Photo- and Aromatic Stacking-Induced Green Emissive Peptidyl

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Biological and Medical Applications of Materials and Interfaces

Photo- and Aromatic Stacking-Induced Green Emissive Peptidyl Nanoparticles for Cell Imaging and Monitoring of Nucleic Acid Delivery Jia Kong, Yuefei Wang, Wei Qi, Rongxin Su, and Zhimin He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03945 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019

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ACS Applied Materials & Interfaces

Photo- and Aromatic Stacking-Induced Green Emissive

Peptidyl

Nanoparticles

for

Cell

Imaging and Monitoring of Nucleic Acid Delivery Jia Kong,† Yuefei Wang,*,†,§ Wei Qi,*,†,‡,§ Rongxin Su,†,‡,§ Zhimin He†

† State

Key Laboratory of Chemical Engineering, School of Chemical Engineering and

Technology, Tianjin University, Tianjin 300072, P. R. China ‡ Collaborative

Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, P. R. China §Tianjin

Key Laboratory of Membrane Science and Desalination Technology, Tianjin

University, Tianjin 300072, P. R. China. *Correspondence

authors: Yuefei Wang (email: [email protected]) and Wei Qi (email:

[email protected]). KEYWORDS: ferrocene-diphenylalanine, photo-induced, aromatic stacking, cell imaging, gene delivery

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ABSTRACT: Owing to their potential applications in biomedicine and biotechnology, peptide nanostructures that exhibit stable intrinsic fluores-cence in the visible range are highly desired. This research proposes a facile strategy to construct peptidyl virus-like nanoparticles (NVPs) that show green luminescence by co-assembly of two bioactive ferrocenediphenylalanine-based (Fc-FF) peptides. The green fluorescence of NVPs was originated from the highly ordered structures assembled by the amphiphilic Fc-FF-based peptides via strong π-π stacking interactions. In the assemblies, Fc-FF chromophore can be hydrolyzed under the natural light irradiation, which eliminates the fluorophore quenching effect of Fc and increases the aromatic stacking interactions, thereby giving rise to strong fluorescent nanoparticles. The NVPs could cross cytomembrane barriers by virtue of the HIV V3 peptide and the nuclear localization signal, and could thus be used for long-term cell imaging with excellent photostability and biocompatibility in physiological condition. In addition, NVPs could package DNA and be used to monitor the delivery of DNA, indicating great potential in the tracking and monitoring of genetic biological processes. INTRODUCTION Peptides have been the focus of many studies in recent years owing to their structural simplicity, biocompatibility, simple synthesis, and capacity to self-assembly into a variety of functional nanostructures.1-7 Just like fluorescent protein,8 self-assembled peptide nanostructures with fluorescent properties in the visible light range are highly desirable due to their promising applications in biomedical fields.9-13 Currently, the construction of fluorescent peptide nanostructures is typically achieved by doping with dyes,14, 15 application of quantum dots (QDs) or conjugation of the peptides directly with an aggregation-induced emission (AIE) chromophore.16-20 However, the photobleaching and cytotoxicity of fluorescent dyes and QDs remains a concern for biomedical applications.21, 22 Accordingly, the construction of peptide nanostructures with tunable visible fluorescent emission that arises from the peptide itself is highly desirable. 2


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Diphenylalanine (FF)-based peptides were first reported to self-assemble into quantumconfined nanotubes or nanowires that exhibit intrinsic fluorescence, mainly in the deep ultraviolet emission (~285 nm) and blue emission (~450 nm) ranges.13, 23-25 These fluorescent nanostructures may show limitations as probes or nanocarriers for cell imaging and targeted drug delivery owing to their large size, lacking of binding peptide sequences and unphysiological assembling conditions. To control the size and broaden the fluorescent spectral region of the peptide nanostructures, cyclization of the aromatic dipeptides or doping with metal ions has been proposed.10,

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The visible emission signal allows these aromatic

peptides to act as promising candidates for biomedical use and opto-electric applications.27, 28 However, construction of multifunctional peptidyl nanoparticles that exhibit strong visible fluorescence, excellent bioactivity, and targeting capability under mild conditions still remains a formidable challenge. In this study, we designed peptidyl virus-like nanoparticles (NVPs) showing stable luminescence in the green spectral range. The NVPs were co-assembled spontaneously using two ferrocene-diphenylalanine-based (Fc-FF) peptides: Fc-FF based nuclear localization signal (Fc-FF-NLS) and Fc-FF based third variable loop (Fc-FF-V3) (Figure S1). The Fc-FF segment acts as a hydrophobic head, which facilitates the self-assembly of the peptides into fluorescent NVPs.29 The V3 peptide, derived from the third variable loop of the human immunodeficiency virus, endowed the NVPs with cell-penetrating capability,30 whereas the NLS peptide of simian virus 40 conferred the NVPs with nuclear localization ability.31 When the NVPs were co-assembled by Fc-FF-NLS and Fc-FF-V3 via noncovalent interactions, the Fc group in the Fc-FF moiety simultaneously hydrolyzed under photo-irradiation, yielding an FF derivative that retained the cyclopentadiene unit at the N-terminus (CPD-FF). This eliminated the quenching effect of Fc, and the NVPs showed strong green luminescence (Scheme 1).17,

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The biocompatible NVPs were photostable for several days under

physiological conditions and could penetrate the cell membrane barriers and be used for cell 3



imaging without cytotoxicity. In addition, the fluorescent NVPs system could efficiently enclose nucleic acid and monitor the delivery of genetic drugs. Compared with previously reported luminescent peptide nanoparticles, which were usually self-assembled via ultra-short aromatic peptides, both of the molecular building blocks used in our system were composed of long bioactive peptide sequences. Molecular dynamic simulations showed that the hydrophobic CPD-FF sequence of the peptide localized in the core of the nanoparticles via strong π-π stacking interactions, which may be responsible for the strong fluorescence induced by AIE.

