Novel Strategy toward AIE-Active Fluorescent Polymeric Nanoparticles

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A novel strategy towards AIE-active fluorescent polymeric nanoparticles from polysaccharides: preparation and cell imaging Qing Wan, Ruming Jiang, Lili Guo, Shengxian Yu, Meiying Liu, Jianwen Tian, Guoqiang Liu, Fengjie Deng, Xiaoyong Zhang, and Yen Wei ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b01908 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 30, 2017

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ACS Sustainable Chemistry & Engineering

A novel strategy towards AIE-active fluorescent polymeric nanoparticles from polysaccharides: preparation and cell imaging Qing Wana,#, Ruming Jianga,#, Lili Guoc, Shengxian Yua, Meiying Liua, Jianwen Tiana, Guoqiang Liub, Fengjie Denga, Xiaoyong Zhanga,*, Yen Weib,* a

b

Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China. Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua

University, Beijing, 100084, P. R. China. c

Department of Physiology, Medical School of Nanchang University, Nanchang 330006, PR China.

# These authors contributed equally to this work Corresponding authors Xiaoyong Zhang, email: [email protected] Yen Wei, email: [email protected]

ACS Paragon Plus Environment


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Abstract Fluorescent polymeric nanoparticles (FPNs) as novel theranostic agents for cancer diagnose and treatment have been widely investigated in recent years. However, most of FPNs were constructed with typical inorganic quantum dots, fluorescent proteins and conventional organic dyes, which have been still suffered from many obstacles such as serious cytotoxicity, easy enzymolysis and vicious aggregation-caused fluorescence quenching (ACQ). Herein, to overcome these problems, we design and synthesize novel FPNs with a unique aggregation-induced emission (AIE) feature using resourceful and cost-effective oxidized sodium alginate (OSA) as natural polymer protected shells of FPNs. Moreover, differing from commercial or synthetic polymers such as PEG, BSA and lecithin, sodium alginate from marine seaweeds is cheaper, abundant source and excellent biocompatibility. Thus-prepared AIE-active OSA-Phe-OSA FPNs by the facile Schiff base condensation have many advantages, such as strong fluorescence, great water dispersity, excellent photostability and desirable biocompatibility and stained performance. These features endow them great potential for various biomedical applications.

Key

words:

Aggregation-induced emission, carbohydrate polymers, fluorescent polymeric

nanoparticles, biomedical applications


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Introduction The biological imaging techniques have been extensively explored for cancer diagnose and therapy applications over the past several decades.1-4 Biological imaging techniques, such as X-ray imaging,5 magnetic resonance imaging,6,

7

radionuclide imaging,8,

9

computed tomography10 and fluorescent

imaging,11-13 have been used for these purposes. Among these techniques, fluorescent imaging has received increasing attention and been widely developed because of its obvious advantages of easy operation, high sensitivity and selectivity, satisfying visualization. Up to now, the appeared fluorescent materials for various biomedical applications were majorly divided into inorganic fluorescent nanomterials (semiconductor quantum dots,14-17 carbon dots,18-20 Ln ion doped nanomaterials,21-23 silicon quantum dots and metallic clusters),24-29 fluorescent proteins30-32 and conventional organic dyes (perylene, fluorescein, rhodamine).33-36 However, it is regrettable that the inherent drawbacks of inorganic quantum dots such as relatively difficult preparation, potential toxicity, poor biodegradability and difficult surface modification limit their promising potential in biomedical applications. As another powerful tool of bioprobes, fluorescent proteins were often limited employment due to their high cost, low molar absorptivity, easy enzymolysis and low photobleaching thresholds. On the other hand, conventional organic dyes have been deemed to a class of ideal luminescent agents, which have been extensively used to biological imaging, target tracing and cancer diagnose. Unfortunately, because of most conventional organic dyes were constructed by abundant hydrophobic aromatic rings, which are usually in the same plane, resulting in the intramolecular accumulation by the π-π interaction when in the states of aggregated or high concentrations. This intramolecular accumulation behavior of fluorescent organic dyes leads to the luminescence quenching effect due to the energy from excited state to ground state emits by a way of nonradiative channel. This common phenomenon of conventional dyes was named as aggregation-caused quenching (ACQ) effect. In other words, conventional fluorescent dyes usually have luminescent effect in single molecular state, while the fluorescence quenched at aggregated state. Because of the natural hydrophobicity of conventional fluorescent dyes, they often aggregated into nanoparticles and caused fluorescence quenching in aqueous and physiological environment. Although adopting hydrophilic polymers to encapsulate hydrophobic organic dyes to enhance their hydrophilicity, the fluorescent dyes are still aggregated in the polymeric micelles, causing luminescence quenching. Reducing concentrations of organic dyes in polymeric micelles could reduce ACQ effect, however, the fluorescence intensity was also decreased.



