Self-Assembling Nonconjugated Poly(amide-imide) into

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Self-assembling Non-conjugated Poly(amide-imide) into Thermoresponsive Nanovesicles with Unexpected Red fluorescence for Bioimaging Jun-Jie Yan, Xin-Yu Wang, Meng-Zhen Wang, Dong-Hui Pan, Run-Lin Yang, Yu-Ping Xu, Li-Zhen Wang, and Min Yang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00051 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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Self-assembling Non-conjugated Poly(amide-imide) into Thermoresponsive Nanovesicles with Unexpected Red fluorescence for Bioimaging Jun-Jie Yan,*,†,§ Xin-Yu Wang,†,§ Meng-Zhen Wang,† Dong-Hui Pan,† Run-Lin Yang,† Yu-Ping Xu,† LiZhen Wang,† and Min Yang*,†

†Molecular

Imaging Center, Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key

Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, P. R. China

KEYWORDS: self-assembly, poly(amide-imide), red-shifted fluorescence, nanovesicles, bioimaging

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ABSTRACT: Non-conjugated red fluorescent polymers have been increasingly studied to improve the biocompatibility and penetration depth over conventional fluorescent materials. However, the accessibility of such polymers remains challenging due to the scarcity of non-conjugated fluorophores and lacking relevant mechanism of red-shifted fluorescence. Herein, we discovered that the combination of hydrogen bonding and π-π stacking interactions provides non-conjugated poly(amideimide) with a large bathochromic shift (> 100 nm) from blue-green fluorescence to red emission. The amphiphilic PEGylated poly(amide-imide) derived from in-situ PEGylation self-assembled into nanovesicles in water, which isolated the aminosuccinimide fluorophore from the solvents and suppressed the hydrogen bonds formation between aminosuccinimide fluorophores and water. Therefore, the fluorescence of PEGylated poly(amide-imide) in water was soundly retained. Furthermore, the strong hydrogen bonding and hydrophobic interactions with water provided PEGylated poly(amide-imide) with a reversible thermoresponsiveness and presented a concentrationdependent behavior. Finally, accompanied with the excellent biostability and photostability, PEGylated poly(amide-imide) exhibited as a good candidate for cell imaging.


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INTRODUCTION The unconventional fluorescence of non-conjugated polymers has been capturing sustained interests in biological applications due to their excellent water solubility, biocompatibility and multiplicity, tremendously expanding the traditional knowledge hierarchy of fluorescent materials.1-6 However, many of these unconventional fluorescent polymers present only blue or green emissions, while it remains a big challenge to achieve red fluorescence as in the cases of carbon dots7-9 and semiconducting polymers,10-13 which ascribes to the different fluorescence mechanism and lacking prerequisites that can lead to the large red-shifted emission.14-16 Poly(amide-imide)s (PAIs) are one kind of outstanding engineering materials with superior thermal stability, mechanical properties and chemical resistance, but the research of their optical properties has been relatively overlooked. Besides, strong intermolecular interactions result in poor solubility and restrict their potential roles in biological applications.17 Recently, we circumvented these issues by employing a functional thiolactonemaleimide monomer via in-situ PEGylation and provided aliphatic PAIs with unique blue/green fluorescence in organic solvents.18-20 However, not only this wavelength emission could be interfered with autofluorescence of biological objects such as cells, tissues and organs, but also the fluorescence of PAIs considerably decreases or completely quenches in protic medium due to the formation of hydrogen bonds between the fluorophores and protic solvents,19, 21 which is disfavored similarly to the notorious aggregation-caused quenching (ACQ) effect and leads to great limitations in cell imaging and protein labelling. Theoretically, eliminating the hydrogen bonding interaction between the fluorophores and water can efficiently suppress the fluorescence quenching of PEG-PAIs in aqueous solutions. Referring to the approaches of loading hydrophobic dyes, we speculate that encapsulating hydrophobic fluorescent segments into self-assembled nanostructures including micelles and vesicles could potentially retain the