Scheme 1. Self-assembly of bioactive peptidyl nanoparticles (NVPs) with strong green fluorescence via simultaneously non-covalent self-assembly and photolysis of the Fc moiety. The Fc-FF moiety could serve as a hydrophobic group to facilitate the co-assembly of bioactive peptides (Fc-FF-NLS and Fc-FF-V3) into well-defined nanoparticles. Under photoirradiation, the Fc group within the nanoparticles will be hydrolyzed in water, giving rise to the fluorescent chromophore CPD-FF at the N terminus of the peptide. In this process, the nanoparticles will not disassemble. Instead, the aromatic stacking of the CDP-FF moiety between peptides within the nanoparticles imparts the system with strong green luminescence. The green emissive NVPs can be used for cell imaging and monitoring of nucleic acid delivery by virtue of its cellpenetrating and DNA-packaging capabilities.

RESULTS AND DISCUSSION Photo-induced green emission of peptidyl nanoparticles.

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To study the optical behaviors of the designed molecules, lyophilized peptides were dissolved in 50 mM phosphate-buffered saline (PBS, pH = 7.2) and then incubated for aggregation for 24 h at 25°C under natural light irradiation. Optical absorption experiments showed that the Fc-FF-NLS and Fc-FF-V3 share similar absorption spectra, with a strong absorption peak at 240–285 nm and three weak absorption peaks at ~308, 352 and 445 nm (Figure S2). The absorption peaks at 240–285 nm could be assigned to the phenylalanine unit, whereas the spectra in the near-UV-visible region (308–445 nm) exhibited a step-like structure, indicating the formation of intermolecular hydrogen bonds.33 The photoluminescent spectra of the peptide solutions were further characterized by excitation at 260, 308, 352, and 445 nm. For both Fc-FF-NLS and Fc-FF-V3 assemblies, fluorescent emission peaks at ∼313 and 426 nm were observed when excited at 260 nm (Figure S3). Intriguingly, the spectra also showed that self-assembled peptides could emit green light with a maximum at 518–525 nm, and this emissive peak became more distinct when excited at 308, 352, and 445 nm (Figure S3). The emission peak of the peptide solution at ~280–360 nm was a fluorescent signature of phenylalanine residues, and the peaks at around 370 and 426 nm may be induced by AIE. The green fluorescent emission of the peptide solutions with a prominent peak at ∼520 nm has not been reported in previous works. The excitation spectra of the two peptide solutions showed a maximum excitation peak at 490 nm when the emission wavelength was set to 520 nm (Figure 1A). The visible fluorescence of the peptide solutions may be induced by the self-aggregation of the Fc-FF-NLS and Fc-FF-V3 peptides.34 To verify this assumption, we investigated the fluorescent emission spectra at 520 nm as a function of time, and the structure of the peptide assemblages in solution using atomic force microscopy (AFM). As shown in Figure 1B, the fluorescent emission intensity of both Fc-FF-NLS and Fc-FF-V3 increased gradually with time, consistent with the self-assembly of Fc-FF-NLS and Fc-FF-V3 peptides, forming spherical nanoparticles (Figure 1C and 1D). To gain further insights into the AIE, we 5



dissolved the Fc-FF-NLS and Fc-FF-V3 peptides in methanol or acetonitrile under photoirradiation and measured their morphologies and corresponding fluorescent emission spectra (Figure S4). AFM images showed that the Fc-FF-NLS or Fc-FF-V3 in methanol and acetonitrile could not aggregate and that the peptide solutions showed no fluorescent emission. These results demonstrated that the visible fluorescent emission peak at 518–525 nm was caused by the aggregation of Fc-FF-NLS and Fc-FF-V3 molecules in water.

Figure 1. (A) Photoluminescence (PL) spectra of Fc-FF-NLS and Fc-FF-V3 peptides. The excitation spectra (dashed line) and emission spectra (solid line) of these two peptides (4 mM) were characterized after incubation for 24 h at 25°C under photoirradiation, respectively. (B) PL intensity of Fc-FF-NLS (4 mM) and Fc-FF-V3 (4 mM) at 520 nm measured as a function of time at 25°C. (C-D) AFM images of FcFF-NLS and Fc-FF-V3 monomeric peptides (C) and assembled spherical nanoparticles (D) at 15 min. (E-F) MALDI-TOF-MS of Fc-FF-NLS (E) and Fc-FF-V3 peptides (F). Mass spectra were measured when the peptides were dissolved in H2O for 0 h (blue line) or 48 h (red line) under photo-irradiation. (G)

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Fluorescent emission spectra of Fc, FF, Fc-FF, NLS, V3, CPD-FF-NLS, and CPD-FF-V3 after incubation in 50 mM PBS (pH 7.2) for 24 h under photo-irradiation. The concentration of the peptides or Fc molecules was 4 mM. (H-I) Fluorescent images of CPD-FF-NLS (H) and CPD-FF-V3 assemblies (I) in green-fields (excitation at 488 nm). (J) FTIR absorption spectrum in the amide I region of CPD-FF-NLS and CPD-FFV3. (K) Schematic illustration showing the self-assembly and photolysis of Fc-FF-NLS and Fc-FF-V3 into green emissive nanoparticles.

