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Biological and Medical Applications of Materials and Interfaces

Self-healing Organic Fluorophore of Cyanine Conjugated Amphiphilic Polypeptide for Near Infrared Photostable Bioimaging Tuanwei Li, Le Liu, Titao Jing, Zheng Ruan, Pan Yuan, and Lifeng Yan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02621 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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ACS Applied Materials & Interfaces

Self-healing Organic Fluorophore of Cyanine Conjugated Amphiphilic Polypeptide for Near Infrared Photostable Bioimaging

Tuanwei Li, Le Liu, Titao Jing, Zheng Ruan, Pan Yuan, Lifeng Yan *

Hefei National Laboratory for Physical Sciences at the Microscale, CAS Key Laboratory of Soft Matter Chemistry, iChEM, and Department of Chemical Physics, University of Science and Technology of China. Hefei, 230026, P.R.China.

Corresponding

author:

Lifeng

Yan,

Fax/Tel:

+86-551-63606853;

[email protected]

1

ACS Paragon Plus Environment

E-mail:

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract: Photobleaching and bio-toxicity are the main bottlenecks for organic fluorescent dyes applied in real-time dynamic monitoring of living cells. Here, an unnatural amino acid (UAA), 4-nitro-3-phenyl-L-alanine (NPA), was used as a scaffold to covalently link a near infrared (NIR) fluorophore Cy5.5, and an amphiphilic

polypeptide

Poly[oligo(ethylene

glycol)

methyl

ether

methacrylate]-block-poly[2-Amino-N4-(2-diisopropylamino-ethyl)-L-Aspartic

acid]

(P(OEGMA)21-P(Asp)16-iPr) was then conjugated for increasing the photostability and improving the biocompatibility simultaneously. The protective agent of NPA can service as an effective triplet state quenching by intramolecular electron transfer between the Cy5.5 and NPA. The less sensitivity of electron transfer process for molecular oxygen makes it an ideal photostabilized strategy for fluorophores applied in live-cell imaging. Bonding to copolymer is a common way for hydrophobic dyes to expand their application in biomedical imaging and increase their functionality depending on the delivery system. The results indicate that Cy5.5-NPA linked polypeptide

copolymer

exhibited

enhanced

photostability

and

excellent

biocompatibility, which means this scaffolding strategy has potential application in fluorescence-guided surgery (FGS), lived-cell imaging and super resolution microscopy.

Keywords: self-healing fluorophores, photostability, photobleaching, bioimaging, polypeptide.

2


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Introduction Extrinsic organic fluorophores can serve as versatile tools for awareness and understanding of bio-systems by transmitting the messages of dynamic and functional information in biological systems at high spatial and temporal resolution.1-2 Organic fluorophores, especially NIR ones, are also frequented used in biological imaging,3 medical diagnostic2 and super-resolution microscopy4 for their modifiable photophysical properties,5-7 linking flexibility1 and small perturbation to the system.8 However, the inherent instability of fluorophores, containing the transient switch between bright and dark states (blinking) and the irreversible photoinduced degradation (photobleaching), limits their applicability in super-resolution imaging and real-time monitoring of the dynamic processes.9-10 Therefore, improving the photostability of organic fluorophores is correspondingly in great demand across various scientific disciplines.11 Photostability of organic fluorophores is prominently influenced by non-fluorescent triplet excited state (T1), which derives from the first singlet excited state (S1) by intersystem crossing process (ISC).12 The fluorophores in their T1, rare but with high energy and long lifetime, can react with molecular oxygen, biomolecules and solvent impurity by electron transfer reactions or triplet-triplet energy transfer process to produce a series of reactive oxygen species (ROS) and then induce photoblinking (non-fluorescent radical species) and photobleaching (degradation).13-14 So, many strategies have been adopted to diminish the influence of T1 to photostability. The utility of buffer additives, for example, reducing and oxidizing system (ROXS) is the 3



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most effective method to suppress the photoblinking and photobleaching.15 Basing on the collision theory of chemical reaction, stabilizers such as 1,3,5,7 cyclooctatetraene (COT), ascorbic acid and trolox can reduce the lifetime of fluorophore triplet states or radical states and then increase photostability.4, 16 However, when it comes to living cells, the methods mentioned above are constrained for their draconian demands on the systems and millimolar concentrations for additives, which are hostile to living systems.17 Hence, the strategy that covalently conjugate photostabilizers to fluorophores

was

introduced18-19

and

expanded

to

photostabilization, naming “self-healing fluorophores”.10,

achieve 20-23

intramolecular

Though the lower

increase in photostability competed with solution-based healing, self-healing fluorophores is still the priority for biological imaging due the reproducible stabilization effects, increased effective local concentration of the stabilizer rather than solution concentration and subdued biotoxicity. In fact, different approaches to improve the photostability of organic fluorophores are used altogether to achieve optimal results. Aggregation in aqueous solutions is another problem encountered by most organic fluorophores by their hydrophobicity.24 But, linking flexibility of the organic fluorophores makes it easy to attach to almost any molecule of interest, such as the polymer, which means it can be endowed different functions depending on the polymers.1, 25-27 Recently, polypeptides has been widely used in bioimaging and smart drug delivery systems for their biocompatibility and biodegradability and could be easily prepared using N-carboxy-anhydride monomer (NCA) monomer via 4


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ring-opening polymerization (ROP). Herein, a polypeptide based polymeric self-healing macro-fluorophore was synthesized by directly linking NPA to Cy5.5 and then bond connecting to an amphiphilic polymer of Poly[oligo(ethylene glycol) methyl

ether

methacrylate]-block-poly[2-Amino-N4-(2-diisopropylamino-ethyl)

-L-Aspartic acid] (P(OEGMA)21-P(Asp)16-iPr). The stabilizer of NPA is considered as an effective triplet state quenching by intramolecular electron transfer processes, and can increase the photostability of the dyes even in the presence of molecular oxygen.28-29 The polymer system can eliminate the disadvantage of Cy5.5 hydrophobic and broaden the application of the cyanine dye. As shown in the MTT and photobleaching experiments upon HepG2 cells, polymeric dyes shows excellent biosecurity and promising potential application in real-time monitoring of living cells.

