibers and could be applied for detection of Cu2+ in vitro and within cells. In a further step, an enzyme-cleavable DEVD peptide was placed between NBD-FFY and the copper binding tripeptide GGH. The resulting self-assembling peptide NBD-FFFDEVDGGH also showed strong fluorescence quenching to Cu2+. Upon the enzymatic cleavage to remove the Cu2+-binding GGH tripeptide from the peptide, the fluorescence was restored. The cellular uptake of nanofibers was better than that of free molecules because of endocytosis. The supramolecular nanofibers with fluorescence turn-on property could therefore be applied for detection of caspase-3 activity in vitro and within cells. We believe that the combination of environment-sensitive fluorescence and fast responses of supramolecular nanostructures would lead to a useful platform to detect many important analytes.
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transitions.30,35,51 These pioneering works indicated the big potential of molecular hydrogels in rapid and specific detection of many important analytes. Recently, Xu and co-workers reported on an assay to detect intracellular phosphatase activity by in situ fluorescent nanofiber formation.36 In the design, an environment-sensitive fluorescent molecule, 4-nitro-2,1,3-benzoxadiazole (NBD), was conjugated to a precursor of gelator. Upon the triggeration of phosphatase, the precursor could be converted to a gelator, resulting in the formation of nanofibers and molecular gels. Though there was no significant fluorescence intensity change between the precursor solution and the nanofiber solution, this system could still be applied for imaging intracellular phosphatase activity by confocal fluorescence microscopy because the in situ nanofiber formation could result in good contrast in fluorescence signal of nanofibers over the background. This study, in combination with other successful examples of using supramolecular nanofibers in molecular gels for detection applications,34,38 inspired us to design environment-sensitive fluorescent supramolecular nanofibers with enhanced cellular uptake by endocytosis and specific, rapid,
nspired by prevailing examples of functional self-assembled systems in biological systems such as lipid bilayers and tubulin assembly, researchers have developed many supramolecular nanostructures with promising properties. The building blocks for constructing these nanostructures are usually amphiphilic, including block copolymers,1−4 protein− polymer hybrids,5−7 macrocycle derivatives,8−13 and small molecules.14−19 Assisted by noncovalent interactions (e.g., hydrophobic interactions, hydrogen bonding, charge interaction, etc.), these amphiphilic molecules can self-assemble into nanostructures with unusual properties beyond single molecules.14,20,21 Due to the good biocompatibility, multivalent display, and fast responses to external stimuli, supramolecular nanostructures of small molecules have attracted extensive recent research interests. They have been applied for drug delivery,22−28 analyte detection,19,29−38 cell culture,39−43 immune response boosting,44,45 cancer cells and bacterial inhibitions,46−50 etc. Among their application in analyte detection, the semiwet environment in molecular hydrogels has provided a good platform to retain protein activity for enzyme detection.37 The self-assembled nanofibers can offer hydrophobic domains to adsorb fluorescent dyes for the detection of metal ions and polyphosphates by fluorescence changes.32,33 With the aim of enzyme-triggered formation of molecular hydrogels, enzymes and their inhibitors can also be identified by the naked eye, depending on the sol−gel phase © 2014 American Chemical Society
Received: November 27, 2013 Accepted: January 27, 2014 Published: January 27, 2014 2193
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and significant fluorescence changes upon contacting with analytes. In this study, we showed that we could integrate the environment-sensitive fluorescent NBD, an enzyme cleavable peptide linker, and a copper ion-binding tripeptide ligand in one self-assembling molecule. At self-assembled stages, NBD would emit brightly. The binding of the metal ion of Cu2+ to the nanofibers could quench the fluorescence of NBD, leading to the formation of fluorescence-quenched nanofibers. The quenching effect was improved at the self-assembled stage. The big difference in fluorescence could therefore be applied for the detection of Cu2+. Adding enzyme to remove Cu2+ binding tripeptide from the self-assembling nanofibers would restore their fluorescence, which could be applied for the detection of enzyme activity. Taken the advantages of enhanced quenching effect in self-assembling nanofibers, improved cellular uptake of nanofibers by endocytosis, brighter fluorescence of NBD in nanofibers, and significant fluorescence changes upon contacting analytes, the environment-sensitive fluorescent supramolecular nanofibers in our study could therefore be applied for detection of important analytes.
affinity.59 Many studies have shown that copper ions can quench the fluorescence of materials, including inorganic quantum dots, fluorescent dyes, and carbon dots.60−62 Therefore, the addition of Cu2+ to solution of these peptides may trigger morphological changes of self-assembled nanostructures and, more importantly, lead to fluorescence quenching, which could be applied for Cu2+ detection. Self-Assembly Behavior. We first prepared NBD-βalanine in one step (Scheme S-1 of the Supporting Information), which could be directly used for solid phase peptide synthesis (SPPS) to produce the designed peptides. Compounds 1−3 formed homogeneous solutions in aqueous solutions at concentrations lower than 3 wt % (Figure 1A, pH =
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RESULTS AND DISCUSSION Molecular Design of a Probe for Cu2+ Detection. We first designed environment-sensitive fluorescent nanofibers that could be applied for the detection of Cu2+. Unlike general fluorescent molecules that usually exhibit fluorescence quenching properties when they aggregate, environment-sensitive fluorescent molecules and aggregation-induced emission (AIE) fluorescent molecules show relatively stronger fluorescence in hydrophobic environments and upon aggregation, respectively. 52,53 This information, in combination with the aforementioned Xu’s report,36 inspired us to use an environment-sensitive fluorescent NBD to construct short peptidebased hydrogelators because NBD would emit stronger yellow fluorescence in the hydrophobic domain of self-assembled nanofibers than in solutions. Aromatic compounds had been widely used as capping groups to generate molecular hydrogelators of short peptides, such as pyren,54 fluorenyl (Fmoc),55 naphthalene (Nap),56,57 and phenothiazine (PTZ)58 groups. However, these aromatic moieties could only emit short wavelength UV lights, hindering their imaging applications in biological systems. We therefore designed the molecules NBD-FFYEnGGH (Scheme 1, compounds 1, 2,
Figure 1. Optical images of (A) a water solution containing 0.5 wt % of (i) 1, (ii) 2, and (iii) 3 (pH = 7.4) and (B) (iv) a gel, (v) a gel, and (vi) a clear solution formed by adding one equivof CuSO4 to the solution of 1, 2, and 3, respectively. Transmission electron microscopy (TEM) images of (C) a solution of 2 and (D) a gel of 2 with one equivof Cu2+.