Photo-irradiation plays crucial roles in the formation of green emissive NVPs. When the peptides self-assembled in 50 mM PBS (pH = 7.2) under darkness, the solutions showed weak fluorescent emission (Figure S5). Fc is a fluorophore quencher, and its hydrolysis occurs in aqueous solution under irradiation by natural light when conjugated with a fluorophore.35-37 Interestingly, the hydrolysis of Fc was also observed in Fc-FF-NLS and Fc-FF-V3 molecules (Figure 1E and 1F). Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of Fc-FF-NLS showed a strong peak at m/z 1447.7, which represented the molecular mass of Fc-FF-NLS, and a weak peak at m/z 1328.6 (Figure 1E). When the Fc-FF-NLS peptides were dissolved in aqueous solution and incubated for 48 h under natural light irradiation, MALDI-TOF-MS of Fc-FF-NLS showed a prominent peak at m/z 1328.6, and the peak at m/z 1447.7 disappeared completely (Figure 1E). The Δm/z for these two peaks matched the gross mass of CPD unit and iron(II), which suggested that the Fc moiety in Fc-FF-NLS molecules may hydrolyze in the presence of both light and H2O (Figure S5).36 Similarly, MALDI-TOF-MS of the Fc-FF-V3 peptide exhibited a peak at m/z 2526.8 initially and a new peak at m/z 2406.8 after the Fc-FF-V3 was incubated in H2O under natural light irradiation for 48 h (Figure 1F). The MS characteristics of these new peaks were further analyzed by evaluating the second-order mass spectrum. The MS results confirmed the Δm/z (~120) origin from the hydrolysis of the Fc group (Figure S6), i.e., the removal of a CPD unit and iron(II) from Fc in Fc-FF-NLS and Fc-FF-V3, yielding new peptide derivatives that retained the other CPD unit at the N-terminus (CPD-FF-NLS and CPD-FF-V3; Figure 1E and 1F). The MS results also explained why the fluorescent emission intensities of both Fc-FF7



NLS and Fc-FF-V3 grew dynamically with time (Figure 1B). As shown in Figure 1D, Fc-FFNLS and Fc-FF-V3 peptides self-assembled into nanoparticles within 15 min in water. However, the visible fluorescent emission intensities of the two peptide assemblies were rather weak at 15 min (Figure 1A and 1B), which may result from the fluorophore quenching effect of Fc. Over time, the reaction of Fc-FF-NLS and Fc-FF-V3 nanoparticles with H2O induced by light irradiation led to the removal of a CPD unit and iron(II) of Fc, eliminating the fluorophore quenching effect of Fc and the increase in the fluorescent intensity of the peptide nanoparticles (Figure 1B). The results of the fluorescent, AFM, and MS analyses demonstrated that both the aggregations and hydrolysis of the Fc-FF-NLS and Fc-FF-V3 peptide are needed for the formation of fluorescent nanoparticles. In addition, the low fluorescent intensity of Fc-FF-V3 compared with that of Fc-FF-NLS may arise from its low hydrolysis efficiency, resulting from its poor solubility, and thus, the intrinsic photoluminescence (PL) was largely quenched by the Fc group (Figure 1F). To investigate the chromophore within the self-assembled peptide nanoparticles, the fluorescent emission spectra of Fc, FF, Fc-FF, NLS, V3, Fc-FF-NLS, and Fc-FF-V3 peptides were measured after incubation in PBS for 24 h under natural light irradiation. The three FcFF-based nanostructures (Fc-FF nanofibrils, Fc-FF-NLS nanospheres, and Fc-FF-V3 nanospheres) shared similar fluorescent excitation/emission spectra in the visible region, with a strong fluorescent emission peak at ∼520 nm (Figure 1G–I and S7). In contrast, Fc, FF, NLS, and V3 molecules do not show any green fluorescence at ∼520 nm when excited at 490 nm (Figure 1G). Therefore, we assumed that the origin chromophore of visible green fluorescence did not solely depend on the Fc molecule or the aromatic residues of FF but was defined by the new hydrolytic product of the CPD-FF sequence that formed during the hydrolysis of the Fc-FF peptide derivatives (Figure S8). To further support these findings, enzyme hydrolysis experiments were conducted using α-chymotrypsin (which preferentially cleaves phenylalanine), trypsin (which preferentially cleaves lysine and arginine), and 8


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protease K (a broad-spectrum protease). The photon emission intensity in the green region of CPD-FF and CPD-FF-NLS solutions showed dramatic reduction after digestion with αchymotrypsin and protease K for 24 h, whereas the fluorescent intensity of the peptide solutions digested with trypsin showed little decrease (Figure S9). Interestingly, due to their poor solubility, CPD-FF-V3 nanoparticles were insensitive to the three enzymes. Nevertheless, the enzyme hydrolysis experiments revealed that aromatic residues in CPD-FF-NLS and CPD-FF-V3 were the origin chromophores of green luminescence, not NLS or V3.

