Saponin-Based Near-Infrared Nanoparticles with Aggregation

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Saponin-Based Near-Infrared Nanoparticles with Aggregation-Induced Emission behavior: Enhancing Cell Compatibility and Permeability Jie Zhang, Qi Wang, Jingkang Liu, Zhiqian Guo, jinfeng yang, Qiang Li, Shaoze Zhang, Chenxu Yan, and Wei-Hong Zhu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00812 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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ACS Applied Bio Materials

Saponin-Based Near-Infrared Nanoparticles with AggregationInduced Emission behavior: Enhancing Cell Compatibility and Permeability Jie Zhang,†,§ Qi Wang,†,§ Jingkang Liu,‡ Zhiqian Guo,*,† Jinfeng Yang,⊥ Qiang Li,† Shaoze Zhang,† Chenxu Yan,† Wei-Hong Zhu*,† †Shanghai

Key Laboratory of Functional Materials Chemistry, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China. E-mail: [email protected]; [email protected] ‡State Key Laboratory of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China ⊥College

of Chemistry and Chemical Engineering, Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, Shihezi University, Shihezi 832000, China §Jie

Zhang and Qi Wang contributed equally.

Supporting Information Placeholder ABSTRACT: Fluorescence imaging is critical for physiological activities and cell biology but limited by the poor solubility, cell compatibility and permeability. Herein, we develop a novel engineering methodology to prepare biocompatible and penetrable aggregationinduced emission (AIE) nanoparticles with the assistance of flash nanoprecipitation (FNP) technology. Based on the donor-π-acceptor (Dπ-A) system, the AIE building block of tricyano-methylene-pyridine (TCM) is fine-tuned to long emission wavelength by modulating the π-conjugation bridge and electron donating group, thereby achieving high solid fluorescent quantum yield, near-infrared (NIR) characteristic, large stokes shift, and excellent photostability. Based on the FNP technology, the amphiphilic saponin solution and TCMN-5 in organic solvent are quickly mixed in the multi-inlet vortex mixer (MIVM), followed by saponin-encapsulation of the hydrophobic AIE nucleation with inhibiting further growth of nanoparticles. The biocompatible amphiphilic saponin such as α-hederin can encapsulate and micellize the AIE TCM fluorophore for efficient cell imaging. The kinetic FNP technology can not only modulate the uniform diameter size, but also distinctly increase the micelle stability when compared to the conventional thermodynamic self-aggregation method, which provides an alternative opportunity for scale-up preparation of drug and probes in delivery vehicles. KEYWORDS: Aggregation-induced emission, Extending emission, Saponin-based micellization, Compatibility and permeability traditional fluorescence probes. Especially, near-infrared (NIR, 650-900 nm) can efficiently increase the penetration depth of bioimaging.14-18 Unfortunately, the AIE NPs still face the challenges of high hydrophobicity, limited cellular compatibility, low permeability and uncontrollable particle size in bioimaging.19-

INTRODUCTION Fluorescence imaging has contributed to fundamental insights into the complex physiological activities, cell biology and medical diagnostics, which requires deep tissue penetration, fast cellular uptake efficiency, as well as long-term stable tracking ability to satisfy the increasingly requirements of spatial and temporal imaging resolution.1-4 Unfortunately, the inherent drawbacks of traditional fluorescent nanoparticles (NPs) are limited to the biological applications, such as undesirable fluorescence properties from aggregation caused quenching (ACQ), short wavelength and poor photostability as well as low cellular uptake efficiency from the improper hydrophobic nature, biocompatibility and uncontrollable particle size.5,6 With the advantages of the aggregation-induced emission (AIE) phenomenon, some advanced efforts have been extensively investigated on tumor bioimaging and therapy,7-13 which has been demonstrated to overcome the specific ACQ effect, improve the sensitivity, and enhance the long-term tracing with respect to

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Recently, we have reported a novel AIE building block of tricyano-methylene-pyridine (TCM) with multi-modification site, bright fluorescence intensity in the aggregation state and tunable fluorescent emission. It is a classical donor-π-acceptor (D-π-A) system that tricyano groups constitute the terminal acceptor group, while the π bridge and electron-donating group are introduced at a symmetrical methyl position. Herein we further adjust the π bridges and the electron donating groups in the TCM building block to efficiently extend emission wavelength with high photostability. Specifically, incorporation of thiophene and methoxytriphenylamine units in TCMN-5 exhibits strong electron donating ability, thus generating 768 nm fluorescence in solid state and 733 nm in solution. By increasing the penetration depth in vivo