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fluorescence of PAIs. PEGylation can transform PAIs from hydrophobic copolymers to amphiphilic copolymers, which can further self-assemble into multiple nanostructures in water due to the repulsion interaction between the hydrophobic and hydrophilic segments.22-25 Prior to this study, the selfassembly of PAIs has never been reported yet. Herein, we serendipitously discovered that nonconjugated PEGylated PAI (PEG-PAI4) with isolated benzene rings spontaneously self-assembled into small nanovesicles in water with a diameter ca. 23 nm. Unexpectedly, the combined intermolecular/intramolecular hydrogen bonding and π-π stacking interactions endowed the nonconjugated PEG-PAI4 with maximal 580 nm red emission. More importantly, the unfavorable fluorescence quenching of PAIs in aqueous solution, which is derived from the formation of hydrogen bonds between aminosuccinimide fluorophores and the water, was simultaneously suppressed and thus facilitated the promise of PAIs in biological applications.

EXPERIMENTAL SECTION Materials. DL-homocysteine thiolactone hydrochloride (Sigma-Aldrich, ≥ 99%), 2-bromoisobutyryl bromide (TCI, >98%), maleic anhydride (TCI, >99%), sodium L-ascorbate (TCI, >98%), copper sulfate pentahydrate (Aladdin, 99.99%), sodium azide (Sangon Biotech, >99%), 1,3-diaminopropane (Amine74, TCI, >98%), p-xylylenediamine (Amine136, Aldrich, 99%), poly(ethylene glycol) methyl ether methacrylate (PEG500) (average Mn ∼ 500, Aldrich), polyethylene glycol monomethylether (average Mn ∼ 1900, Alfa), methacryloyl chloride (Alfa, 97%), rhodamine 6G (Acros, 99%), Fluorescein isothiocyanate isomer I (Sigma-Aldrich, ≥ 97.5%), ER-TrackerTM Red (Beyotime Biotechnology). 2,2-azobis(isobutyronitrile) (AIBN, TCI, >98%), methyl sulfoxide (Acros, extra pure, >99.8%), triethylamine (Acros, extra pure, 99.7%), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Aldrich, ≥98%). Other regents were purchased from SCRC and used as received. Functional


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thiolactone maleimide monomer with 1, 2, 3-triazole linker (TMM) was synthesized according to our previous work.19 Methods. 1H NMR and

13C

NMR spectra were performed on a Bruker Avance 400 spectrometer

(resonance frequency of 400 MHz for 1H and 100 MHz for

13C).

Fluorescence measurement was

recorded on a PE LS55 Fluorescence Spectrometer (Perkin Elmer). UV was carried on a UV-2601 UVvis spectrophotometer (SHIMADZU). The molecular weights and PDI were tested on a Viscotek HT GPC/SEC (Marlvern) with a Model-1122 injection pump and a Model 350 triple detector array (RI, viscosity and small angle laser light scattering). DMF was utilized as an eluent with flow rate of 1 mL min-1 at 30 °C and polystyrenes (Polymer Laboratories, Inc., MA) were used for calibration. Fluorescence lifetime was recorded on a FLS980 Fluorescence Spectrometer (Edinburgh Instruments). TEM was performed on a JEM-2100 (JEOL Ltd.) or on a Tecnai G2 F20 (FEI) analytical electron microscope at 200 kV. Atomic force microscope (AFM) was recorded on a Bruker Dimension Icon with ScanAsyst and the data was processed using NanoScope Analysis. The determination of hydrodynamic diameters was performed on a Malvern DLS Zetasizer Nano ZS90 with a He–Ne laser. Cell imaging was conducted on a TCS SP8 confocal laser scanning microscope (CLSM, Leica, Germany). Synthesis of MPEG2k. Polyethylene glycol monomethylether (Mn ∼ 1900, 9.51 g, 5.0 mmol) and triethylamine (1.02 g, 10.0 mmol) were mixed in 130 mL dichloromethane (DCM), then methacryloyl chloride (1.05 g, 10.0 mmol) were slowly added drop-wise into the mixture with 15 min and stirred overnight at 40 °C. After the reaction, the mixture was filtered, washed with deionized water (10 mL × 3) and extracted with DCM (20 mL × 3). The organic phase was dried with anhydrous Na2SO4, filtered, concentrated and purified by silica chromatography to generate white powders. Yield: 49.7%. 1H NMR