Figure 2. (A) Fluorescent emission spectra and corresponding AFM images of the nanoparticles coassembled by Fc-FF-NLS and Fc-FF-V3 at different molar ratios. (B) The fluorescent emission spectra and corresponding morphology were analyzed to investigate the effects of pH on the optical properties of the NVPs. (C) Time-resolved emission spectra and the lifetime of 0.2 mM Fc-FF-NLS, Fc-FF-V3, and NVPs in 50 mM PBS at pH 7.2 after photoirradiation. (D) Quantum yields (QYs) of 0.2 mM Fc-FF-NLS, Fc-FF-

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V3, and NVPs in 50 mM PBS at pH 7.2. The ultraviolet absorption of the sample at the excitation peak was controlled at 0.07–0.08. (E) Photostability evaluation of NVPs and the organic dye R6G. The NVPs and R6G were irradiated (once per s) for 1000 s under light. (F) Cytotoxicity evaluation and comparison for NVPs and R6G against Magi cells (CD4+/CCR5+) using MTT assays. There were three technical replicates. (G) In vitro fluorescence cellular imaging of Magi cells (CD4+/CCR5+) treated with NVPs (0.1 mM) for 4– 72 h. The green color represents the fluorescence signal of the NVPs. Scale bars: 23 μm.

To gain insights into the intermolecular interactions within green fluorescent nanoparticles, Fourier transform infrared (FTIR) spectroscopy was performed to identify the molecular arrangements of CPD-FF, CPD-FF-NLS, and CPD-FF-V3. The FTIR spectra showed bands centered around 1,650 and 1,678 cm−1 in the amide I region, indicating that aggregation occurred via strong intramolecular hydrogen bonding (Figure 1J).10 The carboxylate peak broadened and was shifted to a lower frequency of 1,536 cm−1 because of salt-bridge formation (Figure 1J).38 The formation of the intramolecular hydrogen bonding and salt-bridge suggested a well-ordered peptide nanostructure.39, 40 Together with the spectral data in the near-UV-visible region presented in Figure S2, these data demonstrated the wellorganized nanostructure for the self-assembled green fluorescent nanoparticles. In summary, there were two major reasons why Fc-FF-NLS and Fc-FF-V3 could emit green luminescence in solution (Figure 1K): 1) Fc-FF-NLS and Fc-FF-V3 self-assembled into nanoparticles driven by hydrophobic interactions and hydrogen bonding, and 2) the Fc-FF chromophore within the self-assembled Fc-FF-NLS and Fc-FF-V3 nanoparticles hydrolyzed in the presence of light and H2O, eliminating the fluorophore quenching effect of Fc and thereby giving rise to strong green emissive peptidyl nanoparticles (Figure S4, S10, 1B, and Table S1). Optical properties for NVPs. To construct NVPs with both strong fluorescence and cell-penetrating capacity, we coassembled Fc-FF-NLS and Fc-FF-V3 molecules in a series of molar ratios and characterized their fluorescent emission spectra and morphology. Due to the good solubility and thorough 10


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hydrolysis, the fluorescent intensity of self-assembled Fc-FF-NLS (4 mM) at 520 nm was about 15-fold stronger than the intensity of Fc-FF-V3 (4 mM) under the same conditions (Figure 2A). Additionally, the fluorescent intensity of the co-assembled NVPs showed a large fluorescence enhancement by increasing the proportion of Fc-FF-NLS until a ratio of 10:1 (blue dashed line). AFM imaging and fluorescent emission spectra showed that the coassembled NVPs (at a molar ratio of 1:10) showed a morphology and fluorescent intensity similar to those of the self-assembled Fc-FF-V3, which exhibited an irregular aggregation state and low fluorescent intensity (Figure 2A and 1D). When the ratio was equal to 1:1, the NVPs showed a smaller size with an average diameter of 206 nm and a large PL intensity enhancement. The final optimal molar ratio of Fc-FF-NLS to Fc-FF-V3 was 10:1 for NVPs, yielding a strong fluorescent intensity (approximately equal to the fluorescent intensity of FcFF-NLS) and uniform size distribution around 66 nm. To determine the main factors for the optical properties of NVPs, a series of experiments were conducted for evaluation of metal ions, pH, solvents, and ion concentrations. A previous study revealed that the metal ion-dipeptide coordination structure resulted in enhanced blue fluorescence.10, 26, 27 Various metal ions, including Cu(II), Zn(II), Fe(II), Ca(II), Mg(II), and Fe(III) were applied to the co-assembled NVPs to study their effects on the fluorescent intensity (Figure S11). As a result, no enhancement of fluorescent emissions with the addition of metal ions was observed; conversely, the fluorescent intensities of these NVPs showed a dramatic decrease. The weak acidity of these metal ion sulfate solutions (pH 11.3) buffer solutions reduced the fluorescent intensity of NVPs, whereas the phosphate buffer at pH 8.1 provided the mostintense fluorescent signal. The AFM images suggested that the NVPs at pH 4.7 or 12.2 showed large particles with diameters of more than 500 nm; however, NVPs at pH 8.1 existed 11