Results and discussion Synthesis and photophyscial properties of NPA-Cy5.5. Covalent linkage of single photostabilizers to small organic fluorophores was an alternative to improve the photophysical performance of fluorophores.22, 28-29 As an excellent scaffold to link two organic molecules with different reaction of functional groups, unnatural amino acids (UAAs) is proposed to synthesize self-healing fluorophore derivatives.28 Here, the NPA was used as a stabilizer to synthesis the self-healing fluorophore. The scaffold of NPA still retains a reactive carboxyl functional group after covalent linkage with Cy5.5, which can be attached to amide groups of polymers to be easily applied in live-cell imaging.28 5



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On the basis of our group previous work,30 the NPA-Cy5.5 was synthesized according to the route presented in Scheme 1. Target products with high purity were obtained and confirmed by the 1H-NMR,

13

C-NMR, and mass spectrometry showing

in the Figure 1 and Supporting Information. As a key intermediate in the synthesis process of NPA-Cy5.5, Cy5.5 is a common cyanine fluorophore used in imaging of tumors for its high molar extinction coefficients (ε= 1.62×105 M-1 cm-1 in DMF), competitive fluorescence quantum yield (Φ = 0.23) and satisfactory optical absorbance and fluorescence emission in NIR region (λmax = 693nm, λem =726nm in dichloromethane, Figure 2). After introducing photostabilizer NPA, the fundamental optical properties of NPA-Cy5.5 (ε= 1.43×105 M-1 cm-1 in DMF, Φ = 0.27, and λmax = 693nm, λem =724nm in dichloromethane, Figure 2) has no noticeably difference compared with Cy5.5, indicating that the addition of NPA has negligible effects on the conjugate structure of fluorophores. The spectral properties of NPA-Cy5.5 and Cy5.5 in representative solvents all showed the nearly uniform profiles, Figure S1. Moreover, the maximum absorption and emission wavelengths of NPA-Cy5.5 and Cy5.5 in DCM, DMF, and methanol show an obvious hypsochromic

shift

with

increased

solvent

polarity,

that

is

negative

solvatochromism.31

Synthesis and Characterization of PC/PN and PC0.05/PN0.05 An amphiphilic polymer of P(OEGMA)21-P(Asp)16 (Scheme 2) was prepared by click reaction of azido modified hydrophilic P(OEGMA)21-APA and hydrophobic 6


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P(Asp)16 initiated with propargylamine.32 FT-IR spectral was used to monitor the completion of the click reaction. As shown in Figure S2, the disappearance of the peak at 2150 cm-1, a characteristic of the antisymmetric stretching vibration of azido group,

confirmed

the

end

of

the

reaction.

1

H-NMR

spectrum

of

P(OEGMA)21-P(Asp)16 in DMSO-d6 was shown in Figure S3. The integration ratio of peak y at 7.26 ppm (protons of the benzyl group) and peak b at 1.25 ppm (protons of dodecyl from the initiator) is 80:19, closed to the theoretical value of 80:18. GPC traces of P(OEGMA)21-APA, P(Asp)16, and P(OEGMA)21-P(Asp)16 were measured to evaluate the molecular weight distribution of these polymers (Figure S4). Narrow molecular distribution (PDI=1.2) and lower elution time of P(OEGMA)21-P(Asp)16 indicted the target product was synthesized effectively. To increase the hydrophilicity of the polymer, an aminolysis with excess N,N-diisopropylethylenediamine was performed. The 1H-NMR spectra contrast of P(OEGMA)21-P(Asp)16 and P(OEGMA)21-P(Asp)16-iPr was shown in Figure S3. The disappearance of the benzyl group signals (7.3 ppm) and the enhancement of terminal tertiary amino (1.0 ppm) for P(OEGMA)21-P(Asp)16-iPr indicated the full conversion. Hydrophobicity of the cyanine fluorophores limited their application in the field of biomedical imaging. Here, the cyanine dyes were conjugated with copolymer to obtain the enhanced solubility and biocompatibility. The 1H-NMR signals of PC/PN were identified by the peak at 7.0-8.0 ppm recognized as the protons of the indole and the benzyl group (Figure S5, S6). However, because the proportion of dyes in polymers is too low for PC0.05/PN0.05, the 1H-NMR signal is hard to identify. The GPC 7



traces using RI detectors were tested to make clear the molecular structure of the polymers, and slightly attenuated elution time for PC/PN indicated that the more dyes were successfully covalently attached to the polymer in comparison with PC0.05/PN0.05 (Figure S7). The fundamental optical properties of PC/PN and PC0.05/PN0.05 in methanol was presented in Table S1. After conjugating with amphiphilic polypeptide, the solubility of the PC/PN and PC0.05/PN0.05 in water increased significantly (Figure S9). The absorption and fluorescence spectra of the dyes in water was showing in the Figure 3. The content of dyes in the polymers was calculated based on UV-vis absorbance measurements, as shown in the Table S2.