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Figure 1. Fine-tuning NIR AIE fluorescence emission, fabricating stable and efficient saponin-based NPs. (A) Rational design to finetune and extend solid emission ranging from 656 to 768 nm. In D-π-A system, TCM with electron-withdrawing tricyano units can be alternatively attached with different bridges (phenyl and thiophene) and electron donors (triphenylamine and methoxytriphenylamine) to obtain the NIR AIE system. (B) Preparation of stable saponin-based AIE nanoparticles by FNP technology for efficiently cellular uptake. THF solution (stream 1) of AIE molecules and saponin, and water (streams 2, 3 and 4) were mixed in the multi-inlet vortex mixer system to prepare NPs. The size and polydispersity index (PDI) of saponin-based AIE nanoparticles can be well controlled by adjusting flow rates of water and THF. The stable and efficient encapsulation system has been demonstrated for rapid imaging of cells.

acceptors, especially focusing on adjusting the conjugation bridges (phenyl and thiophene) and the electron donors (triphenylamine and methoxytriphenylamine) to efficiently modulate the emission wavelength (Figure 1A).35-43 It is noted that, the electron donating groups and electron withdrawing groups were placed on both ends of the molecular skeleton. With the symmetric modification in the building block of TCM,44-46 the different π bridges and electron donating groups were introduced to obtain the N-substituted TCM derivatives. That is, different π bridges (phenyl and thiophene) were introduced into TCM to obtain TCMN-1 and TCMN-2, then the electron-donating groups such as triphenylamine and methoxytriphenylamine were introduced to obtain TCMN-3 and TCMN-4, respectively. Incorporation of both thiophene and methoxytriphenylamine into TCM resulted in TCMN-5. These TCM derivatives are supposed to exhibit different fluorescence properties. The molecular structures of all these luminogens and intermediates were fully characterized in the Supporting Information. NIR AIE-Based TCM Derivatives with Tunable Emission. Much light has been shed on exploring long wavelength AIEgens for penetrating deep into tissues and avoiding auto-fluorescence in bioimaging. In this regard, the photophysical properties of TCM derivatives were recorded in THF and water mixtures, where the maximum absorption was found at 500 nm (TCMN-1), 540 nm (TCMN-2), 500 nm (TCMN-3), 545 nm (TCMN-4), and 545 nm (TCMN-5), indicative of long excitation wavelengths with low bio-photo toxicity (Figure S1). The fluorescence wavelength of TCMN-2 with thiophene bridge (690 nm) is much longer than TCMN-1 with phenyl bridge (625 nm) in Figure 2A, Table S1 and Table S2, because electron-rich thiophene groups are more inclined to donate electrons than benzene groups. Meanwhile, the obvious

with NIR emission, TCMN-5 can greatly promote the potential application in biological imaging (Figure 1A). Currently, fluorophores are always coated with peptides, antibodies and biomolecules to improve the biocompatibility and cell permeability with increasing the solubility of the aqueous imaging system.25,26 Among these, the saponin-based (α-hederin) carrier can not only increase the uptake rate, but also improve the cellular compatibility due to its specific amphiphilic nature.27 However, the saponin-based carrier faces the shortcomings of large diameter distribution, high polydispersity and poor micelle stability. Therefore, we borrow the flash nanoprecipitation (FNP) to prepare stable and uniform saponin-based TCMN-5 nanoparticles, for satisfying cellular internalization speed and fast biological imaging (Figure 1B).28-34 Specifically, the amphiphilic saponin solution and TCMN-5 in organic solvent were quickly mixed within micro-/milliseconds in the multi-inlet vortex mixer (MIVM), followed by saponin-encapsulation of the hydrophobic AIE nucleation with inhibiting further growth of nanoparticles. As consequence, we can well adjust the size and polydispersity of NPs by changing flow rate and solvent ratio with respect to conventional self-assembly methods. Significantly, with the help of FNP technology, scale-up preparation of NPs is expected to be applied to development of new drugs and probe vehicles.