in uniform sized nanospheres (~95 nm). Similar to Fc-FF-NLS and Fc-FF-V3, NVPs coassembled by Fc-FF-NLS and Fc-FF-V3 at a molar ratio of 10:1 could not emit green fluorescence in methanol, but show an enhancement of fluorescent intensity at 520 nm by increasing the solvent ratio of PBS (50 mM, pH 7.2) over 40%, indicating the green PL of coassembled NVPs also arose from aggregation (Figure S12). In addition to the factors discussed above, the effects of ion concentrations were also considered (Figure S13). The results demonstrated that ion concentrations of 50–500 mM enhanced the fluorescent intensity of NVPs, whereas concentrations that were too low or too high weakened the fluorescent intensity. Overall, the above results demonstrated that the NVPs could be used in alkaline (7.0 < pH < 11.2) and hypohaline solutions (50–500 mM), indicating that NVPs could emit strong fluorescent under physiological conditions. To further evaluate the properties of NVPs, the time-resolved fluorescence decay kinetics and quantum yields were determined. As shown in Figure 2C, the emission lifetimes of the self-assembled CPD-FF-NLS, CPD-FF-V3, and NVPs were 5.48, 2.71, and 4.92 ns on average at 520 nm, respectively. Because the NVPs were mainly composed of Fc-FF-NLS, the lifetime of NVPs was more approximate to that of Fc-FF-NLS. The quantum yields (QYs) of the assembled CPD-FF-NLS, CPD-FF-V3, and NVPs were 19.96%, 32.77%, and 25.6%, respectively (Figure 2D), which were higher than the recently published QYs of aromatic peptide nanostructures.10, 26 In order to evaluate the photostability and biocompatibility of NVPs, a comparison between the NVPs and the organic dye rhodamine 6G (R6G) was conducted. As shown in Figure 2E, the fluorescent intensity of the NVPs remained stable after irradiation (once per s) for 1000 s, whereas the fluorescent intensity of R6G showed a ~10% reduction, indicating better photostability for NVPs. We next evaluated the cytotoxicity of NVPs by performing 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays in Magi cells (CD4+/CCR5+) in vitro. The MTT assay results showed that NVPs, CPD-FF-NLS, and CPD12


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FF-V3 had almost no toxicity compared with that of R6G (Figure 2F), suggesting that NVPs may be sufficiently safe for further biomedical applications. To demonstrate the suitability of the NVPs for bioimaging, confocal laser scanning microscopy (CLSM) was used to evaluate the localization of NVPs. As shown in Figure 2G, the significant green fluorescence of the NVPs was observed from Magi cells (CD4+/CCR5+) after 4 h of incubation. Notably, strong fluorescence was observed at 72 h post-incubation due to the good photostability and biocompatibility of the NVPs (Figure 2E-G). Overall, our experiments demonstrated that the NVPs exhibited photostability and biocompatibility as a fluorescent nanoprobe and may be good candidates for cell-imaging in the visible light range. Real-time monitoring of nucleic acid delivery. We finally evaluated the feasibility of the NVPs for monitoring genetic drug delivery in real time. As we previously reported, V3 and NLS viral peptides can encapsulate plasmid DNA through strong electrostatic interactions, forming viral-like particles.41 Meanwhile, FcFF-V3 and Fc-FF-NLS can be labeled with a fluorescein isothiocyanate (FITC) without disrupting the peptide assembly, thus we first compared the cytotoxicity between green NVPs and FITC labeled peptidyl virus-like particles (pVLPs). The result showed that both NVPs and FITC labeled pVLPs have good biocompatibility (Figure A1). However, the photostability between pVLPs and NVPs is quite different. As shown in Figure A2, the fluorescent intensity of the NVPs remained stable after irradiation for 1000 s, whereas the fluorescent intensity of FITC-pVLPs showed a ~65% reduction, indicating this system may be more desirable than the previous work in cell imaging and nucleic acid monitoring due to its excellent photostability. In this work, we found that Fc-FF-NLS and Fc-FF-V3 could completely retard the DNA (Cy5-labeled branched DNA, 471-mer) at N/P ratios above 4 (Figure 3A and 3B) via strong electrostatic interactions.41 Agarose gel electrophoresis revealed that the co-assembled NVPs could retard DNA when the N/P ratio of NVP/DNA was 6 and the molar ratio of Fc-FF-NLS/Fc-FF-V3 was 10:1 (Figure 3C). 13



AFM images showed the formation of uniform nanospheres of NVP@DNA with an average size of 82 nm, when Fc-FF-NLS and Fc-FF-V3 were mixed with DNA for 25 min (Figure 3D). In addition, CLSM was used to evaluate the co-localization of NVP@DNA when NVPs hydrolyzed for 2 h under natural light irradiation. CLSM images suggested that green NVPs and pink Cy5-DNA colocalized in solution, indicating that the NVP peptides could package DNA efficiently (Figure 3E). To investigate DNA delivery and monitor the capacity of NVPs, Cy5-DNA and NVP@DNA were incubated with Magi cells (CD4+/CCR5+). Compared with Magi cells (CD4+/CCR5+) treated with Cy5-DNA, an obvious cell uptake of NVP@DNA was observed in the cells treated with the NVP@OLD (Figure 3F). However, the Cy5-DNA could only bind to the cellular membranes. These results indicated that the green NVPs could efficiently enclose nucleic acid and serve as an optical nanoprobe to monitor gene-based drug delivery.

Figure 3. (A-B) Agarose gel electrophoresis of Fc-FF-NLS@OLD (A) and Fc-FF-V3@OLD (B) complexes at different N/P ratios. M: DNA marker. (C) Electrophoretic mobility shift assay for NVP@OLD. The molar ratio of Fc-FF-NLS/Fc-FF-V3 was 10:1, and the N/P ratio of NVP/OLD was 6. (D) AFM images of NVP@OLD. (E) CLSM images showed the colocalization of NVPs (green particles)

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and Cy5-labeled OLD (pink dots). Scale bars: 9 μm. (F) CLSM images of Magi cells (CD4+/CCR5+) treated with Cy5-OLD and NVP@OLD. Scale bars: 9 μm.