7.4). Upon the addition of one equiv of CuSO4 to a water solution containing 0.5 wt % of these peptides, we observed the formation of a gel (Figure 1B, iv), a gel (Figure 1B, v), and a clear solution (Figure 1B, vi) within 5 min for compounds 1, 2, and 3, respectively (Figure 1B). These observations indicated the different self-assembly properties of the three compounds in the presence of Cu2+ and also implied that the three compounds might show different fluorescence responses to Cu2+. The sol−gel phase transition could not be observed for the control peptide of NBD-FFYEEGGK (Figure S-15 of the Supporting Information). Adding one equiv of other metal ions, including Mn2+, Ni2+, Co2+, Fe2+, Zn2+, Ca2+, Sr2+, and Ba2+, respectively, to the solution of 2 could not result in hydrogelations (Figure S-16 of the Supporting Information). The critical micelle concentration for 2 was 0.47 and 0.056 mg mL−1 in the absence and presence of one equiv of Cu2+, respectively (Figure S-17 of the Supporting Information). These observations indicated that the hydrogelation was due to the selective binding of GGH to Cu2+, which was similar to other examples of metal ion-induced molecular hydrogelations.63−65 The gelation of 2 with Cu2+ was due to the elongation of self-assembled nanofibers. As shown in Figure 1
Scheme 1. Chemical Structure of NBD-FFYEnGGH (1, 2, and 3 when n was 1, 2, and 3, respectively)
and 3 for n = 1, 2, and 3, respectively). The rationales of the design are described as following: (i) many short peptides based on FF capped with aromatic groups have been demonstrated as excellent gelators.19 We therefore believe that peptides based on NBD-FFY may also possess an excellent self-assembly ability. (ii) The En are used to adjust the amphiphilicity and fluorescence property of peptides. (iii) The tripeptide GGH can selectively bind to Cu2+ with a high 2194
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(panels C and D), solution of 2 exhibited short fibers with lengths less than 1 μm, while gel of 2 with Cu2+ showed fiber networks and the length of the fibers was longer than several micrometers. The rheological measurements indicated a mechanically weak hydrogel of 2 with Cu2+ (Figures S-18 of the Supporting Information). Fluorescence Response to Metal Ions. We opted to determine which compound among compounds 1−3 would show the most significant fluorescence change to Cu2+. The solution of 2 emitted the strongest fluorescence upon excitation, compared with solutions of 1 and 3 at the same concentration (Figure 2A and Figure S-19 of the Supporting
upon the addition of more and more Cu2+ to the solution of 2 (0.05 wt %, 410 μM) at the Cu2+ concentration range from 0 to 410 μM (0−1 equiv). Upon excitation by a UV lamp (330 nm), we observed bright fluorescence from the solution of 2 while nearly no fluorescence from solution of 2 with one equiv of Cu2+ (Figure 2B, insets). We also recorded the I0/I values of solution of 2 in the presence of 200 μM of different metal ions. The results in Figure 2B showed that this value in the presence of Cu2+ was the biggest (about 10.1), compared to those in the presence of other metal ions (2.0, 3.3, 3.8, 3.2, 4.9, 1.1, 1.0, 1.0, 1.1, 1.1, and 1.1 for Mn2+, Ni2+, Co2+, Fe2+, Zn2+, Mg2+, Ca2+, Sr2+, Ba2+, Na+, and K+, respectively). These observations suggested that 2 could be applied for detection of Cu2+. Intracellular Imaging of Cu2+. MTT assay indicated that the addition of 410 μM of 2 with 100 μM of Cu2+ to HeLa cells showed no obvious toxicity to the cells (Figure S-24 of the Supporting Information), suggesting its good biocompatibility. The good biocompatibility and selective fluorescence quenching of 2 to Cu2+ ensured its application for imaging the intracellular copper ion. As shown in Figure 2C and Figure S-25 of the Supporting Information, HeLa cells incubated with 0.05 wt % (410 μM) of 2 showed a bright fluorescence at the 2 h time point. If pretreating the cells with 100 μM of Cu2+ for 6 h and then treating with 2, there was nearly no detectable fluorescence in HeLa cells even at the 6 h time point (Figure 2D and Figure S-26 of the Supporting Information) probably due to the fluorescence quenching by Cu2+. Adding 100 μM of Cu2+ to HeLa cells treated with 0.05 wt % of 2 for 2 h would also lead to fluorescence quenching (Figure S-27 of the Supporting Information). These observations, in combination with the results in Figure 2 (panels A and B), indicated that 2 could be applied for in vitro and in vivo detection of Cu2+. However, since other metal ions could also quench the fluorescence of 2, our probe could only be applied for approximate detection of Cu2+. Design of a Probe for Imaging Caspase-3 Activity. The selective binding of Cu2+ to the peptide of GGH could strongly quench the fluorescence of supramolecular nanofibers. This information inspired us to design fluorescence turn-on probes using enzyme cleavable peptides to connect NBD-FF and GGH. If so, enzymatic cleavage would remove the Cu2+ binding tripeptide of GGH from the peptides and therefore restore the fluorescence of supramolecular nanofibers. This process might be applied to detect enzyme activity in vitro and within cells. In order to test our hypothesis, we designed and synthesized the NBD-FFFDEVDGGH (5 in Scheme 2) with a Caspasecleavable DEVD peptide. Compound 5 was also synthesized by SPPS and then purified by HPLC.
Figure 2. (A) Fluorescence values of aqueous solution containing 0.05 wt % of 2 in the presence of different equiv of Cu2+, (B) effect of different metal ions (200 μM) on I0/I of solution of 2 (0.05 wt %) (insets in B: fluorescence images of a solution of 2 (0.05 wt %) in the absence (left) and presence (right) of one equiv of Cu2+), and confocal images (bright field + fluorescence) of (C) HeLa cells treated with 2 (0.05 wt %) at a 2 h time point and (D) HeLa cells pretreated with 100 μM of Cu2+ and then treated with 2 (0.05 wt %) at a 6 h time point.
Information), indicating that the number of E in NBDFFYEnGGH could adjust the fluorescence intensity of the peptides in solutions. The I0/I value for 3 to one equiv of Cu2+ was 6.3, which was smaller than that for 2 to one equiv of Cu2+ (21.8, Figure S-20 of the Supporting Information). The solution of 1 would change to a precipitate (or a partial gel) in the presence of Cu2+ (Figure S-21 of the Supporting Information), which was unsuitable for monitoring its fluorescence change by a fluorescence reader. These observations indicated that 2 was the best candidate among compounds 1−3 for detection of Cu2+. We also synthesized compound 4 of NBD-EEGGH lacking FFY (Scheme S-6 of the Supporting Information). Compound 4 could not self-assemble into nanofibers at the concentration of 410 μM (Figure S-22 of the Supporting Information), and the solution of 4 (410 μM) emitted a weaker signal than the solution of 2 at the same concentration (Figure S-23 of the Supporting Information), indicating that the environment-sensitive fluorophore of NBD could emit stronger fluorescence at self-assembled stages. The stronger emission of NBD in nanofibers than at unassembled stages suggested the advantages of supramolecular nanofibers in the design of bright environment-sensitive fluorescent materials. We then monitored the fluorescence change of solution of 2 with the addition of Cu2+ and other metal ions. As shown in Figure 2A, we observed a continuous fluorescence quenching
Scheme 2. Chemical Structure of NBD-FFFDEVDGGH (5)
Self-Assembly and Fluorescence Properties. Similar to 2, compound 5 could form nanofibers in the presence of one equiv of Cu2+ (Figure 3A), and it possessed a similar CMC value (0.156 mg mL−1) to 2 (0.056 mg mL−1) in the presence of Cu2+ (Figure S-28 of the Supporting Information). We then monitored its fluorescence response. As shown in Figure 3 2195
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activity within cells because the complex showed no obvious toxicity to HeLa cells at this concentration (Figure S-32 of the Supporting Information). As shown in Figure 4 (panels A1−
Figure 3. (A) A TEM image of solution of 5 (0.5 wt %, pH = 7.4) with one equiv of Cu2+. (B) Fluorescence intensity of solution of 5 (0.05 wt %) with or without Cu2+ and a solution of 5 (0.05 wt %) with one equiv of Cu2+ and then treated with caspase-3 (1 unit/mL) for 1 h. (C) Optical images of solution of 5 under a UV lamp (i: without Cu2+, ii: with one equiv of Cu2+, and iii: with Cu2+ and then treated with Caspase-3 for 1 h). (D) Fluorescence spectra of a solution of 5 with one equiv of Cu2+ treated with different concentrations of caspase-3 at 37 °C for 40 min.
Figure 4. Confocal images of HeLa cells treated with the 5:Cu2+ complex for 2 h (A1: bright field, A2: fluorescence, and A3: overlap) and HeLa cells treated with the 5:Cu2+ complex for 2 h and then exposed to UV light for an additional 2 h (B1: bright field, B2: fluorescence, and B3: overlap) (before exposure to UV light, the cells were washed with PBS 3 times to remove the extracellular 5:Cu2+ complex).