RESULTS AND DISCUSSION Rational Design. Extending emission wavelength to NIR region is very attractive in the field of in vivo optical bioimaging due to its deep penetration depth.18 Given that the specific AIE advantages, we rationally designed AIE-active TCM derivatives with D-π-A system, using the tricyano groups as the electron

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Figure 2. NIR AIE-based TCM derivatives with tunable emission. (A) Normalized fluorescent spectra in the aggregation state of TCMN-1 (625 nm), TCMN-2 (690 nm), TCMN-3 (627 nm), TCMN-4 (684 nm) and TCMN-5 (733 nm). Photoluminescence spectra and intensity of (B) TCMN-1, (C) TCMN-2, (D) TCMN-3, (E) TCMN-4 and (F) TCMN-5 (10-5 M) in THF/H2O mixtures with different volume fractions of water (fw), λex = 500 nm. (G) Cyclic voltammograms of TCMN-4 and TCMN-5 measured in CH2Cl2. (H) Schematic diagram in energy levels of TCMN-4 and TCMN-5. (I) Normalized fluorescent spectra of TCM derivatives in 95% water. TCMN-4 and TCMN-5 both have two potential peaks with reversible reduction waves. Obviously, the cyclic voltammetry and theoretical calculations reveal that the band gap between HOMO and LUMO of TCMN-5 is less than that of TCMN-4 (Figure 2H and Figure S2), demonstrating that the electron-donating bridge can reduce the band gap between HOMO and LUMO, and extend the emission wavelength. Therefore, we can successfully modulate the NIR emission from 627 nm (TCMN-4) to 733 nm (TCMN-5) just by inserting a thiophene group, which is highly preferable for bioimaging (Figure 2I). AIE Mechanism of TCMN-5 with Single Crystals. In order to understand the role of the intermolecular interaction in solid-state emission and AIE mechanism, we performed single crystal X-ray diffraction measurement of TCMN-5.48 As shown in Figure 3A, there are large torsional angles of 61.3°-81.1° between the phenyl rings and the pyridine unit in the backbone, thus TCMN-5 undergoes high-frequency motions, leading to nonradiative decay in dissolved state. Furthermore, the moderate interplanar angles of 7.5°-26.2° are observed between the ethylene and TCM units, in which the propeller-like nonplanar conformation would lead to weak π-π interactions (Table S4 in the Supporting Information).25 As a result, the aggregation state brought forth the specific enhanced emission.

AIE effects were observed in both TCMN-1 and TCMN-2 along with molecular aggregation and fluorescence enhancement upon adding water (Figure 2B and 2C). Specifically, the emission density of TCMN-1 decreased at fw > 90%, because the amorphous agglomerate leaded to the non-radiative decay of energy.25 To illustrate the effect of electron donating groups, the triphenylamine and methoxytriphenylamine units were introduced into TCMN-3 and TCMN-4, with extending the emission band to about 627 and 684 nm, respectively (Figure 2D and 2E). For TCMN-4, an enhanced fluorescence was observed with fluorescence quantum yield of about 24-fold in the mixed THF/H2O solution (fw = 95%). To further extend the NIR emission, we introduced the stronger electron donating ability of thiophene and methoxytriphenylamine units as TCMN-5 (Figure 2F). Similarly, TCMN-5 redshifted from 690 to 733 nm when increasing the water fraction. To further understand the conjugation-bridge effect on the luminogens wavelength, the cyclic voltammetry (CV) and theoretical calculations was employed to evaluate the band gap between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) (Figure 2G and Table S3 in the Supporting Information).47 As shown in Figure 2G,

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Figure 3. Explanation of single crystals for AIE characteristic. (A) The single crystal structures of TCMN-5. (B) Top view and (C) side view of molecular stacked structures. (D) Normalized solid-state fluorescent spectra of TCM derivatives. spontaneously nanoclusters in water (Figure S3). Tang group has reported that saponin-based AIE NPs have the superior cellular uptake, compared to other delivery NPs.26 In order to facilitate the cellular uptake, α-hederin with unique permeabilization mechanism and amphiphilic properties was utilized to fabricate saponin@TCMN-5 NPs. First, a traditional solvent displacement method (TSDM) was used to prepare saponin@TCMN-5@TSDM NPs. As shown in Figure 4A, TCMN-5 and α-hederin (1 mg mL−1) were dissolved in THF to form a homogeneous red mixture, followed by adding into water with vigorously stirring for 10 min. Then the amphiphilic αhederin molecules easily self-assembled, forming micelles in aqueous solution. In the light of hydrophobic nature, TCMN-5 aggregates were encapsulated within the α-hederin micelles.