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(400 MHz, CDCl3, ppm), δ 6.13 (s, 1H), 5.58 (s, 1 H), 4.34 – 4.25 (m, 2 H), 3.41-3.87 (m, 166 H), 3.38 (s, 3 H), 1.95 (s, 3 H). Synthesis of PEG-PAIs. The synthesis of PEG-PAI was referred to our previous work.19 The TMM (36.3 mg, 0.1 mmol) and PEG500 (75.2 mg, 0.15 mmol) were dissolved in 1.0 mL DMSO and the solution was purged with argon for 10 min. Then, diamine (0.1 mmol) was added into the mixture, which gradually became orange red. The reaction mixture was stirred and monitored by 1H NMR and 13C

NMR. After the polymerization, the product was purified by precipitating from mixed solvents of

acetone/diethyl ether (1/2, v/v) twice, and the product was dried at 30 °C for 3 h. Yield: 91.6%. Synthesis of PEG-co-PSUC1. According to previously reported procedure,20 PEG500 (995.1 mg, 1.99 mmol), 2-(butylamino)-maleimide (117.2 mg, 0.70 mmol) and AIBN (6.4 mg, 0.039 mmol) were dissolved in 1.0 mL THF and then the solution was degassed by three freeze-evacuate-thaw cycles. After the tube was flame-sealed under vacuum, the mixture was left at 60 °C preheated oil for 4 h. After the polymerization, the reaction mixture was purified by precipitating into diethyl ether and dried to obtain a slight yellow viscous solid. Self-assembly of PEG-PAIs. The self-assembly procedure of PEG-PAIs was performed by a nanoprecipitation method. Typically, PEG-PAI2 (2 mg) was dissolved in 0.2 mL DMSO, and then was added dropwise into DI water (2 mL) under stirring for 30 min. Afterwards, the resultant solution was dialysed against DI water for 6 h. The nanoparticles of PEG-PAIs were concentrated via ultrafiltration centrifugal tube (Millipore, MWCO ~ 5 kDa) at 2000 rpm for 5 min. The self-assembly of PEG-PAIs with different concentration was performed with a similar procedure. In vitro fluorescence imaging. For cell imaging in vitro, BEL-7402 cells and 4T1 cells were seeded and grown in Lab-Tek chambered coverglass wells at a density of 1×104 cells per mL medium (DMEM, Hyclone, Utah, USA) with 10% fetal bovine serum (FBS, Sijiqing Company Ltd., Hangzhou, China)


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for 24 h at 37 °C in 5% CO2 before the cellular uptake study. The coverglass wells were incubated in PEG-PAI4 (2.5 mg/mL) DMEM for 2 h at 37 °C. Afterwards, the cells were washed with PBS (×3), fixed with paraformaldehyde and the cell nucleus were labelled DAPI. The imaging experiments were performed with a TCS SP8 CLSM. Biocompatibility Assay. The cell biocompatibility assay of PEG-PAI4 nanovesicles against 4T1, Hela and BEL-7402 cells were assessed by the standard CCK-8 assay. Typically, the cells were cultivated in a 96-well plate, and the density was 8×103 cells/well. Afterwards, PEG-PAI4 nanovesicles at various concentrations of 0.1, 0.5, 1, 2.5 and 5 mg/ml for 24 h, after washing with DMEM or RPMI1640 (×2), then 100 μl DMEM and 10 μl CCK-8 solution was added into each well and incubated for additional 1 h. The absorbance was detected by a microplate reader (Model 680, Bio-Rad, USA) at the wavelength of 450 nm.