(panels A−C), a water solution of 5 (0.05 wt %, 356 μM) showed strong fluorescence upon excitation at about 330 nm by a hand-held UV lamp (Figure 3C, i). However, the solution could only emit very weak fluorescence upon the addition of one equiv of Cu2+ (Figure 3C, ii). The I0/I value for fluorescence intensity of solution of 5 without Cu2+ over a solution of 5 with one equiv of Cu2+ was 13.4 (Figure 3B), indicating that Cu 2+ could also strongly quench the fluorescence of 5. The fluorescence was restored when caspase-3 was added to the solution in Figure 3C, ii (Figure 3, iii). The LC−MS result indicated that the GGH tripeptide was removed from 5 by the enzyme (Figure S-29 of the Supporting Information). There was no significant difference in fluorescence intensity between the solution of 5 before and after adding caspase-3 in the absence of Cu2+ (Figure S-30 of the Supporting Information). We also recorded the fluorescence spectra of 5:Cu2+ with the addition of different concentrations of caspase-3. The results in Figure 3D indicated that higher fluorescence signals could be observed when higher concentrations of enzyme were used. These observations clearly demonstrated the success of our design and the complex of 5:Cu2+ could be applied for in vitro detection of caspase-3 activity. Intracellular Detection of Caspase-3. Higher cellular uptakes were recorded for the 5:Cu2+ complex than 5 itself (Figure S-31 of the Supporting Information) at 356 μM (0.5 mg mL−1) and 37 °C. Since the CMC value of 5 in the absence of Cu2+ was higher than 356 μM (1.73 mM or 2.43 mg mL−1, Figure S-28 of the Supporting Information), these observations indicated that nanofibers of the 5:Cu2+ complex could penetrate into cells more easily than the free molecule of 5, highlighting the enhanced cellular uptake of self-assembled nanomaterials. A much lower concentration of 5 within cells treated with nanofibers of the 5:Cu2+ complex was observed at 4 °C than that at 37 °C (Figure S-31 of the Supporting Information), indicating that the uptake of nanofibers by cells was due to endocytosis. We fixed the concentration of the complex of 5:Cu2+ to be 100 μM for imaging the caspase-3
A3), HeLa cells exhibited weak fluorescence upon the treatment of the 5:Cu2+ complex, suggesting the weak caspase-3 activity in cells. UV light could induce apoptosis of cells. We therefore exposed the HeLa cells treated with the 5:Cu2+ with UV light for an additional 2 h (the extra-cellular 5:Cu2+ complex was removed before UV irradiation). We observed bright yellow fluorescence within cells (Figure 4, panels B1−B3), suggesting the high caspase-3 activity within the cells. These observations indicated that the complex of 5:Cu2+ could be applied for the detection of caspase-3 activity within cells. Design of a Probe for GSH Detection. In order to prove the universality of self-assembled nanofibers in detection applications, we designed and synthesized NBD-FFFEE-ssEGGH with the glutathione (GSH)-reducible disulfide bond (compound 6 in Figure 5A). Nanofibers could also be observed through TEM (Figure S-33 of the Supporting Information). The complex of 6:Cu2+ could be applied for detection of GSH because the solution of 6 showed much weaker fluorescence in the presence of one equiv of Cu2+, and the addition of GSH could turn on the fluorescence of 6:Cu2+ (insets in Figure 5B) due to the removal of the copper ion binding GGH peptide. Higher concentrations of GSH would result in bigger fluorescence intensity for solution of 6:Cu2+ (Figure 5B). We also detected the changes of fluorescence values of 6:Cu2+ treated with different concentrations of GSH at 0−60 min. The results showed that higher concentrations of GSH would restore the fluorescence of the complex faster (Figure S-34 of the Supporting Information). These observations suggested the potential of the complex of 6:Cu2+ in the detection of GSH.
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CONCLUSION In summary, we have introduced a supramolecular nanofiber system with environment-sensitive fluorescence property. This system shows a selective copper ion-induced fluorescence quenching phenomenon, which can be applied for detection of copper ion in vitro and within cells. What’s more, through 2196
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Peptide Synthesis. The peptide derivative was prepared by solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding N-Fmoc protected amino acids with side chains properly protected by a tertbutyl group. Piperidine (20%) in anhydrous N,N′-dimethylformamide (DMF) was used during deprotection of the Fmoc group. Then the next Fmoc-protected amino acid was coupled to the free amino group using O-(benzotriazol-1-yl)-N,N,N′,N′tetramethyluroniumhexafluorophosphate (HBTU) as the coupling reagent. The growth of the peptide chain was according to the established Fmoc SPPS protocol. After the last coupling step, excessive reagents were removed by a single DMF wash for 5 min (5 mL per gram of resin), followed by five steps of washing using DCM for 2 min (5 mL per gram of resin). The peptide derivative was cleaved using 95% of trifluoroacetic acid with 2.5% of trimethylsilane (TMS) and 2.5% of H2O for 30 min. Twenty milliliters per gram of resin of ice-cold diethylether was then added to the cleavage reagent. The resulting precipitate was centrifuged at 4 °C for 10 min at 10000 rpm. Afterward the supernatant was decanted, and the resulting solid was dissolved in DMSO for HPLC separation using MeOH and H2O containing 0.1% of TFA as eluents. Characterization of the Peptides. The figures of 1H NMR and HR-MS spectra were in the ESI. NBD-FFYEGGH. 1H NMR (400 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.67 (s, 1H), 8.48 (d, J = 8.9 Hz, 1H), 8.26 − 8.09 (m, 5H), 7.98 (d, J = 6.1 Hz, 1H), 7.28 − 6.96 (m, 13H), 6.63 (d, J = 8.4 Hz, 2H), 6.32 (d, J = 8.7 Hz, 1H), 4.52 (d, J = 6.9 Hz, 4H), 4.31 (d, J = 5.7 Hz, 1H), 3.79 − 3.68 (m, 4H), 3.13 (s, 3H), 3.01 − 2.89 (m, 4H), 2.80 − 2.66 (m, 3H), 2.63 (d, J = 13.5 Hz, 1H), 2.33 (s, 1H), 2.29 − 2.20 (m, 2H), 1.93 (s, 1H), 1.78 (d, J = 7.6 Hz, 1H). HR-MS: Calcd M+ = 1089.39, obsd (M + H)+ = 1090.4021. NBD-FFYEEGGH. 1H NMR (400 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.77 (s, 1H), 8.48 (d, J = 8.9 Hz, 1H), 8.13 (m, 8H), 7.30 − 7.01 (m, 13H), 6.63 (d, J = 8.4 Hz, 2H), 6.32 (s, 1H), 4.50 (dd, J = 12.6, 12.2 Hz, 4H), 4.29 (d, J = 7.5 Hz, 2H), 3.78 (dd, J = 16.6, 4.1 Hz, 4H), 3.66 (d, J = 5.7 Hz, 1H), 3.11 (s, 2H), 3.02 − 2.87 (m, 5H), 2.75 (d, J = 14.1 Hz, 2H), 2.65 (m, 2H), 2.34 − 2.22 (m, 4H), 1.93 (d, J = 5.9 Hz, 2H), 1.78 (d, J = 6.8 Hz, 2H). HR-MS: calcd M+ = 1218.44, obsd (M + H)+ = 1219.4432. NBD-FFYEEEGGH. 1H NMR (400 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.57 (s, 1H), 8.48 (d, J = 9.1 Hz, 1H), 8.24 − 8.01 (m, 8H), 7.13 (m, 15H), 6.63 (d, J = 8.5 Hz, 2H), 6.33 (s, 1H), 4.49 (d, J = 5.1 Hz, 4H), 4.40 − 4.20 (m, 4H), 3.80 (m, 2H), 3.74 − 3.68 (m, 3H), 2.95 (m, 5H), 2.82 − 2.59 (m, 5H), 2.29 (d, 5H), 1.90 (s, 3H), 1.77 (s, 3H). HR-MS: calcd M+ = 1347.48, obsd (M + H)+ = 1348.4864 NBD-FFYEEGGK. 1H NMR (400 MHz, DMSO-d6): δ 9.19 (s, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.24 − 8.03 (m, 9H), 7.10 (dd, 13H), 6.64 (d, J = 8.4 Hz, 2H), 6.33 (d, J = 8.9 Hz, 1H), 4.50 (s, 3H), 4.29 (s, 3H), 4.19 (d, J = 4.7 Hz, 2H), 3.82 − 3.67 (m, 7H), 3.02 − 2.88 (m, 3H), 2.76 (m, 4H), 2.27 (d, J = 8.0 Hz, 4H), 1.93 (s, 2H), 1.78 (s, 3H), 1.53 (s, 2H), 1.30 (dd, 3H). HR-MS: calcd M+ = 1209.47, obsd (M + H)+ = 1210.4813. NBD-EEGGH. 1H NMR (400 MHz, DMSO-d6): δ 9.40 (s, 1H), 8.84 (s, 1H), 8.52 (d, J = 9.0 Hz, 1H), 8.16 (m, 4H), 7.30 (m, 1H), 7.09 (d, 1H), 6.44 (d, J = 8.9 Hz, 1H), 4.54 (td, J = 8.7, 5.3 Hz, 1H), 4.33 − 4.21 (m, 2H), 3.79 − 3.65 (m, 6H), 2.99 (m, 2H), 2.64 (m, 2H), 2.21 (m, 4H), 2.00 − 1.64 (m, 4H). HR-MS: calcd M+ = 761.24, obsd (M + H)+ = 762.2439.
Figure 5. (A) Chemical structure of compound 6 and (B) fluorescence spectra of solution containing 0.05 wt % of 6 with one equiv of Cu2+ treated with different concentrations of GSH at 37 °C for 40 min ([insets in B: fluorescence images of a solution of 6 (0.05 wt %) with one equiv of Cu2+ (left) and treated with 1 mM of GSH (right)].
placing an enzyme-cleavable linker between the NBD-FFY and GGH:Cu2+ complex, this system can be applied for detection of enzyme of caspase-3 by fluorescence turn on. Besides the caspase-3 cleavable DEVD peptide, other peptide substrates of enzymes may also be used to yield supramolecular nanofibers for the detection of activities of other enzymes. The environment-sensitive fluorescent probe of NBD can emit stronger at self-assembled stages than in its free form, and the supramolecular nanofibers can easily penetrate into cells by endocytosis. Besides, unlike free probes that will diffuse easily in the whole cell, supramolecular nanofibers may be more difficult to move in the viscous solution within cells. Therefore, environment-sensitive fluorescent nanofibers may be applied for real time and in situ detection of enzyme activity. Due to the presence of a large pool of ligands that can selectively bind to important analytes and ease of design of self-assembling molecules based on these ligands and short peptides capped with aromatic environment-sensitive fluorophore, we believe our system can be expanded to other environment-sensitive fluorophores and other ligands. These efforts may lead to the development of highly selective sensors for detection of many important analytes.