In the molecular packing structure of TCMN-5, the two antiparallel TCM planes have no obvious overlap with large spacing of 3.647 Å, suggestive of poor π-π interactions (Figure 3B and C). The intramolecular motion can be well restricted, resulting in red shift emission and high solid emission of the TCMN-5. As a consequence, we can successfully achieve the modulation of AIEactive luminescence wavelength in TCM derivatives from 656 to 768 nm in their solid state (Figure 3D). FNP Technology for Stable and Uniform Saponin-Based AIE Nanoparticles. Due to the excellent AIE properties and nearinfrared fluorescence emission, TCMN-5 was investigated in bioimaging fields. However, the hydrophobic surface of TCMN-5 aggregation leads poor cellular uptake during the incubation period. Specifically, hydrophobic TCMN derivatives easily form

Figure 4. FNP technology for stable and uniform saponin AIE nanoparticles. (A) TEM images of saponin@TCMN-5, prepared by traditional nanoprecipitation method. (B) Schematic illustration of FNP and TEM images of saponin@TCMN-5. (C) Dynamic laser scattering (DLS) results of saponin@TCMN-5 prepared by FNP with different flow rates of water and THF.

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Compared with TCMN-5 NPs (Figure S3), the saponin@TCMN5@TSDM have irregular spherical shapes with broad size distribution by transmission electron microscopy (TEM, Figure 4A). However, this size inhomogeneous saponin@TCMN5@TSDM is not desirable in bioimaging application.49 Comparably, in order to fabricate uniform- and size-controllable nanoparticles, the kinetic FNP technology was introduced to prepare saponin@TCMN-5 NPs.28-34 As shown in Figure 4B, TCMN-5 and α-hederin in THF solution (stream 1), and water (streams 2, 3 and 4) were vigorously mixed within an MIVM system.27 TEM results show that the optimized saponin@TCMN5 NPs have uniform spherical shapes, even being capable of well controlling the size as small as 30 nm (Figure 4B). As shown in Figure 4C, in order to obtain different particle sizes and polydispersity index, the flow rate was modulated from 12 to 24 mL min−1 for THF stream and from 24 to 96 mL min−1 for water streams. Finally, THF was removed by dialyzing against water for 12 hours before further characterization and cellular experiments. With advantage of FNP technology, the DLS results revealed that prepared saponin@TCMN-5 NPs have small diameter of about 104.7 nm and small polydispersity index (PDI) of 0.158 (Figure 4C), which can be enriched in tumor tissues and further facilitate in vivo tumor-targeted imaging due to the enhanced permeability and retention (EPR) effect.17 Moreover, the controllable size and polydispersity of NPs could be achieved by modulating the flow rate of THF and water streams and volume fraction of water (Figure 4C). When the volume ratio of was changed 3:1 to 5:1 for water to THF, the NPs size and PDI decreased remarkably. Meantime, when the volume ratio was set to 3:1 for water to THF, the NPs size became smaller with the increscent of flow rate because the more nuclei and compact NPs were more likely to form under vigorously mixing process. The negative zeta potential of saponin@TCMN-5 (-20 mV) was lower than that of TCMN-5 NPs (-5 mV), providing the electrostatic stabilization of nanoparticles. Therefore, the uniform saponin NPs can be very conveniently prepared by FNP technology. Excellent Colloidal Stability and Photostability of Saponin@TCMN-5. The excellent colloidal stability and photostability are critical for long-term biological tracing and cellular retention. We compared the size stabilities of saponin@TCMN-5 NPs fabricated by FNP technology, saponin@TCMN-5@TSDM NPs and TCMN-5 NPs. The particle size of saponin@TCMN-5@TSDM and TCMN-5 NPs increased rapidly within 5 days (Figure 5A). In contrast, saponin@TCMN-5 NPs fabricated by FNP remained at a constant size for at least 15 days. Consequently, the kinetic FNP technology can not only modulate the diameter size, but also distinctly increase the colloidal stability when compared to the conventional thermodynamic selfaggregation method, which provides an alternative opportunity for scale-up preparation of drug and probes in delivery vehicles. The excellent photostability of probes is very critical to avoid photobleaching during prolonged bioimaging.50-59 Here the photostability of saponin@TCMN-5 NPs was compared with commercial ICG. The absorption intensity of saponin@TCMN-5 remains 87% of its initial value after light irradiation (Figure 5B). However, the absorption intensity of ICG lost quickly to 50% within 50 s and dropped sharply to 12% after 200 s. Overall, the outstanding photostability of saponin@TCMN-5 NPs can greatly benefit for bioimaging. Saponin-Based AIE Nanoparticles for Excellent Biocompatibility and Rapid Cell Imaging. The efficient aggregate emission and the delivery properties of saponin@TCMN-5 NPs prompt us to explore its bioimaging applications.59-63 The cells incubated with TCMN-5 showed weak