RESULTS AND DISCUSSION The red fluorescent PAI was synthesized by the polycondensation of p-xylylenediamine (Diamine136), thiolactone-maleimide monomer and poly(ethylene glycol) methyl ether methacrylate (PEG500) (Mn ∼ 500) at a mole ratio of 1:1:1.5, excess PEG500 was used to ensure the complete consumption of in-situ generated thiol intermediate (Figure 1a). Before the polycondensation, neither thiolactonemaleimide monomer nor PEG500 is fluorescent, except for the intrinsically weak emission of diamine136 (Em ~ 418 nm in DMSO). Combining amine-maleimide Michael addition, aminolysis of a thiolactone and thiol-methacrylate Michael addition yielded PEG-PAI4 with a molecular weight of 49 kDa and a polydispersity index (PDI) of 1.74, which was verified by 1H,

13C

nuclear magnetic

resonance (NMR) (Figure S2) and gel permeation chromatography (GPC) (Figure 2b). Similar to our previous work, PEG-PAI4 showed excellent solubility in various organic solvents (including acetone


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(Ace), methylene dichloride (DCM), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), N,Ndimethylformamide (DMF) and methanol) and water with solvent-dependent fluorescence and quantum yield (ϕ) (Figures 1b, 1c), which were ascribed to the formation of aminosuccinimide fluorophores. Also, PEG-PAI4 presented an excitation-dependent emission, which was very similar to carbon dots (Figure 1e, 1f). But strangely, the maximum emission of PEG-PAI4 was at ~ 570 nm in DMSO, almost 100 nm red-shift compared to the typical emission of a polymer containing an aminosuccinimide fluorophore (Figure 2c). By contrast, diamine-136 did not show an excitation-dependent emission and its maximum excitation in DMSO is around 320 nm, while excitation above 450 nm gave no detectable fluorescence (Figure 1g). This excludes the contribution of diamine-136 to the fluorescence of PEGPAI4 since its maximum excitation wavelength is around 500 nm (Figure 1f), thus the fluorescence center of PEG-PAI4 is from the aminosuccinimide fluorophore. More significantly, the red fluorescence of PEG-PAI4 in water was soundly retained with a quantum yield of 5.8% (Em ~ 580 nm), utilizing rhodamine 6G as a standard (Figure 1c). This was nearly 60-fold fluorescence enhancement of PAIs in water (typical ϕ < 0.1%).18, 19, 21


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Figure 1. Synthesis and optical properties of PEG-PAI4. (a) Synthesis of red fluorescent PEG-PAI4. (b) Optical images of PEG-PAI4 in various solvents under UV light. (c) Quantum yields of PEG-PAI4 in various solvents (the reference used in acetone, DMF and methanol was quinine sulfate, while the reference used in DCM, THF, DMSO and H2O was rhodamine 6G due to the difference in maximal fluorescence wavelength). (d) Absorbance spectra of PEG-PAI4 in various solvents. (e) Normalized fluorescence spectra of PEG-PAI4 in various solvents. (f) Excitation-dependent fluorescence of PEG-


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PAI4 in water. (g) Excitation-dependent fluorescence of diamine-136 in DMSO. Excitation and emission slit widths: 5 nm, 5 nm.

Various strategies and factors can result in red-shifted emission, which primarily comprise π-π stacking,26 electrostatic interactions,27 hydrogen bond,28 peripheral substitution,29-31 annulation,32,

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trans-cis isomerization34 and excited-state intramolecular proton transfer (ESIPT).35 Looking into the structure of PEG-PAI4, it is indeed composed of four segments, namely PEG, aminosuccinimide fluorophore, PAI and diamine-136. The potential driving force for red-shifted emission of PEG-PAI4 could probably be related with hydrogen bonding and π-π stacking interactions. To probe the possible reasons for this large red-shifted emission of PEG-PAI4, we designed and synthesized another two control polymers PEG-co-PSUC1 and PEG-PAI1 with similar molecular weights to that of PEG-PAI4 (Figures 2a-2c, S3 and S4). Specifically, PEG-co-PSUC1 has PEG and aminosuccinimide fluorophore two segments, only very weak hydrogen bonding (hydrogen bonding with solvents is not included here) interaction exists. PEG-PAI1 has similar composition as that of PEG-PAI4 except that diamine136 was replaced by 1,3-diaminopropane, the hydrogen bonding interaction among PAI chains is much stronger than that in PEG-co-PSUC1. As shown in Figure 2a and 2c, diamine-136 has a maximum emission at 418 nm and PEG-co-PSUC1 has intrinsic fluorescence of the aminosuccinimde fluorophore around 462 nm in DMSO, and both of their fluorescent units are the basic constituents of PEG-PAI4. The strong intermolecular hydrogen bonding interaction among the rigid PAI chains gave PEG-PAI1 blue-green emission (Em ~ 494 nm in DMSO) with a 32 nm red-shift compared to that of PEG-coPSUC1. In the case of PEG-PAI4, the involvement of diamine-136 yielded non-conjugated PAI backbones with three isolated benzene rings in every repeating unit, which satisfied the necessity for