Photostability of Cy5.5 and NPA-Cy5.5 in solution To evaluate the photostability of Cy5.5 and NPA-Cy5.5, a series of photobleaching experiments were implemented in the mixed solution of DMF and PBS buffer under ambient conditions. As shown in Figure 3, the photobleaching curves of the dyes at a concentration of 10µM were measured using a 635nm laser in a gradient DMF solutions. In 10% volume percent DMF solvent, the two dyes shared similarity in their dramatic photobleaching characteristic: a 90% decrease of absorbance was observed within 300s. When increasing the content of DMF in solvent from 10% to 40%, the difference of NAP-Cy5.5 and Cy5.5 in photostability becomes more pronounced. As shown in Figure 3b, the NPA-Cy5.5 still retained 66% of its absorption intensity, while only 11% for Cy5.5 after being irradiated for 600s by laser, showing superior photostability of NPA-Cy5.5. But, as the DMF content continuing to 8


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increase, the photostability of Cy5.5 increased faster than NPA-Cy5.5. The two dyes still maintained high absorbance (34% for Cy5.5 and 43% for NPA-Cy5.5) even if they were irradiated for 1500s. These suggest that the photobleaching properties of Cy5.5 and NPA-Cy5.5 have a strong dependence on solvents. The photostability was enhanced with the increase of DMF in mixed solvent, indicating a remarkable dependence on organic solvent. Nevertheless, the effect of organic solvent on NPA-Cy5.5 isn’t the main influencing factor of photobleaching as the photostability has no significant improvement after the proportion of DMF in the solvent reached to 30% For a quantitatively calculating of the relative increase of photostability, the absorbance of Cy5.5 and NPA-Cy5.5 at different irradiation time was recorded and fit to an exponential decay. Here, a mono-exponential decay [Eq. 1] was plotted and the characteristic bleaching time constantτbleach was defined as: τbleach = (-1/R0) for a quantitative comparison on the photostability of the dyes23. y=y0+A*expR0*x

[1]

The bleaching time constant of Cy5.5 and NPA-Cy5.5 in different mixed solvents was presented in Figure 4. As stated before, the triplet excited state (T1) of the dyes is a key stage in photobleaching, in which the fluorophore tended to recover the ground state by energy transfer process (closely correlated with the concentration of O2) or electron transfer reaction (closely related to the solvents), as shown in the Scheme 3. When increasing the polarity of solvents, reducing the ration of DMF in our experiment, the electron transfer reaction was accelerated and radical states of the 9



fluorophore increased. The long-lived and highly reactive radical states of the fluorophore can pronounce photobleaching, which is agreed with the reduction of the bleaching time constant of Cy5.5 when reducing the content of DMF in solvents (Figure 3a). However, theτbleach of the NPA-Cy5.5 has a slight reduction even if the content of DMF reduced to 30% (Figure 3b). That is because the stabilizer of NPA can recover the fluorophore from radical states to the ground state by intramolecular electron transfer processes and impair the influence of radical states to the photostability of the dyes. The poor photostability of NPA-Cy5.5 in the mixed solvents with 10% and 20% DMF cannot be explained by the mechanism of self-healing depicted in Scheme 3. It is possible that the dissolving state of the dyes, which varying depending on the solvents for the hydrophobic property of the organism dyes, influences the photostability. So, the absorbance of the dyes was used as an indirect criteria to evaluate the solubility in different solvents (Figure S8). In 30% DMF content solvent, the absorbance of NPA-Cy5.5 reached the maximum and maintains stable even with an increase of DMF content (Figure S8b), which hints the sufficiently dissolved of the dyes. However, when reducing the adding of DMF in solvent, a significantly reduces of absorbance was observed, which is consistent with the poor photostability of NPA-Cy5.5. Then, we can speculate that the photobleaching of NPA-Cy5.5 may be closely related to the dissolving state. Intramolecular photostabilization by introducing triple-state quenching agents, such as NPA, is a feasible method to improve the photostability of dyes. This strategy 10


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appears to be a favorite choice in biological fields for its special demands of water although the beneficial effect of NPA is suppressed in the organic solvent for its charge transfer mechanism of triple-state quenching.33

Photostability of Cy5.5 and NPA-Cy5.5 in living cells Photostability experiments of Cy5.5 and NPA-Cy5.5 in living cells were studied by means of confocal laser scanning microscopy (CLSM), HepG2 cells were plated on 20 mm glass petri dishes at a density of 6000 cells per dish for 24 h. After discarding the culture medium, fresh DMEM containing 2% DMSO and 3 µM Cy5.5 and NPA-Cy5.5 was added and incubated for 5 h. As shown in Figure 5, the both dyes showed a relatively weak fluorescent intensity at a longer exposure time (713 ms) and rapid photobleaching behavior (about 120 s), which was consistent with the photobleaching experiment in solvent (Figure 3a). The photostability of the NPA-Cy5.5 is tied to the solubility and improved the water solubility of the dyes is an effective way to release the basic photostability instincts of the NPA-Cy5.5.

Photostability of PC/PN and PC0.05/PN0.05 in living cells For hydrophobic fluorescent dyes, conjugated with polymers is an effective way to expand its application in living cells. However, it's inevitable even for polymeric dyes that aggregation induced quenching (AIQ) maybe highlighted along with the increasing concentration of dyes.34 Just as is depicted in Figure S9, an anomaly that strong fluorescence enhancements as the photobleaching time went on was observed 11



at a concentration of 50µg/mL for PC/PN. As shown in Figure 6, the fluorescence intensity of PC increased quickly at the first five measurement points (about 27s) and then weakened in the subsequent 200s (Figure 6a and 6c). In contrast to this, a slow increasing process (about the first 130s) and mitigating photobleaching (25% decrease of fluorescence intensity after the subsequent 400s irradiation) for PN was observed (Figure 6b and 6d), indicating conjugating amphiphilic copolymer can promote the photostability of the dye, especially for the NPA-Cy5.5. It was also found in our studies that fluorescence recovery from the fluorescence quenching state induced by homo Förster resonance energy transfer (homoFRET) is a common phenomenon, especially for the supramolecular self-assembly.35 Size change of the micelles under light irradiation is slight measured by DLS, indicates that the fluorescence enhanced phenomenon in the begin stages of the irradiation cannot be construed as increasing distance among the dye molecules (which diminishing the influence of FRET) caused by the volume expansion of polymer systems. Here, a photobleaching-induced fluorescence enhanced mechanism was put forward to account for this phenomenon, as shown in Scheme 4. The fluorescence molecules were in very crowded status before photobleaching, which induced very serious fluorescence quenching by homoFRET at the beginning (weak fluorescence signal). But, after partial dye molecules were bleaching, the less crowded status among the dyes eased the fluorescence quenching (stronger fluorescence signal). In order to verify the hypothesis, a sample photobleaching experiment was implemented in DMEM media. As shown in Figure 7, the PC/PN at a concentration of 12