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EXPERIMENTAL SECTION Chemicals and Materials. Fmoc-amino acids were obtained from GL Biochem (Shanghai, China). All the other starting materials were obtained from Alfa (Beijing, China). Commercially available reagents and solvents were used without further purification, unless noted otherwise. The synthesized compounds were characterized by 1H NMR (Bruker ARX-300) using DMSO-d6 as the solvent. 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 at the LCMS-20AD (Shimadzu) system. HR-MS was performed at the Agilent 6520 Q-TOF LC/MS using the ESI-L low concentration tuning mix (Lot no. LB60116 from the Agilent Tech.). 2197
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solution containing 0.05 mg mL−1 of 2 was added to the HeLa cells. The images were recorded at different time points (excitation wavelength = 488 nm). For Copper ion pretreated HeLa cells, the cells were treated with 100 μM of CuSO4 in DMEM solution for 6 h first. The cells were then washed with PBS 3× before the treatment of DMEM solution containing 0.05 mg mL−1 of 2. The images were recorded at different time points under the same detected conditions (excitation wavelength = 488 nm). This part of the experiment was performed on a laser scanning confocal microscope (OLYMPUS FV1000S-IX81). Laser Scanning Confocal Microscopy for Imaging Caspase-3 Activity in HeLa Cells. HeLa cells were incubated in 24-well plates at a density of 10000 cells per well for 24 h. The DMEM solution containing 0.05 mg mL−1 of 5 with 100 μM of CuSO4 was then added to the HeLa cells. Two hours later, the DMEM solution was removed and cells were washed for 3 times with PBS. Cells were then exposed to 254 nm UV irradiation at 10000 mJ cm−2 for additional 2 h. Images were recorded by confocal microscopy. For the control group, cells were not exposed to the UV irradiation. The images were taken by a laser scanning confocal microscopy (Leica TSC SP8) . Determination of Peptide Concentration in HeLa Cells by LC−MS. HeLa cells were incubated in 24-well plates at a density of 2 × 106 cells per well for 24 h. A stock solution containing 10 mg mL−1 of 5 was prepared (sodium carbonate was used to adjust the pH value to 7.4). Fifty microliters of peptide solution and 950 μL DMEM with 10% FBS were added to cells and the cells were then incubated at 37 or 4 °C (final concentration of peptide was 0.5 mg mL−1). Four hours later, DMEM containing peptide was removed and cells were washed with PBS 3×. Five hundred microliters of DMSO was added to each well to dissolve compounds in cells. After being treated with sonication for 15 min, the solutions were collected and centrifuged at 1570g for 10 min. The amount of peptides in the HeLa cells was determined by LC−MS.
NBD-FFFDEVDGGH. 1H NMR (400 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.83 (s, 1H), 8.52 − 8.33 (m, 3H), 8.22 − 8.00 (m, 5H), 7.91 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.35 − 6.96 (m, 16H), 6.32 (d, J = 8.2 Hz, 1H), 4.61 − 4.46 (m, 5H), 4.35 (d, J = 5.2 Hz, 1H), 4.15 − 4.09 (m, 1H), 3.69 (d, J = 6.5 Hz, 4H), 3.05 − 2.63 (m, 13H), 2.26 (m, 3H), 2.06 − 1.84 (m, 3H), 1.76 (s, 1H), 1.26 (dd, J = 11.2, 5.4 Hz, 2H), 0.82 (dd, J = 6.6, 3.6 Hz, 6H). HR-MS: calcd M+ = 1402.52, obsd (M + H)+ = 1403.5300. NBD-FFFEE-ss-EGGH. 1H NMR (400 MHz, DMSO-d6): δ 8.84 (s, 1H), 8.48 (d, J = 8.7 Hz, 1H), 8.25 − 7.94 (m, 12H), 7.26 − 7.01 (m, 17H), 6.32 (d, J = 9.2 Hz, 1H), 4.66 − 4.44 (m, 5H), 4.26 (m, 4H), 3.71 (t, J = 7.1 Hz, 6H), 3.06 − 2.95 (m, 5H), 2.91 − 2.84 (m, 2H), 2.81 − 2.72 (m, 6H), 2.68 − 2.58 (m, 2H), 2.36 (m, 4H), 2.29 − 2.16 (m, 7H), 1.92 (dd, J = 13.9, 6.8 Hz, 3H), 1.75 (dd, J = 14.8, 7.5 Hz, 3H). HR-MS: calcd M+ = 1565.5340, obsd (M + H)+ = 1566.5439. Hydrogel Formation. Peptides were prepared at a final concentration of 5 mg mL−1 in water solution (Na2CO3 was used to adjust the pH value to 7.4). One equiv of CuSO4 was then added to the solution to initiate hydrogelation. A transparent gel would form within 3 min. Rheology. Rheology was done on an AR 1500ex (TA Instruments) system, 25 mm parallel plates were used during the experiments at the gap of 400 μm. Dynamic strain sweep was performed and the strain values within the linear range were chosen for the following dynamic frequency sweep. The gels were also characterized by the mode of dynamic frequency sweep in the region of 0.1−100 rad/s at the strain value of 1%. Critical Micelle Concentration (CMC). The CMC values of peptides in water solution (pH = 7.4) were determined by dynamic light scattering (DLS). Solutions containing different concentration of peptides were tested, and the light scattering intensity was recorded for each concentration analyzed. Dynamic light scattering (DLS) was performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) at 532 nm under room temperature (22−25 °C). Transmission Electron Microscopy (TEM). TEM samples (5 mg mL−1 NBD-FFYEEGGH and NBD-FFFDEVDGGH with one equiv of Cu2+) were prepared at 25 °C. A micropipet was used to load 5 μL of sample solution to a carbon-coated copper grid. The excess solution was removed by a piece of filter paper. The samples were dried overnight in a desiccator and then conducted on a Tecnai G2 F20 system, operating at 200 kV. MTT Assay. The cytotoxicity of the solutions of peptides and peptide: Cu2+ complex was evaluated by MTT assay. HeLa cells were seeded in 96-well plates at a density of 5000 cells per well and incubated for 24 h. The peptides or peptide: Cu2+ complex in DMEM (Dulbecco’s Modified Eagle Medium) solutions were added into the cells (final concentrations were 0.5 mg mL−1) for different times (2, 4, and 6 h). One hundred microliters of MTT solution (5 mg mL−1 in PBS) was added to each well. Four hours later, the MTT solution was removed and the samples in the wells were air-dried. Fifty microliters of DMSO was added to dissolve the formed crystals; the optical density of the solution was measured at 490 nm using a microplate reader (Bio-RAD iMarkTM, America). The Hela cells without any treatments were used as the control. Laser Scanning Confocal Microscopy for Imaging Cu2+ in HeLa Cells. After being incubated for 24 h in 24-well plates at a density of 10000 cells per well, 1 mL of DMEM
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ASSOCIATED CONTENT
* Supporting Information S
1
H NMR and HR-MS spectra, CMC of peptides in the presence and absence of Cu2+, cell inhibition curves, confocal images of 2 in the absence and presence of Cu2+, and LC−MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. *E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge the financial support from NSFC (Grants 51222303, 51373079, and 81301311). We thank Prof. Bing Xu at Brandeis University and Prof. Yi Cao at Nanjing University for their valuable suggestions. We also thank Ms. Nannan Xiao for her kind help with confocal fluorescence microscopy measurements.