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the formation of stable bilayer assemblies of amphiphiles.36 Therefore, π-π stacking could probably occur in PEG-PAI4 and lead to further red-shifted fluorescence. The optimized structure of repeating units of PEG-PAI4 was calculated via MM2 energy minimization (BioChem3D) as shown in Figure 2h-2j, which theoretically supported the potential hydrogen bonding and π-π stacking interactions among PEG-PAI4 chains. To verify our hypothesis, we first used transmission electron microscope (TEM) to measure the sizes of these three polymers, which should have similar sizes because of similar molecular weights (Table S1). As shown in Figure 2d-2f, the size of PEG-co-PSUC1 is 14.6 ± 2.8 nm, while PEG-PAI1 and PEG-PAI4 have larger sizes of 21.2 ± 4.0 nm and 23.4 ± 3.9 nm respectively, indicating the obvious size increasements of PAIs by hydrogen bonding interaction. Also, dynamic light scattering (DLS) was utilized to measure their corresponding hydrodynamic sizes, and they were 17.9 ± 6.3 nm, 23.9 nm ± 8.5 nm and 25.4 ± 8.0 nm for PEG-co-PSUC1, PEG-PAI1 and PEG-PAI4 respectively at concentrations below 2 mg/mL (Figure S5), which were consistent with the sizes from TEM measurement. Moreover, Fourier transform infrared spectroscopy (FTIR) was utilized to record hydrogen bonds among these polymers. In Figure 2g, PEG-co-PSUC1 has an absorbance at 3444 cm-1 in the range of 3200-3500 cm-1, which is the typical peak of free N-H stretch band.37 In the case of PEG-PAI1, besides the free N-H stretch band at 3426 cm-1, a new band at 3313 cm-1 appeared, indicating the existence of hydrogen-bonded N-H stretch band. As for PEG-PAI4, only hydrogen-bonded N-H stretch band at 3312 cm-1 was observed, which revealed that all N-H bonds in PEG-PAI4 participated in the hydrogen bonding interaction. These results verify that hydrogen bonding interaction facilitates molecular aggregation and results in the redshifted fluorescence.


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Figure 2. Chemical structures and characterizations of PEG-co-PSUC1, PEG-PAI1 and PEG-PAI4. (a) Chemical structures. (b) GPC curves in DMF. (c) Fluorescence spectra in DMSO. (d-f) TEM images of (d) PEG-co-PSUC1, (e) PEG-PAI1 and (f) PEG-PAI4, 2 mg/mL. (g) FTIR measurement. (h) Optimized structure of complete amide-imide repeating unit ((ab)x(abba)y style) via MM2 energy minimization (BioChem3D). (i) Intermolecular hydrogen bond among PEG-PAI4 (sub-repeating unit: (ab) style). (j) π-π stacking interaction among PEG-PAI4.

On the other hand, π-π stacking can also lead to dynamic molecular aggregation. However, the intermolecular interaction via π-π stacking depends on the concentration and a high concentration is usually required to facilitate this non-covalent interaction.38 If there is π-π stacking in PEG-PAI4, the size of PEG-PAI4 would be much bigger than that of PEG-PAI1 under the same condition. In general, it is not appropriate to measure the sizes of nanostructures via DLS in a concentrated solution due to