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300µg/mL showed the similar photobleaching characteristics as in the above experiments. When cutting the portions of dyes in the copolymer (PC0.05/PN0.05, the polymer bonding without to dye could be treated as bonding to a photobleaching dye), the fluorescence intensity of the system decayed with the irradiation time, which is consistent with the assumption. Therefore, it’s necessary to minimize the use of dyes by reducing the adding of PC/PN or decreasing the ratio of dyes to polymer as PC0.05/PN0.05 to mitigate effects caused by AIQ. Photobleaching is a common challenge for most organic dyes during prolonged monitoring of dynamic events inside living cells in the presence of oxygen.36 To evaluate the photostability of the dyes in living cells, an Olympus IX71 fluorescence microscope was used to qualitatively compare the photostability of the PC/PN or PC0.05/PN0.05 in a large region, and a CLSM was selected to quantitatively evaluate the photobleaching of the dyes in a single cell. HepG2 cells were incubated for 12 h with PC/PN or PC0.05/PN0.05 before washing with PBS for photobleaching testing using an Olympus IX71 fluorescence microscope. Figure 8 shows fluorescence microscope images of PC/PN acquired at different irradiance time. It can be found that, both PC and PN had very high fluorescent intensity, even if at low dyes concentration (25µg/mL) and short exposure time (100 ms) before performing photobleaching experiments. However, as the photobleaching experiments went on, the fluorescent intensity of PC rapidly reduced and fluorescence practically disappeared after irradiated for 2 minutes (Figure 7a), exhibiting poor photostability. By comparison, the fluorescence signal of PN remained can be distinguish even after irradiated for 13



longer than 6 minutes (Figure 7b), indicating an increased photobleaching resistance. The synchronous experiment for PC0.05/PN0.05 was presented in the Figure S11. For CLSM, a shorter culture time about 5 h for HepG2 cells with the dyes was essential. As shown in Figure 9, the representative photobleaching images were presented with exposure times of 968 ms and 25 bleaching iterations between the two scans. PC0.05 and PN0.05 are dyes staining in the cytoplasm of living cells and there is negligible fluorescence in the cell nucleus (Figure 9). The fluorescence of PC becomes too faint to detect after 48 repeated cycles of consecutive bleaching, indicating the end of the bleaching. In the meantime, the brightness of PN0.05 weakens slightly and obvious bleaching was recorded until 100 repeated cycles, which mean enhanced photostability of PN0.05. 2.5-dimensional (2.5D) fluorescence intensity profiles at different bleaching time were presented in Figure 9d and 9e, showing the photobleaching process, visually. The mean fluorescence intensity of the region of interest (ROI) over time was autonomously extracted by LSM 710, and the photobleaching curves were shown in Figure 9c. As shown, 250s later, the bleaching experiment of PC0.05 was automatically stopped for the fluorescence intensity reaching the threshold (fallen below 5% of initial intensity). PN0.05, however, still retained 60% fluorescence till 500s later, demonstrating the significant advantage than PC0.05 for bioapplications. Similar result for PC/PN can be found in Figure S10. In addition, it is worth noting that AIQ happened every once in a while, although in a low concentration of 250µg/mL for PC0.05/PN0.05 or 15µg/mL for PC/PN, for the active transport of living cells to the macromolecular. When scanning the area near 14


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the cells, the photobleaching characterization of PC0.05/PN0.05 in PBS media can also be acquired by CLSM (Figure 10). The counting of the emitters in the images represented the fluorescence intensity at the image capture time points. It can be found that the reduction in the number of fluorescence emitters was obvious with the repeated scanning of the same area of the PBS media for PC0.05, while there was only slightly reduced for PN0.05, indicating improved photobleaching resistance. The improvement in photostability via an intramolecular electron transfer reactions highlights the potential applications of super-resolution stimulated emission depletion (STED) microscopy in living samples for self-healing fluorescence probe without need of the chemical additive and extra oxygen scavenging systems.

Cytotoxicity The MTT method was used to evaluate the biocompatibility of PC/PN and PC0.05/PN0.05 against the HepG2 cells. As shown in the Figure 12, more than 90% cell viability for PC/PN and PC0.05/PN0.05 at the tested concentration for photobleaching shows their excellent biosecurity. Meanwhile, the dyes were still low-toxic even at a higher concentration, indicating an excellent application prospect in biomedical imaging.

Conclusions A self-healing small-molecule fluorophore NPA-Cy5.5 was synthesized by covalent linkage a triplet state quenching NPA to a widely used cyanine dyes Cy5.5. The 15



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NPA-Cy5.5 performs excellent photostability in the mixed solvents of PBS and DMF. However, the NPA-Cy5.5 was bleached rapidly at relatively low content of DMF for its poor solubility and negligible improvement in photostability at high content of DMF for its suppressed charge transfer process in organic solvent. After bonding to an amphiphilic polypeptide copolymer, the macro-fluorophore can effectively improve biocompatibility and imaging contrast, which is desirable for biomedical imaging. Photobleaching experiments were carried out in living cells, a significantly improved photostability was recorded. In conclusion, conjugated with copolymer for self-healing macro-fluorophore is an efficient way to expand its application in biological imaging, and may be a potential candidate for fluorescence-guided surgery, super resolution microscopy and real-time dynamic monitoring of living cells.

Experimental Section Materials Unless otherwise noted, all reagents (AR purity) were used as obtained without any further purification. 1,1,2-Trimethyl-1H-benz[e]indole, 6-Bromohexanoic acid, methyl iodide, CuBr,

N-hydroxysuccinimide ( HOSu ), Dicyclohexylcarbodiimide (DCC),

4-nitro-3-phenyl-L-alanine,

malondialdehyde

bis(phenylimine)

monohydrochloride, Poly[oligo(ethylene glycol) methyl ether methacrylate (OEGMA, Mw= 450), N,N,N’,N’,N’-pentamethyldiethylene triamine (PMDETA) were purchased from Aladdin Corporation ( China ). Fetal bovine serum (FBS) and dulbecco modified eagle medium (DMEM) were purchased from Sangon Corporation (China). Dialysis 16


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bag (cutoff Mw = 3000) was obtained from Bomei Biotechnology Corporation. Other organic solvents and chemicals were purchased from Sigma / Sinoreagent Corporation. Tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and n-hexane were dried with CaH2. Milli-Q Synthesis System (18.2 M, Millipore, USA) was applied in the preparation of ultrapure water.