Mechanisms for green fluorescence. Recent studies have shown that aromatic peptidyl nanostructures can emit intrinsic fluorescent signals in the visible range when forming cyclized aromatic dipeptides or when doped with metal ions.10, 26-28 Accordingly, the underlying mechanisms were interpreted by quantum confinement effects, shallow radiative traps,42 and electron delocalization.33 In our work, the relationship between the green fluorescence and aggregate state of NVPs was very clear from our experimental research.

Figure 4. Possible mechanisms for fluorescence. (A) Molecular diagram of the CPD-FF peptide. (B) Representative coarse-grained molecular dynamics self-assembly process of 600 CPD-FF-NLS molecules leading to the formation of spheres in H2O. (C) Restriction of intramolecular motions (RIMs) leading to AIE phenomenon. IM, intermolecular motion. The upward pointing black arrows indicate UV absorption. The down arrows and red arrows represent the transition probability. The non-radiative transition can be ascribed to the intramolecular motion, thus resulting in weak PL. (D, E) The free energy surface of CPD-

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FF-NLS assemblies as a function of the angle and the centroid distance between two inter- (D) and intramolecular (E) aromatic rings. (F, G) Simulation structure from an all-atom MD run (G) starting from our reconstructed atomistic structure of the CPD-FF-NLS sphere from CG beads (F). (H) All-atom MD simulations of a fine-grained structure of a sphere showed that two adjacent aromatic rings in the assembled nanostructures adopted a T-shaped orientation. O atoms are shown in red, N atoms are shown in blue, and C atoms are shown in yellow.

As shown in Figure 4A, a single CPD-FF molecule had one cyclopentadienyl and two phenyl residues linked through single bonds. When monomeric peptides are dissolved in organic solvent, i.e., methanol and acetonitrile, the phenyl rings and cyclopentadienyl ring rotate or vibrate against the stator on the single bond axes to dissipate energy in three conformations (gauche+, trans, and gauche-).43 Thus, most of the energy of the excited monomeric CPD-FF will be consumed through vigorous intramolecular motions before radiative transition, resulting in weak emission. However, when CPD-FF or its derivatives self-assemble into nanospheres (Figure 1D, 2A-2B, and 3D), the aromatic groups would aggregate into a three-dimensional aromatic stacking nanostructure with rigid restriction of intramolecular motions (RIMs) (Figure 4B),17 which blocks the nonradiative pathway and then turns on the fluorescent emission in the visible range (Figure 4C). To further elucidate the luminescence mechanism of self-assembled CPD-FF-based peptide architectures, we performed coarse-grained molecular dynamics (CG-MD) simulations of a system consisting of 600 CPD-FF-NLS peptides in water owing to its excellent luminescent properties.38, 44, 45 Starting from a disordered state, CPD-FF-NLS selfassembled into clusters by rapid aggregation in water within 124 ns (Figure 4B). Following this aggregation, the clusters started to fuse and merge into one large sphere. Finally, a stable solid spherical structure formed in water after 1250 ns calculations. This self-assembly process was consistent with the process observed by scanning electron microscopy (SEM) analysis that the peptide monomer first self-assembled into small-sized spheres and these spherical nanoparticles further aggregated into large spheres with increased incubation time 16


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(Figure S16). In the final simulation structure, most of the phenyl rings of CPD-FF-NLS assembled into the core of the peptidyl nanostructure. We calculated the intra- and interpeptide aromatic ring angles using the CG model to character the aromatic stacking mode of CPD-FF-NLS. The calculation result showed that the interpeptide aromatic ring angle was distributed mainly from 40° to 90°, with a high probability around 90° at a minimum distance of ∼0.57 nm between the centers of the two benzene rings, suggesting that aromatic stacking preferred to adopt a T-shaped stacking mode. The distribution of the intrapeptide aromatic ring angle showed a similar feature with a distribution mainly in the range from 30° to 90°, demonstrating the crucial role of aromatic ring stacking in stabilizing self-assembled CPDFF-NLS nanoparticles (Figure 4D-E). To accurately examine the role of aromatic stacking, we use GROMACS to generate a fine-grained structure based on the CG-MD trajectories.46 To save computational time, we selected a sphere consisting of 31 CPD-FF-NLS peptides as the start model (Figure 4F) and performed a 100-ns all-atom MD simulation. The total feature, hydrophobic, and hydrophilic solvent accessible surface areas of the spheres did not change much during MD simulation (Figure 4G). The final simulation results showed that the phenyl rings adopted a T-shaped stacking pattern between two inter-/intra-molecular CPD-FF-NLS peptides (Figure 4H). In addition, we further calculated the free energy surface as a function of the angle and the centroid distance between intermolecular aromatic rings and cyclopentadiene rings. The results revealed characteristics similar to those of the intrapeptide aromatic ring stacking mode, with a main distribute in the range from 40° to 90° with a low energy of around 90° at a minimum distance of ∼0.57 nm, indicating the crucial role of the cyclopentadiene ring in stabilizing self-assembled CPD-FF-NLS nanoparticles (Figure S17). The simulation results supported that stacking of aromatic rings and cyclopentadiene rings contributed to RIM, which may be responsible for the AIE phenomenon of CPD-FF based peptides.