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the multiple scattering, restricted diffusion and particle-particle interactions (e.g., particle-particle collisions, long range electrostatic interactions, etc.).39,40 Therefore, atomic force microscope (AFM) and TEM were employed to measure the sizes of PEG-PAI1 and PEG-PAI4 in their concentrated aqueous solutions. As characterized by AFM (Figure 3a), PEG-PAI1 and PEG-PAI4 are well dispersed and have close size around 30 nm in diluted aqueous solutions (~ 2 mg/mL). However, when determined in concentrated solutions (~ 5 mg/mL), sizes of PEG-PAI1 and PEG-PAI4 increased to about 42.1 ± 14.0 nm and 85.2 ± 11.4 nm, respectively. The TEM measurement also revealed that the size of PEG-PAI1 slightly increased to 38.3 ± 7.9 nm and the size of PEG-PAI4 significantly increased to 64.0 ± 14.8 nm (Figure 3b). By contrast, the size of PEG-co-PSUC1 in concentrated solution did not change obviously (Figure S6). These results confirmed the role of hydrogen bonding interaction in the molecular aggregation of PEG-PAIs, and this interaction is also dependent on solute concentration.41 Moreover, additional π-π stacking interaction could further contribute in the molecular aggregation of PEG-PAIs. Therefore, with combined hydrogen bonding and π-π stacking interactions, PEG-PAI4 achieved the most molecular aggregation and obtained the largest red-shifted fluorescence. As demonstrated in our previous work, the fluorescence of PEG-PAI1 quenches in aqueous solution due to the hydrogen bonds between aminosuccinimide fluorophores and water.19 Unexpectedly, in this work, the fluorescence of PEG-PAI4 in water is well retained. PAIs are typically hydrophobic and water-insoluble because the amide bonds participate in intermolecular hydrogen bonding interactions. PEGylation not only enhances the solubility of PAIs, but also transforms from hydrophobic PAIs to amphiphilic PEG-PAIs, which can self-assemble into various nanostructures in water. Generally, hydrogen bonding interaction has a significant impact on self-assembled morphologies of polymers.42, 43

For instance, Duan et al. demonstrated that rigid polyimide (PI) and its hydrogen bonding attaching

to poly(4-vinylpyridine) (PVPy) chains drove these rigid/coil polymer pairs self-assemble into hollow


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spheres in solvents of chloroform or THF.44 Also, π-π interaction is a fundamental driving force for the polymer self-assembly and plays a synergistic role for the generation of a hollow sphere.45, 46 However, the submicrometer size of the resultant hollow structures from these interactions was too large to be used in biological applications.47, 48 Herein, TEM measurement as shown in Figure 3b revealed that PEG-PAI1 and PEG-PAI4 self-assembled into micelles and vesicles in water (2 mg/mL), respectively. This possibly resulted from the π-π stacking among the aromatic units in PEG-PAI4 backbones, which rendered PEG segments as the hydrophilic layers and led to the formation of vesicles. These results were consistent with previous studies that block copolymers49, 50 and graft polymers51, 52 could selfassemble into vesicles (Figure 3c). The rough surface of vesicles possibly resulted from the characteristics of step-growth polymerization (wide PDI) and PAI (labile to crystallinity). Incorporating aromatic segments into PAI actually increased the hydrophobicity of PAI, which could aid in increasing the surface roughness of vesicles.53 Additionally, multiple potential crystalline domains in the chains of PEG-PAI4 also can contribute to the construction of rough vesicle surface.54 Moreover, in a concentrated aqueous solution (5 mg/mL), vesicles tended to further undergo adhesion and fusion to decrease the interfacial potential and resulted in the generation of multicompartment vesicles.43, 55-57 Similar to crosslink enhanced emission (CEE) in non-conjugated polymer dots,58 more rigid structure of PEG-PAI4 nanovesicles obtained decreased vibration and rotation of aminosuccinimide fluorophore. Overall, the combined hydrogen bonding and π-π stacking interactions among PEG-PAI4 nanovesicles encapsulated aminosuccinimide fluorophores in a more isolated environment from water than that of PEG-PAI1 nanomicelles, therefore strikingly retaining their intrinsic fluorescence. PEGylation of PAI4 with longer PEG chains (PEG2k) presented a better effect to shield aminosuccinimide fluorophores from water and accomplished another 10 nm red-shifted emission (Figure S7), and the quantum yield of PEG-PAI4 slightly increased to 6.1%.