General Methods All 1H-NMR and

13

C-NMR spectra were recorded on Bruker AV300 or Bruker

400M NMR spectrometer in the fourier transform mode. The CDCl3 or DMSO-d6 containing 0.03 v/v% tetramethylsilane (TMS) as internal standard were used to dissolve the samples. FT-IR spectra in a range of 400 - 4000 cm-1 were recorded on a Bruker EQUINOX 55 spectrometer using a KBr disk as a reference background. The molecular weight of the samples was measured using a LC-20AD Gel permeation chromatography (GPC, Shimadzu). DMF( HPLC grade ) was used as the mobile phase ( 1.0 mL/min ) at 60 oC.

Synthesis of 1,1,2,3-tetramethyl-1H-benzoindolium iodide 1.56g (7.45 mmol, 1.0 equiv.) of 1,1,2-Trimethyl-1H-benz[e]indole was dissolved in 20 mL toluene. Then 0.46 mL (8.94 mmol, 1.25 equiv.) of methyl iodide was added to the solution and the mixture was refluxed at 110 oC overnight. After cooling to room temperature, the precipitate was separated from the solution by filtration, and washed with diethyl ether to obtain the target product as gray powder (1.92g, 73.5% 17



yield). 1

H NMR (300 MHz, DMSO-d6):δ 8.38 (d, J = 8.3 Hz, 1H), 8.30 (d, J = 8.9 Hz, 1H),

8.22 (d, J=8.1Hz,1H), 8.12 (d, J = 8.9 Hz, 1H), 7.83-7.69 (m, 2H), 4.10 (s, 3H), 2.88 (s, 3H), 1.76 (s, 6H). 13

C NMR (101 MHz, DMSO-d6):δ 196.37 , 139.93 , 136.96 , 133.48 , 130.98 ,

130.21 , 128.84 , 127.60 , 127.58 , 123.88 , 113.62 , 55.72 , 35.57 , 21.74 , 14.46.

Synthesis of 3-(5-carboxypentyl)-1,1,2-trimethyl-1H-benzoindolium bromide 2.88g (13.8 mmol, 1.0 equiv.) of 1,1,2-Trimethyl-1H-benz[e]indole was dissolved in 15 mL 1,2-dichlorobenzene. Then 3.0g (15.4 mmol, 1.1 equiv.) of 6-bromohexanoic acid was added to the solution and the mixture was refluxed at 110 o

C for 16h. After cooling to room temperature, the precipitate was washed with

diethyl ether and dried under vacuum to obtain the target product as aubergine solid (3.87g, 69.4% yield). ESI-MS m/z: [M-I]+ calcd for C40H43N2O2, 583.33; found, 583.33075. 1

H NMR (400 MHz, DMSO-d6):δ 8.39 (d, J = 8.1 Hz, 1H), 8.30 (d, J = 8.9 Hz, 1H),

8.23 (d, J = 7.7 Hz, 1H), 8.17 (d, J = 8.9 Hz, 1H), 7.82-7.70 (m, 2H), 4.59 (t, J = 7.7 Hz, 2H), 2.96 (s, 3H), 2.24 (t, J = 7.2 Hz, 2H), 1.97-1.86 (m, 2H), 1.77 (s, 6H), 1.63-1.53 (m, 2H), 1.53-1.42 (m, 2H). 13

C NMR (101 MHz, DMSO-d6):δ 196.84 , 174.80 , 138.96 , 137.43 , 131.15 ,

130.19 , 128.87 , 127.74 , 123.90 , 113.82 , 55.96 , 48.12 , 33.84 , 27.64 , 25.87 , 24.53 , 22.07 , 14.27 . 18


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Synthesis of Cy5.5-NHS 1.0g (2.85 mmol, 1.0 equiv.) of 1,1,2,3-tetramethyl-1H-benzoindolium iodide was dissolved in 30 mL of acetic anhydride. Then, 0.96g (3.70 mmol, 1.3 equiv.) of malondialdehyde bis(phenylimine) monohydrochloride was added, and the mixture was refluxed in nitrogen at 110 oC for 2 h. After cooling to room temperature, 1.5g (3.71 mmol, 1.3 equiv. ) of 3-(5-carboxypentyl)-1,1,2-trimethyl-1H-benzoindolium bromide dissolved in 15 mL of pyridine was added dropwise. The reaction was stirred at room temperature overnight before removing the solvent under reduced pressure. The crude product was purified using silica gel column chromatography with a gradient elution from petroleum ether to ethyl acetate (EtOAc) to EtOAc/methanol (10:1) to afford the desired product (1.54g, 76.1% yield). 1

H NMR (400 MHz, DMSO-d6):δ 12.00 (s, 1H), 8.47 (t, J = 13.1 Hz, 2H),

8.31-8.21 (m, 2H), 8.16-8.00 (m, 4H), 7.79-7.65 (m, 4H), 7.55-7.49 (m, 2H), 6.63 (t, J = 12.3 Hz, 1H), 6.36 (dd, J = 15.7, 13.9 Hz, 2H), 4.24 (t, J = 7.2 Hz, 2H), 3.74 (s, 3H), 2.21 (t, J = 7.2 Hz, 2H), 1.97 (s, 6H), 1.96 (s, 6H), 1.82-1.70 (m, 2H), 1.56 (q, J = 7.4 Hz, 2H), 1.48-1.37 (m, 2H). 13

C NMR (101 MHz, Chloroform-d):δ 174.71 , 152.98 , 152.82 , 140.08 , 139.30 ,

133.94 , 133.68 , 131.75 , 131.71 , 130.58 , 130.50 , 129.98 , 128.17 , 128.11 , 127.68 , 126.51 , 124.94 , 122.33 , 110.55 , 103.90 , 103.24 , 51.18 , 51.09 , 44.30 , 34.29 , 32.66 , 27.79 , 27.65 , 27.10 , 26.20 , 24.34.