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Environment-Sensitive Fluorescent Supramolecular Nanofibers for Imaging Applications Yanbin Cai,† Yang Shi,† Huaimin Wang,† Jingyu Wang,† 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), ‡College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, P. R. China S Supporting Information *
ABSTRACT: The combination of an environment-sensitive fluorophore, 4-nitro-2,1,3-benzoxadiazole (NBD), and peptides have yielded supramolecular nanofibers with enhanced cellular uptake, brighter fluorescence, and significant fluorescence responses to external stimuli. We had designed and synthesized NBD-FFYEEGGH that can form supramolecular nanofibers and emit brighter than its counterpart of NBDEEGGH without the self-assembling property. The nanofibers of NBD-FFYEEGGH could specifically bind to Cu2+, leading to the formation of fluorescence quenched elongated nanofibers. This fluorescence quenching property was enhanced in self-assembling nanofibers and could be applied for detection of Cu2+ in vitro and within cells. In a further step, an enzyme-cleavable DEVD peptide was placed between NBD-FFY and the copper binding tripeptide GGH. The resulting self-assembling peptide NBD-FFFDEVDGGH also showed strong fluorescence quenching to Cu2+. Upon the enzymatic cleavage to remove the Cu2+-binding GGH tripeptide from the peptide, the fluorescence was restored. The cellular uptake of nanofibers was better than that of free molecules because of endocytosis. The supramolecular nanofibers with fluorescence turn-on property could therefore be applied for detection of caspase-3 activity in vitro and within cells. We believe that the combination of environment-sensitive fluorescence and fast responses of supramolecular nanostructures would lead to a useful platform to detect many important analytes.
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transitions.30,35,51 These pioneering works indicated the big potential of molecular hydrogels in rapid and specific detection of many important analytes. Recently, Xu and co-workers reported on an assay to detect intracellular phosphatase activity by in situ fluorescent nanofiber formation.36 In the design, an environment-sensitive fluorescent molecule, 4-nitro-2,1,3-benzoxadiazole (NBD), was conjugated to a precursor of gelator. Upon the triggeration of phosphatase, the precursor could be converted to a gelator, resulting in the formation of nanofibers and molecular gels. Though there was no significant fluorescence intensity change between the precursor solution and the nanofiber solution, this system could still be applied for imaging intracellular phosphatase activity by confocal fluorescence microscopy because the in situ nanofiber formation could result in good contrast in fluorescence signal of nanofibers over the background. This study, in combination with other successful examples of using supramolecular nanofibers in molecular gels for detection applications,34,38 inspired us to design environment-sensitive fluorescent supramolecular nanofibers with enhanced cellular uptake by endocytosis and specific, rapid,
nspired by prevailing examples of functional self-assembled systems in biological systems such as lipid bilayers and tubulin assembly, researchers have developed many supramolecular nanostructures with promising properties. The building blocks for constructing these nanostructures are usually amphiphilic, including block copolymers,1−4 protein− polymer hybrids,5−7 macrocycle derivatives,8−13 and small molecules.14−19 Assisted by noncovalent interactions (e.g., hydrophobic interactions, hydrogen bonding, charge interaction, etc.), these amphiphilic molecules can self-assemble into nanostructures with unusual properties beyond single molecules.14,20,21 Due to the good biocompatibility, multivalent display, and fast responses to external stimuli, supramolecular nanostructures of small molecules have attracted extensive recent research interests. They have been applied for drug delivery,22−28 analyte detection,19,29−38 cell culture,39−43 immune response boosting,44,45 cancer cells and bacterial inhibitions,46−50 etc. Among their application in analyte detection, the semiwet environment in molecular hydrogels has provided a good platform to retain protein activity for enzyme detection.37 The self-assembled nanofibers can offer hydrophobic domains to adsorb fluorescent dyes for the detection of metal ions and polyphosphates by fluorescence changes.32,33 With the aim of enzyme-triggered formation of molecular hydrogels, enzymes and their inhibitors can also be identified by the naked eye, depending on the sol−gel phase © 2014 American Chemical Society
Received: November 27, 2013 Accepted: January 27, 2014 Published: January 27, 2014 2193
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and significant fluorescence changes upon contacting with analytes. In this study, we showed that we could integrate the environment-sensitive fluorescent NBD, an enzyme cleavable peptide linker, and a copper ion-binding tripeptide ligand in one self-assembling molecule. At self-assembled stages, NBD would emit brightly. The binding of the metal ion of Cu2+ to the nanofibers could quench the fluorescence of NBD, leading to the formation of fluorescence-quenched nanofibers. The quenching effect was improved at the self-assembled stage. The big difference in fluorescence could therefore be applied for the detection of Cu2+. Adding enzyme to remove Cu2+ binding tripeptide from the self-assembling nanofibers would restore their fluorescence, which could be applied for the detection of enzyme activity. Taken the advantages of enhanced quenching effect in self-assembling nanofibers, improved cellular uptake of nanofibers by endocytosis, brighter fluorescence of NBD in nanofibers, and significant fluorescence changes upon contacting analytes, the environment-sensitive fluorescent supramolecular nanofibers in our study could therefore be applied for detection of important analytes.
affinity.59 Many studies have shown that copper ions can quench the fluorescence of materials, including inorganic quantum dots, fluorescent dyes, and carbon dots.60−62 Therefore, the addition of Cu2+ to solution of these peptides may trigger morphological changes of self-assembled nanostructures and, more importantly, lead to fluorescence quenching, which could be applied for Cu2+ detection. Self-Assembly Behavior. We first prepared NBD-βalanine in one step (Scheme S-1 of the Supporting Information), which could be directly used for solid phase peptide synthesis (SPPS) to produce the designed peptides. Compounds 1−3 formed homogeneous solutions in aqueous solutions at concentrations lower than 3 wt % (Figure 1A, pH =
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RESULTS AND DISCUSSION Molecular Design of a Probe for Cu2+ Detection. We first designed environment-sensitive fluorescent nanofibers that could be applied for the detection of Cu2+. Unlike general fluorescent molecules that usually exhibit fluorescence quenching properties when they aggregate, environment-sensitive fluorescent molecules and aggregation-induced emission (AIE) fluorescent molecules show relatively stronger fluorescence in hydrophobic environments and upon aggregation, respectively. 52,53 This information, in combination with the aforementioned Xu’s report,36 inspired us to use an environment-sensitive fluorescent NBD to construct short peptidebased hydrogelators because NBD would emit stronger yellow fluorescence in the hydrophobic domain of self-assembled nanofibers than in solutions. Aromatic compounds had been widely used as capping groups to generate molecular hydrogelators of short peptides, such as pyren,54 fluorenyl (Fmoc),55 naphthalene (Nap),56,57 and phenothiazine (PTZ)58 groups. However, these aromatic moieties could only emit short wavelength UV lights, hindering their imaging applications in biological systems. We therefore designed the molecules NBD-FFYEnGGH (Scheme 1, compounds 1, 2,
Figure 1. Optical images of (A) a water solution containing 0.5 wt % of (i) 1, (ii) 2, and (iii) 3 (pH = 7.4) and (B) (iv) a gel, (v) a gel, and (vi) a clear solution formed by adding one equivof CuSO4 to the solution of 1, 2, and 3, respectively. Transmission electron microscopy (TEM) images of (C) a solution of 2 and (D) a gel of 2 with one equivof Cu2+.