Figure 5. Excellent colloidal stability and photostability of saponin@TCMN-5. (A) Long-term micelle stability of different AIE NPs in phosphate buffer saline (pH = 7.4). (B) Time courses of absorption intensity change of TCMN-5 (10 μM) in phosphate buffer saline (pH = 7.4). ICG (10 μM) (Indocyanine Green) is used for comparison. fluorescence, while the intensity of saponin@TCMN-5 treated cells gradually strengthen upon the increasing incubation time, suggesting the excellent internalization efficiency of the saponin@TCMN-5 (Figure 6A). Also, the 40.6 times enhancement was observed for emission intensity of saponin@TCMN-5 treated cells, compared to TCMN-5 (Figure 6B). The results indicated that saponin functionalized saponin@TCMN-5 NPs facilitated the cellular internalization of NPs. Remarkably, even just after 5 min incubation, the emission of saponin@TCMN-5 NPs was also bright within the HeLa cells, suggestive of fast cell imaging. In addition, a colocalization test illustrated saponin@TCMN-5 NPs are mainly lysosome localization (Figure S4). Also, the cell flow experiment was performed to evaluate the cellular retention efficiency of the saponin@TCMN-5 NPs. The Hela cells were incubated with TCMN-5 and saponin@TCMN-5 NPs overnight at 37 °C. The flow cytometry was used to record the fluorescence profiles of the labeled cells that were subcultured for designated time intervals. As shown in Figure 6D and 6E, saponin@TCMN-5 NPs can effectively stain cells compared to TCMN-5, which can be ascribed to the specific membrane penetration mechanism of saponin. Cytotoxicity assessment is very of importance for the development of biological probes, particularly for membrane permeabilization or modulation.63-71 The methylthiazolyldiphenyltetrazolium bromide (MTT) assay was applied to determine the cytotoxicity of TCMN-5 and saponin@TCMN-5 NPs. The viabilities of Hela cells showed negligible difference and remained above 95% and 85% after incubation with TCMN-5 and

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Figure 6. Saponin-based AIE nanoparticles for excellent biocompatibility and rapid cell imaging. (A) Confocal images of Hela cells incubated with TCMN-5 (10 μM) and saponin@TCMN-5 (10 μM) from 5 to 30 min. (B) Fluorescence intensity of the TCMN-5 and saponin@TCMN-5 stained HeLa cells with different incubation times. (C) Cell viability after incubating TCMN-5 and saponin@TCMN-5 at different concentrations with Hela cells for 24 h. (D and E) Flow cytometry histograms for Hela cells incubation with TCMN-5 and saponin@TCMN-5 for varied durations. saponin@TCMN-5 NPs, respectively, suggestive of low cytotoxicity (Figure 6C). Therefore, the high efficiency cellular uptake and low toxicity of saponin@TCMN-5 NPs is ideal for clinical applications.

Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ORCID Wei-Hong Zhu: 0000-0001-9103-166X Zhiqian Guo: 0000-0002-2192-825X

CONCLUSIONS All good permeability, high brightness, long wavelength, uniform morphology, superior stability and satisfy biocompatibility are required in fluorescence bioimaging. We have developed a series of NIR AIE fluorophores ranging from 656 to 768 nm in solid state, and followed by preparing highly efficient and stable saponinbased NPs with FNP technology. The wavelength of TCM probes can be fine-tuned by modulating the π bridges and the electron donating groups within D-π-A system. Saponins (α-hederin) performance biocompatible amphiphilicity and specific membrane penetration mechanism, then can encapsulate AIE lumimogens to obtain hydrophilic nanoparticles. In view of the simplicity of the kinetic controllable FNP method, the size-controlled and micellarstabilized saponin-based NIR AIE delivery system was obtained, which proved to be an effective tool for cancer imaging. More importantly, the delivery system exhibits advantages, such as scaleup fabrication, fast delivery, excellent photo- and micelle-stability.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by NSFC Science Center Program (21788102) and Creative Research Groups (21421004), National key Research and Development Program (2016YFA0200300 and 2017YFC0906900), NSFC/China (21636002, 21622602 and 81602718), Shanghai Municipal Science and Technology Major Project (Grant No.2018SHZDZX03), and Fundamental Research Funds for the Central Universities, and Program of Introducing Talents of Discipline to Universities (B16017).

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. This shows the materials, synthesis process, measurement, and characterization, spectral properties, single crystal, DLS and TEM images, and confocal imaging.

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Saponin-Based Near-Infrared Nanoparticles with Aggregation

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