19



Synthesis of NPA-Cy5.5-NHS 100mg (0.14 mmol, 1.0 equiv.) of Cy5.5 and 20mg (0.17 mmol, 1.2 equiv.) of N-hydroxysuccinimide (NHS) were dissolved in 10 mL dichloromethane (DCM) at 0 o

C Then 36mg (0.17 mmol, 1.2 equiv.) of dicyclohexylcarbodiimide (DCC) dissolved

in DCM was added dropwise. After stirring at ambient temperature overnight, the precipitate was filtered and the filtrate was evaporated under reduced pressure to yield a blue solid. The crude compound was directly used for the next reaction without any further purification.

Synthesis of NPA-Cy5.5 35mg (0.15 mmol, 1.1 equiv.) of 4-nitro-3-phenyl-L-alanine and 56mg (0.43 mmol, 3.0 equiv.) of K2CO3 in 5 mL of H2O was added to a solution of Cy5.5-NHS in 3 ml of DMF in ice bath. Then this mixture was stirred at 30 oC for 24 h before quenching the reaction with 5 mL of 1 M HCl. The mixture was extracted by DCM (3 × 50 ml), and the combined organic phases was washed with brine. After drying over Na2SO4, the solvent was evaporated and the crude product was isolated using silica gel column chromatography (EtOAc/methanol gradient) to afford the desired product (43mg, 34% yield). ESI-MS m/z: [M-I]+ calcd for C49H51N4O5, 775.39; found, 775.38428. 1

H NMR (400 MHz, Chloroform-d):δ 8.15-8.05 (m, 2H), 8.05-7.90 (m, 6H),

7.90-7.70 (m, 2H), 7.68-7.57 (m, 2H), 7.54-7.40 (m, 4H), 7.30 (d, J = 8.8 Hz, 1H), 7.21 (d, J = 5.3 Hz, 1H), 6.77 (d, J = 13.7 Hz, 1H), 6.53 (t, J = 12.5 Hz, 1H), 5.96 (d, J = 13.4 Hz, 1H), 4.59 (q, J = 5.1 Hz, 1H), 3.96 (s, 3H), 3.59-3.46 (m, 2H), 2.24 (dt, J = 20


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9.5, 4.5 Hz, 2H), 2.10-1.90 (m, 12H), 1.89-1.66 (m,4H), 1.57-1.45 (m, 2H), 1.26 (m, 2H). 13

C NMR (75 MHz, Chloroform-d):δ 175.06 , 173.05 , 171.90 , 151.94 , 150.77 ,

148.06 , 146.26 , 140.02 , 139.29 , 133.36 , 131.97 , 131.74 , 130.88 , 130.73 , 130.14 , 128.16 , 128.01 , 127.83 , 127.76 , 126.38 , 125.18 , 124.95 , 122.87 , 122.04 , 121.94 , 110.59 , 110.30 , 104.93 , 102.58 , 55.70 , 51.03 , 50.83 , 43.84 , 37.72 , 35.96 , 32.22 , 27.77 , 27.64 , 27.60 , 26.61 , 25.81 , 24.64.

Synthesis of P(OEGMA)21 and P(Asp)16 P(OEGMA)21 and P(Asp)16 were prepared by the method as described previously by our group.32 While the feed ratio of OEGMA to RAFT-APA was 35:1 for the synthesis of P(OEGMA)21 and Asp-NCA to 2-Propynylamine was 20:1 for the synthesis of P(Asp)16.

Synthesis of P(OEGMA)21-P(Asp)16 via Click Reaction. 1.12g (107µmol, 1.0 equiv.) of P(OEGMA)21, 356mg (1.07µmol, 1.0 equiv.) of P(Asp)16 and 20mg (140µmol,1.3 equiv.) of CuBr were dissolved in 5 mL of dry DMF in an inert gas purged Schlenk tube. Then 40µL (192µmol, 1.7 equiv.) of PMDETA was added rapidly to the solution, and the mixture was stirred for 3 days at 30 oC. After the termination of the reaction, the mixture was purified by dialysis against EDTA aqueous solution and ultrapure water successively. A yellow solid was get when removing the water by freeze-drying (1.23g, 83.5% yield). 21



Aminolysis of P(OEGMA)21-P(Asp)16 716mg (52µmol, 1.0 equiv.) of polymer was dissolved into dry DMF in a flame-dried and inert gas purged Schlenk tube. 300µL (1.73 mmol, 33.3 equiv.) of DIPEA was added rapidly to the solution, and the mixture was stirred for 3 days at 45 o

C. The product was purified by dialysis with ultrapure water, and the water was

removed using freeze-drying (640mg, 69% yield).

Conjugation of Cy5.5 and NPA-Cy5.5 5.5mg (7.45µmol, 1.1 equiv.) of Cy5.5 or 7.0mg (7.45µmol, 1.1 equiv.) of NPA-Cy5.5 dissolved in dry DMF was added to a dry DMF solution containing 1.2mg (10.43µmol, 1.2 equiv.) of HoSu and 100mg (7.0µmol, 1.0 equiv.) of polymer. After stirring 10 min, 2.2mg (10.67µmol, 1.5 equiv.) of DCC was added and the mixture was reacted for 24 h in the dark. For purification, dialysis and freeze-drying technique were used to obtain the final product as blue viscose solid (named PC and PN respectively). The PC0.05 and PN0.05 were synthesized as the above method,and the feed ratio of dye and polymer were 1:20.