7.4). Upon the addition of one equiv of CuSO4 to a water solution containing 0.5 wt % of these peptides, we observed the formation of a gel (Figure 1B, iv), a gel (Figure 1B, v), and a clear solution (Figure 1B, vi) within 5 min for compounds 1, 2, and 3, respectively (Figure 1B). These observations indicated the different self-assembly properties of the three compounds in the presence of Cu2+ and also implied that the three compounds might show different fluorescence responses to Cu2+. The sol−gel phase transition could not be observed for the control peptide of NBD-FFYEEGGK (Figure S-15 of the Supporting Information). Adding one equiv of other metal ions, including Mn2+, Ni2+, Co2+, Fe2+, Zn2+, Ca2+, Sr2+, and Ba2+, respectively, to the solution of 2 could not result in hydrogelations (Figure S-16 of the Supporting Information). The critical micelle concentration for 2 was 0.47 and 0.056 mg mL−1 in the absence and presence of one equiv of Cu2+, respectively (Figure S-17 of the Supporting Information). These observations indicated that the hydrogelation was due to the selective binding of GGH to Cu2+, which was similar to other examples of metal ion-induced molecular hydrogelations.63−65 The gelation of 2 with Cu2+ was due to the elongation of self-assembled nanofibers. As shown in Figure 1
Scheme 1. Chemical Structure of NBD-FFYEnGGH (1, 2, and 3 when n was 1, 2, and 3, respectively)
and 3 for n = 1, 2, and 3, respectively). The rationales of the design are described as following: (i) many short peptides based on FF capped with aromatic groups have been demonstrated as excellent gelators.19 We therefore believe that peptides based on NBD-FFY may also possess an excellent self-assembly ability. (ii) The En are used to adjust the amphiphilicity and fluorescence property of peptides. (iii) The tripeptide GGH can selectively bind to Cu2+ with a high 2194
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(panels C and D), solution of 2 exhibited short fibers with lengths less than 1 μm, while gel of 2 with Cu2+ showed fiber networks and the length of the fibers was longer than several micrometers. The rheological measurements indicated a mechanically weak hydrogel of 2 with Cu2+ (Figures S-18 of the Supporting Information). Fluorescence Response to Metal Ions. We opted to determine which compound among compounds 1−3 would show the most significant fluorescence change to Cu2+. The solution of 2 emitted the strongest fluorescence upon excitation, compared with solutions of 1 and 3 at the same concentration (Figure 2A and Figure S-19 of the Supporting
upon the addition of more and more Cu2+ to the solution of 2 (0.05 wt %, 410 μM) at the Cu2+ concentration range from 0 to 410 μM (0−1 equiv). Upon excitation by a UV lamp (330 nm), we observed bright fluorescence from the solution of 2 while nearly no fluorescence from solution of 2 with one equiv of Cu2+ (Figure 2B, insets). We also recorded the I0/I values of solution of 2 in the presence of 200 μM of different metal ions. The results in Figure 2B showed that this value in the presence of Cu2+ was the biggest (about 10.1), compared to those in the presence of other metal ions (2.0, 3.3, 3.8, 3.2, 4.9, 1.1, 1.0, 1.0, 1.1, 1.1, and 1.1 for Mn2+, Ni2+, Co2+, Fe2+, Zn2+, Mg2+, Ca2+, Sr2+, Ba2+, Na+, and K+, respectively). These observations suggested that 2 could be applied for detection of Cu2+. Intracellular Imaging of Cu2+. MTT assay indicated that the addition of 410 μM of 2 with 100 μM of Cu2+ to HeLa cells showed no obvious toxicity to the cells (Figure S-24 of the Supporting Information), suggesting its good biocompatibility. The good biocompatibility and selective fluorescence quenching of 2 to Cu2+ ensured its application for imaging the intracellular copper ion. As shown in Figure 2C and Figure S-25 of the Supporting Information, HeLa cells incubated with 0.05 wt % (410 μM) of 2 showed a bright fluorescence at the 2 h time point. If pretreating the cells with 100 μM of Cu2+ for 6 h and then treating with 2, there was nearly no detectable fluorescence in HeLa cells even at the 6 h time point (Figure 2D and Figure S-26 of the Supporting Information) probably due to the fluorescence quenching by Cu2+. Adding 100 μM of Cu2+ to HeLa cells treated with 0.05 wt % of 2 for 2 h would also lead to fluorescence quenching (Figure S-27 of the Supporting Information). These observations, in combination with the results in Figure 2 (panels A and B), indicated that 2 could be applied for in vitro and in vivo detection of Cu2+. However, since other metal ions could also quench the fluorescence of 2, our probe could only be applied for approximate detection of Cu2+. Design of a Probe for Imaging Caspase-3 Activity. The selective binding of Cu2+ to the peptide of GGH could strongly quench the fluorescence of supramolecular nanofibers. This information inspired us to design fluorescence turn-on probes using enzyme cleavable peptides to connect NBD-FF and GGH. If so, enzymatic cleavage would remove the Cu2+ binding tripeptide of GGH from the peptides and therefore restore the fluorescence of supramolecular nanofibers. This process might be applied to detect enzyme activity in vitro and within cells. In order to test our hypothesis, we designed and synthesized the NBD-FFFDEVDGGH (5 in Scheme 2) with a Caspasecleavable DEVD peptide. Compound 5 was also synthesized by SPPS and then purified by HPLC.
Figure 2. (A) Fluorescence values of aqueous solution containing 0.05 wt % of 2 in the presence of different equiv of Cu2+, (B) effect of different metal ions (200 μM) on I0/I of solution of 2 (0.05 wt %) (insets in B: fluorescence images of a solution of 2 (0.05 wt %) in the absence (left) and presence (right) of one equiv of Cu2+), and confocal images (bright field + fluorescence) of (C) HeLa cells treated with 2 (0.05 wt %) at a 2 h time point and (D) HeLa cells pretreated with 100 μM of Cu2+ and then treated with 2 (0.05 wt %) at a 6 h time point.
Information), indicating that the number of E in NBDFFYEnGGH could adjust the fluorescence intensity of the peptides in solutions. The I0/I value for 3 to one equiv of Cu2+ was 6.3, which was smaller than that for 2 to one equiv of Cu2+ (21.8, Figure S-20 of the Supporting Information). The solution of 1 would change to a precipitate (or a partial gel) in the presence of Cu2+ (Figure S-21 of the Supporting Information), which was unsuitable for monitoring its fluorescence change by a fluorescence reader. These observations indicated that 2 was the best candidate among compounds 1−3 for detection of Cu2+. We also synthesized compound 4 of NBD-EEGGH lacking FFY (Scheme S-6 of the Supporting Information). Compound 4 could not self-assemble into nanofibers at the concentration of 410 μM (Figure S-22 of the Supporting Information), and the solution of 4 (410 μM) emitted a weaker signal than the solution of 2 at the same concentration (Figure S-23 of the Supporting Information), indicating that the environment-sensitive fluorophore of NBD could emit stronger fluorescence at self-assembled stages. The stronger emission of NBD in nanofibers than at unassembled stages suggested the advantages of supramolecular nanofibers in the design of bright environment-sensitive fluorescent materials. We then monitored the fluorescence change of solution of 2 with the addition of Cu2+ and other metal ions. As shown in Figure 2A, we observed a continuous fluorescence quenching
Scheme 2. Chemical Structure of NBD-FFFDEVDGGH (5)
Self-Assembly and Fluorescence Properties. Similar to 2, compound 5 could form nanofibers in the presence of one equiv of Cu2+ (Figure 3A), and it possessed a similar CMC value (0.156 mg mL−1) to 2 (0.056 mg mL−1) in the presence of Cu2+ (Figure S-28 of the Supporting Information). We then monitored its fluorescence response. As shown in Figure 3 2195
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activity within cells because the complex showed no obvious toxicity to HeLa cells at this concentration (Figure S-32 of the Supporting Information). As shown in Figure 4 (panels A1−
Figure 3. (A) A TEM image of solution of 5 (0.5 wt %, pH = 7.4) with one equiv of Cu2+. (B) Fluorescence intensity of solution of 5 (0.05 wt %) with or without Cu2+ and a solution of 5 (0.05 wt %) with one equiv of Cu2+ and then treated with caspase-3 (1 unit/mL) for 1 h. (C) Optical images of solution of 5 under a UV lamp (i: without Cu2+, ii: with one equiv of Cu2+, and iii: with Cu2+ and then treated with Caspase-3 for 1 h). (D) Fluorescence spectra of a solution of 5 with one equiv of Cu2+ treated with different concentrations of caspase-3 at 37 °C for 40 min.
Figure 4. Confocal images of HeLa cells treated with the 5:Cu2+ complex for 2 h (A1: bright field, A2: fluorescence, and A3: overlap) and HeLa cells treated with the 5:Cu2+ complex for 2 h and then exposed to UV light for an additional 2 h (B1: bright field, B2: fluorescence, and B3: overlap) (before exposure to UV light, the cells were washed with PBS 3 times to remove the extracellular 5:Cu2+ complex).