17



CONCLUSION We demonstrated that Fc-FF peptide derivatives, including Fc-FF-V3 and Fc-FF-NLS, could self-assemble into green fluorescent NVPs with excellent photostability and biocompatibility. The Fc-FF moiety initially served as a hydrophobic head that could promote the self-assembly of the peptides in water into well-defined nanoparticles. Intriguingly, after photo-irradiation, the Fc moiety within the nanoparticles will be hydrolyzed, leaving a cyclopentadienyl ring at the N-terminus of the peptides. This process not only eliminated the fluorescent quenching effect of the Fc moiety but also increased the π-π stacking interactions within the self-assembled nanoparticles, leading to the formation of NVPs with strong green luminescence as a result of the AIE effect.29,

36

Unlike the ultraviolet emission or blue

emission of nanostructures self-assembled by short aromatic FF derivatives, the NVPs had a narrow emission bandwidth and visible fluorescent spectrum in the green range under physiological conditions. Moreover, the bioactive V3 and NLS peptide sequences imparted the NVPs with excellent cell-penetrating30 and nuclear localization capabilities.31 The NVPs could penetrate the cytomembrane barriers for long-term cell imaging without cytotoxicity, package nucleic acids, and deliver DNA into vulnerable cells. Further studies are needed to evaluate the biomedical applications of NVPs for the tracking and monitoring of other biological and genetic processes.

EXPERIMENTAL SECTION Chemicals and Materials: Ferrocene-diphenylalanine (Fc-FF), Fc-FFGPKKKRKV (Fc-FFNLS), and Fc-FFGCRKSIHIGPGRAFYTTGC (Fc-FF-V3) were synthesized by GL Biochem Ltd. (China). 4',6-Diamidino-2-phenylindole (DAPI) and 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation and cytotoxicity assay kits were purchased from KeyGen Biotech. Co., Ltd. (China). HEK293T cells were provided by Professor Mi Lizhi (Tianjin University). Magi cells (CD4+/CCR5+) were a gift from Dr. Liang 18


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Weizi (Tianjin University). All other chemicals were purchased from Sigma-Aldrich (China) and used without further purification Preparation of Fc-FF-based peptidyl nanoparticles and NVPs: Lyophilized Fc-FF and FcFF-NLS peptides were dissolved in PBS (50mM, pH 7.2) at a concentration of 4 mM and then incubated for 15 min at 25°C under light irradiation, resulting in the formation of selfassemblies. The lyophilized Fc-FF-V3 peptide was dissolved in ddH2O at a concentration of 40 mM. and then diluted in PBS (50mM, pH 7.2) at a final concentration of 4 mM, which was then incubated for 15 min at 25°C under light irradiation. For the formation of NVPs, dissolved Fc-FF-V3 was diluted in Fc-FF-NLS at different molar ratios and was further incubated for 15 min at 25°C under light irradiation. To enclose nucleic acids, DNA was added to the NVP solution at predefined N/P ratios and incubated for 25 min at 25°C. Fluorescence spectroscopy: The fluorescence signal of peptide solutions (4 mM, 100 μL) was measured in 96-well plates on an EnSpire Multilabel Reader (PerkinElmer). The step widths were set to 2 nm, and the measurement height was set to 9.5 mm. For time-resolved fluorescence, the intensity was recorded at 30-min intervals. Atomic Force Microscopy: The morphology of the peptide nanostructures (0.8 mM) was characterized using tapping mode AFM on an AFM5500 system (Agilent Technologies) with silicon probes (PPP-NCL-20, Nanosensors; resonant frequency 146–236 kHz, force constant 21–98 N/m). The samples were prepared by depositing a drop (10 μL) of the peptide solution on a fresh silicon wafer for a 30-s adsorption, and the excess buffer was removed by purging with nitrogen gas. Images were then recorded using Picoscan software and were processed using Gwyddion software. Scanning electron microscopy: A drop (10 μL) of peptide solution was placed onto a clean glass slide and allowed to adsorb for 30 s. The excessive liquid was removed with filter paper. The slide was coated with Au for 90 s and observed under a S-4800 field-emission scanning electron microscope (Hitachi, Tokyo, Japan) operated at 3 kV. 19



Identification of peptides by MALDI-TOF-MS/MS: A MALDI-TOF/TOF target mass spectrometer (MTP 384; Bruker Daltonics, Billerica, MA, USA) equipped with a Bruker Daltonics Autoflex TOF/TOF III was used for MALDI-TOF-MS/MS analysis. The samples (Fc-FF based peptides) were hydrolyzed in water under the light. Spectra were measured and processed using mMass software. Fluorescence imaging: The fluorescence images of peptide nanoparticles were characterized using an IX73 system (Olympus). The solution of self-assembled peptidyl nanoparticles (0.8 mM) was placed on a 6-well culture plate and then removed using a pipette. Fluorescence image were recorded in bright field and dark field. FTIR spectroscopy: The samples (3 mg/mL, 1 mL) were prepared and vacuum dried in Eppendorf tubes. Spectra were recorded on a Nicolet 6700 (Thermo Nicolet Corporation) and corrected for absorption from a phosphate-buffered blank sample. Spectra were measured at 4 cm−1 resolution and by averaging 32 scans. The spectra were then processed using EZ OMNIC software. Quantum yield: The absolute quantum yield of peptidyl nanoparticles was measured using the integrating-sphere method in Fluorolog (Horiba Scientific). The absorption of the sample at maximum excitation wavelength was adjusted to 0.07–0.08. The excitation range was set to 480–500 nm, and the luminescence range was set to 499.8–750 nm. Fluorescence lifetime: The time-resolved PL of Fc-FF based peptides was evaluated on a Fluorolog3 instrument (HORIBA JOBIN YVON, Japan) with excitation at 350 nm. The lifetimes of 0.2 mM Fc-FF-NLS, Fc-FF-V3, and NVPs were measured in 50 mM PBS (pH 7.2). Cell culture: Magi cells (CD4+/CCR5+) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere containing 5% CO2 using 25 cm2 tissue-culture flasks.