Characterization Absorbance and emission spectra Bulk absorption and emission spectra were recorded on a UV1700PC ultraviolet spectrophotometer (Shanghai AuCy Scientific Instrument Co.Ltd) and a F97pro 22


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fluorescence

spectrophotometer

(Shanghai

Lengguang

Industrial

Co.

Ltd),

respectively. The dyes were dissolved in DCM at 5µM and the resulting spectra normalized to their maxima.

Fluorescence quantum yield Methylene blue trihydrate in methanol was chosen as the standard for quantum yield measurements for the spectral matching. The Cy5.5, NPA-Cy5.5, PC, PN, PC0.05, PN0.05 dissolved in methanol were excited at the wavelength of 680nm and the fluorescence integral intensity were measured to calculate the fluorescence quantum yield.

Photobleaching To evaluate the photostability of Cy5.5 and NPA-Cy5.5, the dyes were dissolved in mixed solvent of 0.2 M phosphate buffered saline PBS buffer/DMF (90:10, 80:20, 70:30, 60:40, 25:75 and 0:100) at a concentration of 10µM. 2 mL of the samples in a square quartz cell (1 cm×1 cm) were irradiated with a 635nm semiconductor laser (Hi-Tech Optoelectronices Co.Ltd) at a distance of 5 cm (160 mW/cm2). The absorption spectra of the dye solutions were monitored for the indicated time under ambient conditions and the decrease at the absorption peak was taken to evaluate the relative rate of fluorophore photobleaching. The photobleaching of PC/PN and PC0.05/PN0.05 in living cells was performed on an Olympus IX71 fluorescence microscope equipped with a high-pressure mercury lamp (Olympus U-HGLGPS) and 23



using a 40× air immersion objective. Fluorescence images of the samples under continuous illumination were recorded with intervals of 1 frame/min, exposure time of 100 ms and frame size of 512×512 pixels. The fluorescence intensity of the intracellular of bleaching time was used to estimate the relative photostability of the dyes. The confocal laser scanning microscopy (CLSM, LSM 710, Axio Observer) was used with an oil immersion objective (63x, NA 1.40, Carl Zeiss). Confocal time series was recorded with a specific interval, exposure time and the laser line of 633nm was used for bleaching and imaging. The mean fluorescence intensity of the bleached region over time was extracted to quantitatively evaluate the photobleaching of the fluorescent dyes.

Cellular Experiments HepG2 cancer cells (the American Type Culture Collection) were cultured in DMEM medium (GE Healthcare Life Sciences China) supplemented with 10% fetal bovine serum (FBS, TianHang Biotechnology Co.Ltd,China) at 37 oC under 5% CO2 atmosphere. To study the photostability using the Olympus IX71 fluorescence microscope, HepG2 cells were seeded in a 6-well plates at a density of 10000 cells per well. After 24 h, the culture medium was discarded and fresh DMEM containing PC/PN at a concentrations of 15µg/mL, 25µg/mL, 50µg/mL or PC0.05/PN0.05 at the concentration of 150µg/mL, 300µg/mL, 500µg/mL were added. The cells were washed with PBS twice after incubating for another 12 h. 24


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For CLSM experiments, the cells were plated on 20 mm glass petri dishes at a density of 6000 cells per dish for 24 h. After discarding the culture medium, fresh DMEM containing 15µg/mL of PC/PN or 250µg/mL of PC0.05/PN0.05 was added and incubated for 5 h.

Cyotoxicity Assays Methyl tetrazolium (MTT) viability assay against HepG2 cells was used to evaluate the cytotoxicity of polymeric dyes (PC/PN and PCµ/PNµ）in vitro. In short, cells at a density of 3500 cells per well were plated in 96-well plates and incubated for 24 h. After replacing the medium with fresh DMEM containing Polymer-dyes at a series of concentrations, the HelpG2 cells were incubated for another 24 h. Then, 20µL MTT (5 mg/mL) was added respectively and incubated for 4 h. 150µL DMSO was added to dissolve the crystal after removing the DMEM medium, and the absorbance per well at 570nm was measured to evaluate the cells viability.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: NMR spectra of Cy5.5, NPA-Cy5.5, and series of polymers, Mass spectra of Cy5.5 and NPA-Cy5.5, GPC traces of PC/PN and PC0.05/PN0.05, Absorption and fluorescence emission spectra of Cy5.5, NPA-Cy5.5 PC/PN and PC0.05/PN0.05, solubility of the PC/PN and Cy5.5/NPA-Cy5.5 in water. Fluorescence images of 25



HepG2 cells incubated in DMEM containing 50µg/mL PC and PN. A time series of images were recorded at different bleaching time.

Author Information Corresponding Authors: Lifeng Yan* E-mail:[email protected] ORCID Lifeng Yan: 0000-0002-6063-270X Tuanwei Li: 0000-0003-3168-8584 Le Liu: /0000-0001-6344-3528 Titao Jing: 0000-0001-6819-046X Zheng Ruan: 0000-0002-2759-0587 Notes The authors declare no competing financial interest.

Acknowledgement The research is supported by the National Natural Science Foundation of China (No. 26


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51673180 and 51373162).

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Captions of Figures Figure 1. 1H-NMR and 13C-NMR spectra of Cy5.5 (a, b) and NPA-Cy5.5 (c, d). Figure 2. Absorption and fluorescence emission spectra of Cy5.5 and NPA-Cy5.5 in DCM. Figure 3. Absorption and fluorescence emission spectra of PC/PN (a) and PC0.05/PN0.05 (b) in water. Figure 4. Photobleaching behavior of Cy5.5 (a) NPA-Cy5.5 (b) in different fraction of DMF in the mixture of DMF and PBS (irradiated under a 635 nm laser). Figure 5. The bleaching time constantτbleach in different gradient solutions of DMF to PBS. Figure 6. a) Time series CLSM images of HepG2 cells incubated in DMEM containing 2% DMSO and 3 µM Cy5.5 and NPA-Cy5.5 (scale bar 15µm); b) Time-course