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Figure 3. PEG-PAI1 and PEG-PAI4 in diluted (2 mg/mL) and concentrated (5 mg/mL) aqueous solution. (a) AFM images. (b) TEM images. (c) The schematic of the self-assembly of PEG-PAI4 in water via combined hydrogen bonding and π-π stacking interactions.

Because of the strong hydrogen bonding interaction among polymer chains, PAI4 were thoroughly PEGylated to enhance their water solubility and stability. Incomplete PEGylation with remaining thiol groups would cause thiol oxidation and lead to the generation of crosslinking networks. In addition, nanostructures of PEG-PAI4 varied from nanovesicle to multicompartment nanovesicles upon increasing the concentration. Therefore, it’s challenging to tune the size of PEG-PAI4 nanovesicles by changing its composition or concentration. Alternatively, we studied the effect of temperature on the size of PEG-PAI4 nanovesicles. Interestingly, PEG-PAI4 exhibited a thermoresponsive behavior with a


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lower critical solution temperature (LCST) (Figure 4a). As shown in Figure 4b, the transmittance of PEG-PAI4 aqueous solution (3 mg/mL) decreased sharply around 39 ºC, but the transmittances of PEG-PAI1 and PEG2k-PAI4 did not change apparently upon increasing the temperature (up to 50 ºC). Moreover, the LCST of PEG-PAI4 in the aqueous solution is concentration dependent, and briefly a higher concentrated solution led to a lower LCST (Figure S8). To better understand the morphological change of PEG-PAI4 above LCST, TEM and DLS were leveraged to characterize the nanostructure transition of PEG-PAI4. Figure 4c indicated that the morphology of PEG-PAI nanovesicles was broken and aggregated into solid nanoparticles with a diameter of 69.8 ± 12.4 nm and a PDI of 0.091. Based on these results, we speculated that the LCST of PEG-PAI4 was possibly related with its hydrophobicity, which resulted from the combined hydrogen bond and π-π stacking interactions. Below LCST, PEG500 shielding self-assembled PEG-PAI4 into nanovesicles and allowed solubilization. Above LCST, PEG-PAI4 nanovesicles collapsed and occurred phase separation, leading to stronger intermolecular hydrogen bonding and π-π stacking interactions than that of nanovesicles. In cases of PEG-PAI1 and PEG2K-PAI4, weaker hydrogen bonding interaction (without π-π stacking interaction) and the longer PEG side chain provided them with better water solubility,59 so no obvious LCST were observed. The thermoresponsiveness of PEG-PAI4 was reversible and the nanovesicle morphology could recover, but its response rate was slower than that of typical thermoresponsive materials such as poly(N-isopropylacrylamide). Interestingly, above LCST, PEG-PAI4 nanoparticles compressed aminosuccinimide fluorophores into a more compact structure, and thus achieved enhanced fluorescence (Figure 4d).


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Figure 4. Reversible thermoresponsiveness of PEG-PAI4 nanoparticles in aqueous solution. (a) Daylight images. (b) Transmittance of PEG-PAI4, PEG-PAI1 and PEG2K-PAI4 at different temperatures, 3 mg/mL. (c) TEM image of PEG-PAI4 nanoparticles after heating the PEG-PAI4 nanovesicle aqueous solution to 40 ºC, 3 mg/mL. Inset: DLS spectrum of PEG-PAI4 nanoparticle aqueous solution at 40 ºC. (d) Fluorescence spectra of PEG-PAI4 before and after heating in the aqueous solution. Ex: 505 nm, excitation and emission slit widths: 5 nm, 5 nm.