(panels A−C), a water solution of 5 (0.05 wt %, 356 μM) showed strong fluorescence upon excitation at about 330 nm by a hand-held UV lamp (Figure 3C, i). However, the solution could only emit very weak fluorescence upon the addition of one equiv of Cu2+ (Figure 3C, ii). The I0/I value for fluorescence intensity of solution of 5 without Cu2+ over a solution of 5 with one equiv of Cu2+ was 13.4 (Figure 3B), indicating that Cu 2+ could also strongly quench the fluorescence of 5. The fluorescence was restored when caspase-3 was added to the solution in Figure 3C, ii (Figure 3, iii). The LC−MS result indicated that the GGH tripeptide was removed from 5 by the enzyme (Figure S-29 of the Supporting Information). There was no significant difference in fluorescence intensity between the solution of 5 before and after adding caspase-3 in the absence of Cu2+ (Figure S-30 of the Supporting Information). We also recorded the fluorescence spectra of 5:Cu2+ with the addition of different concentrations of caspase-3. The results in Figure 3D indicated that higher fluorescence signals could be observed when higher concentrations of enzyme were used. These observations clearly demonstrated the success of our design and the complex of 5:Cu2+ could be applied for in vitro detection of caspase-3 activity. Intracellular Detection of Caspase-3. Higher cellular uptakes were recorded for the 5:Cu2+ complex than 5 itself (Figure S-31 of the Supporting Information) at 356 μM (0.5 mg mL−1) and 37 °C. Since the CMC value of 5 in the absence of Cu2+ was higher than 356 μM (1.73 mM or 2.43 mg mL−1, Figure S-28 of the Supporting Information), these observations indicated that nanofibers of the 5:Cu2+ complex could penetrate into cells more easily than the free molecule of 5, highlighting the enhanced cellular uptake of self-assembled nanomaterials. A much lower concentration of 5 within cells treated with nanofibers of the 5:Cu2+ complex was observed at 4 °C than that at 37 °C (Figure S-31 of the Supporting Information), indicating that the uptake of nanofibers by cells was due to endocytosis. We fixed the concentration of the complex of 5:Cu2+ to be 100 μM for imaging the caspase-3
A3), HeLa cells exhibited weak fluorescence upon the treatment of the 5:Cu2+ complex, suggesting the weak caspase-3 activity in cells. UV light could induce apoptosis of cells. We therefore exposed the HeLa cells treated with the 5:Cu2+ with UV light for an additional 2 h (the extra-cellular 5:Cu2+ complex was removed before UV irradiation). We observed bright yellow fluorescence within cells (Figure 4, panels B1−B3), suggesting the high caspase-3 activity within the cells. These observations indicated that the complex of 5:Cu2+ could be applied for the detection of caspase-3 activity within cells. Design of a Probe for GSH Detection. In order to prove the universality of self-assembled nanofibers in detection applications, we designed and synthesized NBD-FFFEE-ssEGGH with the glutathione (GSH)-reducible disulfide bond (compound 6 in Figure 5A). Nanofibers could also be observed through TEM (Figure S-33 of the Supporting Information). The complex of 6:Cu2+ could be applied for detection of GSH because the solution of 6 showed much weaker fluorescence in the presence of one equiv of Cu2+, and the addition of GSH could turn on the fluorescence of 6:Cu2+ (insets in Figure 5B) due to the removal of the copper ion binding GGH peptide. Higher concentrations of GSH would result in bigger fluorescence intensity for solution of 6:Cu2+ (Figure 5B). We also detected the changes of fluorescence values of 6:Cu2+ treated with different concentrations of GSH at 0−60 min. The results showed that higher concentrations of GSH would restore the fluorescence of the complex faster (Figure S-34 of the Supporting Information). These observations suggested the potential of the complex of 6:Cu2+ in the detection of GSH.
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CONCLUSION In summary, we have introduced a supramolecular nanofiber system with environment-sensitive fluorescence property. This system shows a selective copper ion-induced fluorescence quenching phenomenon, which can be applied for detection of copper ion in vitro and within cells. What’s more, through 2196
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Peptide Synthesis. The peptide derivative was prepared by solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding N-Fmoc protected amino acids with side chains properly protected by a tertbutyl group. Piperidine (20%) in anhydrous N,N′-dimethylformamide (DMF) was used during deprotection of the Fmoc group. Then the next Fmoc-protected amino acid was coupled to the free amino group using O-(benzotriazol-1-yl)-N,N,N′,N′tetramethyluroniumhexafluorophosphate (HBTU) as the coupling reagent. The growth of the peptide chain was according to the established Fmoc SPPS protocol. After the last coupling step, excessive reagents were removed by a single DMF wash for 5 min (5 mL per gram of resin), followed by five steps of washing using DCM for 2 min (5 mL per gram of resin). The peptide derivative was cleaved using 95% of trifluoroacetic acid with 2.5% of trimethylsilane (TMS) and 2.5% of H2O for 30 min. Twenty milliliters per gram of resin of ice-cold diethylether was then added to the cleavage reagent. The resulting precipitate was centrifuged at 4 °C for 10 min at 10000 rpm. Afterward the supernatant was decanted, and the resulting solid was dissolved in DMSO for HPLC separation using MeOH and H2O containing 0.1% of TFA as eluents. Characterization of the Peptides. The figures of 1H NMR and HR-MS spectra were in the ESI. NBD-FFYEGGH. 1H NMR (400 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.67 (s, 1H), 8.48 (d, J = 8.9 Hz, 1H), 8.26 − 8.09 (m, 5H), 7.98 (d, J = 6.1 Hz, 1H), 7.28 − 6.96 (m, 13H), 6.63 (d, J = 8.4 Hz, 2H), 6.32 (d, J = 8.7 Hz, 1H), 4.52 (d, J = 6.9 Hz, 4H), 4.31 (d, J = 5.7 Hz, 1H), 3.79 − 3.68 (m, 4H), 3.13 (s, 3H), 3.01 − 2.89 (m, 4H), 2.80 − 2.66 (m, 3H), 2.63 (d, J = 13.5 Hz, 1H), 2.33 (s, 1H), 2.29 − 2.20 (m, 2H), 1.93 (s, 1H), 1.78 (d, J = 7.6 Hz, 1H). HR-MS: Calcd M+ = 1089.39, obsd (M + H)+ = 1090.4021. NBD-FFYEEGGH. 1H NMR (400 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.77 (s, 1H), 8.48 (d, J = 8.9 Hz, 1H), 8.13 (m, 8H), 7.30 − 7.01 (m, 13H), 6.63 (d, J = 8.4 Hz, 2H), 6.32 (s, 1H), 4.50 (dd, J = 12.6, 12.2 Hz, 4H), 4.29 (d, J = 7.5 Hz, 2H), 3.78 (dd, J = 16.6, 4.1 Hz, 4H), 3.66 (d, J = 5.7 Hz, 1H), 3.11 (s, 2H), 3.02 − 2.87 (m, 5H), 2.75 (d, J = 14.1 Hz, 2H), 2.65 (m, 2H), 2.34 − 2.22 (m, 4H), 1.93 (d, J = 5.9 Hz, 2H), 1.78 (d, J = 6.8 Hz, 2H). HR-MS: calcd M+ = 1218.44, obsd (M + H)+ = 1219.4432. NBD-FFYEEEGGH. 1H NMR (400 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.57 (s, 1H), 8.48 (d, J = 9.1 Hz, 1H), 8.24 − 8.01 (m, 8H), 7.13 (m, 15H), 6.63 (d, J = 8.5 Hz, 2H), 6.33 (s, 1H), 4.49 (d, J = 5.1 Hz, 4H), 4.40 − 4.20 (m, 4H), 3.80 (m, 2H), 3.74 − 3.68 (m, 3H), 2.95 (m, 5H), 2.82 − 2.59 (m, 5H), 2.29 (d, 5H), 1.90 (s, 3H), 1.77 (s, 3H). HR-MS: calcd M+ = 1347.48, obsd (M + H)+ = 1348.4864 NBD-FFYEEGGK. 1H NMR (400 MHz, DMSO-d6): δ 9.19 (s, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.24 − 8.03 (m, 9H), 7.10 (dd, 13H), 6.64 (d, J = 8.4 Hz, 2H), 6.33 (d, J = 8.9 Hz, 1H), 4.50 (s, 3H), 4.29 (s, 3H), 4.19 (d, J = 4.7 Hz, 2H), 3.82 − 3.67 (m, 7H), 3.02 − 2.88 (m, 3H), 2.76 (m, 4H), 2.27 (d, J = 8.0 Hz, 4H), 1.93 (s, 2H), 1.78 (s, 3H), 1.53 (s, 2H), 1.30 (dd, 3H). HR-MS: calcd M+ = 1209.47, obsd (M + H)+ = 1210.4813. NBD-EEGGH. 1H NMR (400 MHz, DMSO-d6): δ 9.40 (s, 1H), 8.84 (s, 1H), 8.52 (d, J = 9.0 Hz, 1H), 8.16 (m, 4H), 7.30 (m, 1H), 7.09 (d, 1H), 6.44 (d, J = 8.9 Hz, 1H), 4.54 (td, J = 8.7, 5.3 Hz, 1H), 4.33 − 4.21 (m, 2H), 3.79 − 3.65 (m, 6H), 2.99 (m, 2H), 2.64 (m, 2H), 2.21 (m, 4H), 2.00 − 1.64 (m, 4H). HR-MS: calcd M+ = 761.24, obsd (M + H)+ = 762.2439.