of

the

normalized

fluorescence

intensity

for

Cy5.5/NPA-Cy5.5. Figure 7. Results from photobleaching experiments of PC and PN obtained from CLSM. A time series CLSM images of HepG2 cells incubated in DMEM containing 50µg/mL PC (a) and PN (b); Time-course of the normalized fluorescence intensity for PC/PN (c and d). Figure 8. Photobleaching behavior of PC/PN (a) and PC0.05/PN0.05 (b) in DMEM at a concentration of 300µg/mL. The PC/PN corresponding to the crowded status of the dyes, while the PC0.05/PN0.05 illustrating the eased status. Figure 9. Fluorescence microscopy images of HepG2 cells incubated in DMEM 33



containing 25µg/mL PC and PN. A time series of images were recorded at different bleaching time (scale bar 15 µm). Figure 10. Results from photobleaching experiments of PC0.05 and PN0.05 obtained from CLSM. A time series CLSM images of HepG2 cells incubated in DMEM containing 250µg/mL PC0.05 (a) and PN0.05 (b); (c) Time-course of the normalized fluorescence intensity for PC0.05/PN0.05; 2.5D fluorescence images for PC0.05 (d) and PN0.05 (e). Figure 11. Photobleaching behavior of PC0.05 (up) and PN0.05 (down) in PBS (scale bars, 1µm). Repeated scanning of the same area near the cells and the counting of the emitters in the images of 1, 11, 21, 31, and 41 indicates enhanced photostability for PN0.05 compared with PC0.05. Figure 12. Cytotoxicity of PC/PN (a) and PC0.05/PN0.05 (b) to HepG2 cancer cells.

Scheme 1. Synthesis route of the Cy5.5 and self-healing NPA-Cy5.5. Scheme 2. Synthesis route of the PC/PN and PC0.05/PN0.05. Scheme 3. Jablonski diagram for NPA-Cy5.5 excitation and deactivation pathways. S0: the singlet ground state of the NPA-Cy5.5; S1: the first singlet excited state after excitation (EX) by photon absorption from S0; T1: the first triplet excited state via intersystem crossing (ISC); The NPA-Cy5.5 in the excited state (S1 and T1) can recover ground state by fluorescence emission (F), phosphorescence emission (P), internal conversion (IC), energy transfer with 1O2 or electron transfer with the NPA. 34


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Scheme 4. The fluorescence enhanced mechanism by photobleaching-induced process. (a, b, c and d represent the fluorescence molecules in eased status, excited state, quenching status by neighboring molecules, and degradation).

35



Scheme 1. Synthesis route of the Cy5.5 and self-healing NPA-Cy5.5.

36


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Figure 1. 1H-NMR and 13C-NMR spectra of Cy5.5 (a, b) and NPA-Cy5.5 (c, d).

37



Figure 2. Absorption and fluorescence emission spectra of Cy5.5 and NPA-Cy5.5 in DCM.

38


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Scheme 2. Synthesis route of the PC/PN and PC0.05/PN0.05

39



Figure 3. Absorption and fluorescence emission spectra of PC/PN (a) and PC0.05/PN0.05 (b) in water.

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Figure 4. Photobleaching behavior of Cy5.5 (a) NPA-Cy5.5 (b) in different fraction of DMF in the mixture of DMF and PBS (irradiated under a 635 nm laser).

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Figure 5. The bleaching time constantτbleach in different gradient solutions of DMF to PBS.

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Scheme 3. Jablonski diagram for NPA-Cy5.5 excitation and deactivation pathways. S0: the singlet ground state of the NPA-Cy5.5; S1: the first singlet excited state after excitation (EX) by photon absorption from S0; T1: the first triplet excited state via intersystem crossing (ISC); The NPA-Cy5.5 in the excited state (S1 and T1) can recover ground state by fluorescence emission (F), phosphorescence emission (P), internal conversion (IC), energy transfer with 1O2 or electron transfer with the NPA.

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Figure 6. a) Time series CLSM images of HepG2 cells incubated in DMEM containing 2% DMSO and 3 µM Cy5.5 and NPA-Cy5.5 (scale bar 15µm); b) Time-course of the normalized fluorescence intensity for Cy5.5/NPA-Cy5.5.

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Figure 7. Results from photobleaching experiments of PC and PN obtained from CLSM. A time series CLSM images of HepG2 cells incubated in DMEM containing 50µg/mL PC (a) and PN (b); Time-course of the normalized fluorescence intensity for PC/PN (c and d).

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Scheme 4. The fluorescence enhanced mechanism by photobleaching-induced process. (a, b, c and d represent the fluorescence molecules in eased status, excited state, quenching status by neighboring molecules, and degradation).

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Figure 8. Photobleaching behavior of PC/PN (a) and PC0.05/PN0.05 (b) in DMEM at a concentration of 300µg/mL. The PC/PN corresponding to the crowded status of the dyes, while the PC0.05/PN0.05 illustrating the eased status.

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Figure 9. Fluorescence microscopy images of HepG2 cells incubated in DMEM containing 25µg/mL PC and PN. A time series of images were recorded at different bleaching time (scale bar 15 µm).

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Figure 10. Results from photobleaching experiments of PC0.05 and PN0.05 obtained from CLSM. A time series CLSM images of HepG2 cells incubated in DMEM containing 250µg/mL PC0.05 (a) and PN0.05 (b); (c) Time-course of the normalized fluorescence intensity for PC0.05/PN0.05; 2.5D fluorescence images for PC0.05 (d) and PN0.05 (e). 49



Figure 11. Photobleaching behavior of PC0.05 (up) and PN0.05 (down) in PBS (scale bars, 1µm). Repeated scanning of the same area near the cells and the counting of the emitters in the images of 1, 11, 21, 31, and 41 indicates enhanced photostability for PN0.05 compared with PC0.05.

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Figure 12. Cytotoxicity of PC/PN (a) and PC0.05/PN0.05 (b) to HepG2 cancer cells.

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Table Of Contents (TOC) graphic

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Self-healing Organic Fluorophore of Cyanine ... - ACS Publications

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