Next, the potential application of PEG-PAI4 nanovesicles as a candidate for cell imaging was thoroughly evaluated. First, we measured the fluorescence lifetime of PEG-PAI4 nanovesicles in water, τ1 is 1.18 ns and τ2 is 5.19 ns (Figure 5a), which is similar to that of conventional organic dyes. Second, the photostability of PEG-PAI4 nanovesicles is much better than that of commercial organic dyes. For instance, PEG-PAI4 nanovesicles retained 96.9 % original fluorescence intensity upon continuous irradiation for 30 min (dynamic mode of Perkin Elmer LS 55, excitation slit: 5 nm,


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emission slit: 5 nm), while the fluorescence intensities of fluorescein isothiocyanate (FITC), rhodamine 6G and ER-Tracker Red decreased to 52.8 %, 64.3 % and 72.6 % respectively (Figure 5b). Even when the continuous illumination times were prolonged to 60 min and 120 min, the fluorescence loss of PEG-PAI4 nanovesicles were only 7.0 % and 10.3 % respectively, which indicates the excellent photostability of PEG-PAI4 nanovesicles. When dispersed in phosphate-buffered saline (PBS) and various culture mediums (RPMI1640 + 10 % FBS, DMEM + 10 % FBS), PEG-PAI4 nanovesicles were stable for over half a year without any precipitation and the fluorescence intensity only slightly decreased (Figure 5d, Figure S9). In addition, the biocompatibility of PEG-PAI4 nanovesicles was tested on several cell lines including 4T1, Hela and BEL-7402, and all cell viabilities were still very high when the concentration of PEG-PAI4 was up to 5 mg/mL. Therefore, we utilized PEG-PAI4 nanovesicles as a red imaging agent to label cells (2.5 mg/mL). As shown in Figure 5e, PEG-PAI4 nanovesicles were mainly distributed in the cytoplasm and presented good imaging results.


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Figure 5. Application of PEG-PAI4 nanovesicles for bioimaging. (a) Fluorescence lifetime of PEGPAI4 nanovesicles. (b) Photostability of PEG-PAI4 nanovesicles compared to different dyes including FITC, Rhodamine 6G and ER-Tracker-Red. (c) Cell viability assays of PEG-PAI4 on Hela, 4T1 and BEL-7402 cell lines. (d) Stability of PEG-PAI4 nanovesicles in water, PBS and different culture medium. (e) Confocal images of PEG-PAI4 nanovesicles (2.5 mg/mL) in 4T1 and BEL-7402 cell lines, nucleus were labeled with DAPI. Scale bar: 25 μm.

CONCLUSIONS In summary, we developed non-conjugated PAI with unexpected red fluorescence. PEGylation transformed from hydrophobic PAIs to amphiphilic PEGylated PAIs and partially shielded PAIs form the interaction with solvents. The combined hydrogen bond and π-π stacking interactions provided PEG-PAI4 with 100 nm red-shift emission compared to the intrinsic fluorescence of aminosuccinimide fluorophore. In addition, the combined interactions also aided in the spontaneous self-assembly process of PEG-PAI4 into nanovesicles, which significantly prohibited the fluorescence quenching in protic solvents and retained the fluorescence of PAIs in water. Finally, PEG-PAI4 exhibited excellent biostability and photostability compared to commercial organic dyes and showed good cell imaging results. The optimization of this PAI-based nanovesicle probe and its potential application in vivo research is under the way.

ASSOCIATED CONTENT Supporting Information


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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ 1H

NMR and/or

13C

NMR of MPEG2k, PEG-PAI4, PEG-PAI1 and PEG-co-PSUC1, GPC and

DLS characterization of PEG-co-PSUC1, PEG-PAI1 and PEG-PAI4, TEM image of PEG-co-PSUC1 in concentration solution (5 mg/mL), fluorescence and absorbance spectra of PEG500-PAI4 and PEG2kPAI4 in DMSO, photostability of PEG-PAI4.

AUTHOR INFORMATION Corresponding Authors *Email: [email protected] *Email: [email protected]

ORCID Junjie Yan: 0000-0001-8016-2277 Xinyu Wang: 0000-0002-9167-2077 Min Yang: 0000-0001-6976-8526

Author Contributions §J.

Yan and X. Wang contributed equally to this work.


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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We acknowledge the support from the National Natural Science Foundation of China (21504034, 31671035 and 51473071), Natural Science Foundation of Jiangsu Province (BK20161137 and BK20170204), Jiangsu Provincial Medical Innovation Team (CXTDA2017024) and National Significant New Drugs Creation Program (2017ZX09304021).

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Self-Assembling Nonconjugated Poly(amide-imide) into

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