Figure 5. (A) Chemical structure of compound 6 and (B) fluorescence spectra of solution containing 0.05 wt % of 6 with one equiv of Cu2+ treated with different concentrations of GSH at 37 °C for 40 min ([insets in B: fluorescence images of a solution of 6 (0.05 wt %) with one equiv of Cu2+ (left) and treated with 1 mM of GSH (right)].
placing an enzyme-cleavable linker between the NBD-FFY and GGH:Cu2+ complex, this system can be applied for detection of enzyme of caspase-3 by fluorescence turn on. Besides the caspase-3 cleavable DEVD peptide, other peptide substrates of enzymes may also be used to yield supramolecular nanofibers for the detection of activities of other enzymes. The environment-sensitive fluorescent probe of NBD can emit stronger at self-assembled stages than in its free form, and the supramolecular nanofibers can easily penetrate into cells by endocytosis. Besides, unlike free probes that will diffuse easily in the whole cell, supramolecular nanofibers may be more difficult to move in the viscous solution within cells. Therefore, environment-sensitive fluorescent nanofibers may be applied for real time and in situ detection of enzyme activity. Due to the presence of a large pool of ligands that can selectively bind to important analytes and ease of design of self-assembling molecules based on these ligands and short peptides capped with aromatic environment-sensitive fluorophore, we believe our system can be expanded to other environment-sensitive fluorophores and other ligands. These efforts may lead to the development of highly selective sensors for detection of many important analytes.
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EXPERIMENTAL SECTION Chemicals and Materials. Fmoc-amino acids were obtained from GL Biochem (Shanghai, China). All the other starting materials were obtained from Alfa (Beijing, China). Commercially available reagents and solvents were used without further purification, unless noted otherwise. The synthesized compounds were characterized by 1H NMR (Bruker ARX-300) using DMSO-d6 as the solvent. 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 at the LCMS-20AD (Shimadzu) system. HR-MS was performed at the Agilent 6520 Q-TOF LC/MS using the ESI-L low concentration tuning mix (Lot no. LB60116 from the Agilent Tech.). 2197
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solution containing 0.05 mg mL−1 of 2 was added to the HeLa cells. The images were recorded at different time points (excitation wavelength = 488 nm). For Copper ion pretreated HeLa cells, the cells were treated with 100 μM of CuSO4 in DMEM solution for 6 h first. The cells were then washed with PBS 3× before the treatment of DMEM solution containing 0.05 mg mL−1 of 2. The images were recorded at different time points under the same detected conditions (excitation wavelength = 488 nm). This part of the experiment was performed on a laser scanning confocal microscope (OLYMPUS FV1000S-IX81). Laser Scanning Confocal Microscopy for Imaging Caspase-3 Activity in HeLa Cells. HeLa cells were incubated in 24-well plates at a density of 10000 cells per well for 24 h. The DMEM solution containing 0.05 mg mL−1 of 5 with 100 μM of CuSO4 was then added to the HeLa cells. Two hours later, the DMEM solution was removed and cells were washed for 3 times with PBS. Cells were then exposed to 254 nm UV irradiation at 10000 mJ cm−2 for additional 2 h. Images were recorded by confocal microscopy. For the control group, cells were not exposed to the UV irradiation. The images were taken by a laser scanning confocal microscopy (Leica TSC SP8) . Determination of Peptide Concentration in HeLa Cells by LC−MS. HeLa cells were incubated in 24-well plates at a density of 2 × 106 cells per well for 24 h. A stock solution containing 10 mg mL−1 of 5 was prepared (sodium carbonate was used to adjust the pH value to 7.4). Fifty microliters of peptide solution and 950 μL DMEM with 10% FBS were added to cells and the cells were then incubated at 37 or 4 °C (final concentration of peptide was 0.5 mg mL−1). Four hours later, DMEM containing peptide was removed and cells were washed with PBS 3×. Five hundred microliters of DMSO was added to each well to dissolve compounds in cells. After being treated with sonication for 15 min, the solutions were collected and centrifuged at 1570g for 10 min. The amount of peptides in the HeLa cells was determined by LC−MS.
NBD-FFFDEVDGGH. 1H NMR (400 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.83 (s, 1H), 8.52 − 8.33 (m, 3H), 8.22 − 8.00 (m, 5H), 7.91 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.35 − 6.96 (m, 16H), 6.32 (d, J = 8.2 Hz, 1H), 4.61 − 4.46 (m, 5H), 4.35 (d, J = 5.2 Hz, 1H), 4.15 − 4.09 (m, 1H), 3.69 (d, J = 6.5 Hz, 4H), 3.05 − 2.63 (m, 13H), 2.26 (m, 3H), 2.06 − 1.84 (m, 3H), 1.76 (s, 1H), 1.26 (dd, J = 11.2, 5.4 Hz, 2H), 0.82 (dd, J = 6.6, 3.6 Hz, 6H). HR-MS: calcd M+ = 1402.52, obsd (M + H)+ = 1403.5300. NBD-FFFEE-ss-EGGH. 1H NMR (400 MHz, DMSO-d6): δ 8.84 (s, 1H), 8.48 (d, J = 8.7 Hz, 1H), 8.25 − 7.94 (m, 12H), 7.26 − 7.01 (m, 17H), 6.32 (d, J = 9.2 Hz, 1H), 4.66 − 4.44 (m, 5H), 4.26 (m, 4H), 3.71 (t, J = 7.1 Hz, 6H), 3.06 − 2.95 (m, 5H), 2.91 − 2.84 (m, 2H), 2.81 − 2.72 (m, 6H), 2.68 − 2.58 (m, 2H), 2.36 (m, 4H), 2.29 − 2.16 (m, 7H), 1.92 (dd, J = 13.9, 6.8 Hz, 3H), 1.75 (dd, J = 14.8, 7.5 Hz, 3H). HR-MS: calcd M+ = 1565.5340, obsd (M + H)+ = 1566.5439. Hydrogel Formation. Peptides were prepared at a final concentration of 5 mg mL−1 in water solution (Na2CO3 was used to adjust the pH value to 7.4). One equiv of CuSO4 was then added to the solution to initiate hydrogelation. A transparent gel would form within 3 min. Rheology. Rheology was done on an AR 1500ex (TA Instruments) system, 25 mm parallel plates were used during the experiments at the gap of 400 μm. Dynamic strain sweep was performed and the strain values within the linear range were chosen for the following dynamic frequency sweep. The gels were also characterized by the mode of dynamic frequency sweep in the region of 0.1−100 rad/s at the strain value of 1%. Critical Micelle Concentration (CMC). The CMC values of peptides in water solution (pH = 7.4) were determined by dynamic light scattering (DLS). Solutions containing different concentration of peptides were tested, and the light scattering intensity was recorded for each concentration analyzed. Dynamic light scattering (DLS) was performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) at 532 nm under room temperature (22−25 °C). Transmission Electron Microscopy (TEM). TEM samples (5 mg mL−1 NBD-FFYEEGGH and NBD-FFFDEVDGGH with one equiv of Cu2+) were prepared at 25 °C. A micropipet was used to load 5 μL of sample solution to a carbon-coated copper grid. The excess solution was removed by a piece of filter paper. The samples were dried overnight in a desiccator and then conducted on a Tecnai G2 F20 system, operating at 200 kV. MTT Assay. The cytotoxicity of the solutions of peptides and peptide: Cu2+ complex was evaluated by MTT assay. HeLa cells were seeded in 96-well plates at a density of 5000 cells per well and incubated for 24 h. The peptides or peptide: Cu2+ complex in DMEM (Dulbecco’s Modified Eagle Medium) solutions were added into the cells (final concentrations were 0.5 mg mL−1) for different times (2, 4, and 6 h). One hundred microliters of MTT solution (5 mg mL−1 in PBS) was added to each well. Four hours later, the MTT solution was removed and the samples in the wells were air-dried. Fifty microliters of DMSO was added to dissolve the formed crystals; the optical density of the solution was measured at 490 nm using a microplate reader (Bio-RAD iMarkTM, America). The Hela cells without any treatments were used as the control. Laser Scanning Confocal Microscopy for Imaging Cu2+ in HeLa Cells. After being incubated for 24 h in 24-well plates at a density of 10000 cells per well, 1 mL of DMEM
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ASSOCIATED CONTENT
* Supporting Information S
1
H NMR and HR-MS spectra, CMC of peptides in the presence and absence of Cu2+, cell inhibition curves, confocal images of 2 in the absence and presence of Cu2+, and LC−MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. *E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge the financial support from NSFC (Grants 51222303, 51373079, and 81301311). We thank Prof. Bing Xu at Brandeis University and Prof. Yi Cao at Nanjing University for their valuable suggestions. We also thank Ms. Nannan Xiao for her kind help with confocal fluorescence microscopy measurements.
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REFERENCES
(1) Kim, H. J.; Kim, T.; Lee, M. Acc. Chem. Res. 2011, 44, 72.
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Analytical Chemistry
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dx.doi.org/10.1021/ac4038653 | Anal. Chem. 2014, 86, 2193−2199