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In vitro cytotoxicity assay: The cytotoxicity of peptide nanoparticles was evaluated in Magi cells (CD4+/CCR5+) using MTT assays. Cells were seeded in 96-well plates for 24 h to allow the cells to attach to the wells and then treated with peptide nanoparticles and R6G at a range of concentrations (0–100 μM, 100 µL). After 48 h of treatment, 50 μL MTT was added, and cells were incubated for an additional 4 h. The resulting formazan crystals were dissolved in 150 μL DMSO, and their absorbance at 570 nm was measured. All assays were performed in triplicate. Gel electrophoretic mobility shift assays (EMSAs): EMSAs were performed at increasing N/P ratios of peptide and plasmid DNA (2 μg). The assembled NVP@DNA was loaded onto 1% (w/v) agarose gels (100 V, 30 min). DNA retardation was then visualized using a UV illuminator with a Gel Doc System (Bio-Rad, Hercules, CA, USA). The N/P ratio was the protonatable nitrogen (N) of an amino acid to the phosphate (P) of deoxyribonucleotides, and the formulas are depicted in Equation (1) and (2).41 𝑚𝐹𝑐 ― 𝐹𝐹 ― 𝑉3(𝜇𝑔)

𝑁/𝑃𝐹𝑐 ― 𝐹𝐹 ― 𝑉3/𝐷𝑁𝐴 = 0.51

(1)

𝑚𝐷𝑁𝐴(𝜇𝑔) 𝑚𝐹𝑐 ― 𝐹𝐹 ― 𝑁𝐿𝑆(𝜇𝑔)

𝑁/𝑃𝐹𝑐 ― 𝐹𝐹 ― 𝑁𝐿𝑆/𝐷𝑁𝐴 = 1.12

(2)

𝑚𝐷𝑁𝐴(𝜇𝑔)

Here, mFc-FF-V3 is the mass of the Fc-FF-V3 peptide, mFc-FF-NLS is the mass of the Fc-FF-NLS peptide, and mDNA is the mass of DNA. The N/P ratio of Fc-FF-NLS@DNA and Fc-FF-V3@DNA were 6. For (Fc-FF-V3 + Fc-FFNLS)@DNA, we set the N/P ratio of Fc-FF-NLS/DNA to 6 and the N/P ratio of Fc-FFV3/DNA to 0.3. In this case, the total DNA were packaged by two peptides, and the molar ratio of Fc-FF-V3 to Fc-FF-NLS (nFc-FF-V3: nFc-FF-NLS) was equal to 1:10. Confocal fluorescence imaging: Magi cells (CD4+/CCR5+) were grown on glass-bottomed dish (NEST) for 24 h and then incubated with 0.1 mM NVPs, NVP@DNA, or Cy5-labled DNA (0.1 μM) for 4–72 h. Cells were then washed three times with PBS and fixed in 4%

21



paraformaldehyde (5 min). Nuclei were stained with DAPI (1 min) and observed by confocal microscopy (UltraView Vox, Perkin Elmer). Coarse-Grained MD simulation: All MD simulations were performed using GROMACS 5.0.4 software.47 CG MARTINI V2.2 force field was used to model the CPD-FF-NLS peptides.38,

46

We have performed 3 MD simulations on a 40×40×40 nm3 lattice model

consisting of 600 CPD-FF-NLS peptides. To reduce computational cost, we used high peptide concentrations (22.5 mg/ mL).48 The simulation time for each MD run was 1250 ns. All MD simulations were carried out in the NPT ensemble with GROMACS software, and the detailed parameters were as follows: time step, 25 fs; pressure and temperature, 1 atm and 298 K, respectively, using coupling constants of 3.0 and 0.3 ps, respectively. van der Waals interactions were calculated using a cutoff of 1.2 nm. The neighbor list was updated every 10 steps with a cutoff distance of 1.2 nm. MD Simulations Analysis ： Analysis was performed using in-house-developed codes and GROMACS facilities.49 The free energy landscapes were constructed using -RTln P(x,y), where P(x,y) is the probability of the particular conformation.46 The two reaction coordinates x and y were the solvent-accessible surface area and the degree of dispersion of the peptides, respectively. The latter was obtained by the equation Ddisp =1–nmax/N, where nmax was the maximum number of FF peptide molecules in a single assembled cluster and N was the total number (600) of CPD-FF-NLS peptides in the lattice. The angle between two benzene rings is calculated by the angle between the surface normal of the two rings. The number of interpeptide hydrogen bonds is also calculated. All the snapshots were drawn using VMD software.50 ACKNOWLEDGMENTS

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Photo- and Aromatic Stacking-Induced Green Emissive Peptidyl

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