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This would influence their sensitivity and visualization in vivo. Therefore, discovering a novel fluorescent agent to overcome aforementioned disadvantages from conventional fluorophores is extremely urgent. Aggregation-induced emission phenomenon (AIE) is exactly opposite to ACQ effect. The AIE-active organic molecules will emit strong fluorescence in aggregated or solid state but weak or non-fluorescence in single molecular state.37-41 Since the first report by Tang et al in 2001,42 more and more endeavors have been devoted to designing and synthesizing novel fluorescent small molecules with unique AIE performance. For this reason, according to the statement by Thomson Reuters in 2015, the synthesis of AIE fluorophores and AIE-based materials in biomedical applications have become one of the research hotspots in top two chemistry frontier fields. AIE-active functional polymeric materials have shown great potentials in the research fields of chem/biosensors,43-45 biomedical applications37, 46-57

and optoelectronic devices.58-64 Thanks to the unique AIE feature, many research works focus on

the design and synthesis of various AIEgens with specific performances such as near-infrared emission wavelength,65, 66 target label67, 68 and photodynamic therapy.69-71 However, these synthetic functional AIEgens have bad solubility or dispersity in aqueous and physiological solution, which hinder their biological applications. Aiming at the water soluble problem of AIEgens, many efforts have been made to resolve it. For example, modifying the ionic groups in AIEgens to enhance their water solubility is effective, but these synthetic AIEgens contained positive charges are toxic to normal cells.72, 73 Other method is that adopting amphiphilic polymers to encapsulate AIEgens to form AIE dye doped fluorescent polymeric nanoparticles (FPNs), which can effectively avoid injuries for normal cells because of great biocompatibility and low cytotoxicity of FPNs. However, these FPNs were easily decomposed by existed degrading enzymes in vivo, which would bring about fast leakage of AIEgens from FPNs before arriving to the targeted position in human body. Considering these problems, fabrication of AIE-active FPNs with great stability is extremely important. Many covalent fabricated strategies of AIE-active FPNs, such as chemical post-modification,54, 74 emulsion polymerization,75 RAFT polymerization76-78 and ring-opening polymerization,79, 80 have been reported in recent years. Thus-prepared AIE-active FPNs possess many merits contained great stability and hydrophilicity, excellent biocompatibility and stained performance for living cells. Nevertheless, the amphiphilic polymers used in previous works are high cost and difficult synthesis, and the experimental conditions are rigorous (oxygen-free, water-free, high temperature). Therefore, the preparation of AIE-active FPNs


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with simple operation procedure using low cost and abundant natural polymers is still desirable in the future. In this contribution, we prepare AIE-active FPNs (OSA-Phe-OSA) with red emission, excellent photostability and biocompatibility for living cells using oxidized sodium alginate (OSA) as polymer protected shells and synthetic AIEgen (Phe-NH2) as luminescent core by a facile Schiff base condensation method (Scheme 1). Sodium alginate (SA) is a resourceful and low cost, nontoxic and natural polysaccharide with a number of negative carboxyl groups.81-84 It can be extracted from brown seaweed and has been regarded as safe by Food and Drug Administration. The SA has been extensively used for fabrication of different functional materials with great interest for various applications, including emulsifier for food additional, formation of hydrogel, indigestion tablets for biological active components.85-87 However, original SA is not well soluble in aqueous solution, to improve its water solubility, the SA can be oxidized by sodium periodate to introduce aldehyde and carboxyl groups, in which the aldehyde group could react with amino group and form Schiff base, while the carboxyl group could enhance its water solubility and be used for carrying anticancer agent (such as cisplatin (CDDP)). Moreover, the Schiff base condensation is a facile conjugation reaction under low temperature, catalyst-free, air atmosphere and in the present of water. Combining superior of original materials and facile method, thus-prepared FPNs have extensive biomedical application value. Through investigating the biological toxicity and imaging capability of OSA-Phe-OSA FPNs for living cells, results demonstrate that OSA-Phe-OSA FPNs have great biocompatibility and strong stained performance for living cells. Based on these outstanding performances of OSA-Phe-OSA FPNs, these prepared FPNs have huge potential in biomedical applications.

THF, H2O RT, 12 h

：

Self-assembly C=N

Remove THF

： PheNH2

OSA

Scheme 1 Figure shows synthesis of OSA and OSA-Phe-OSA polymers.



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Experimental procedures Materials and characterization All agents and solvents were provided from commercial sources and used directly without further purification. The aqueous solution used in the experimental process was deionized water. Sodium periodate and sodium alginate were purchased from Aladdin company. The AIEgen (PheNH2) was synthesized from our previous work.80 1H nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance-400 spectrometer with D2O as the solvents. The synthetic materials were characterized by Fourier transform infrared spectroscopy (FT-IR) using KBr pellets, The FT-IR spectra were supplied from Nicolet5700 (Thermo Nicolet corporation). Transmission electron microscopy (TEM) images were recorded on a Hitachi 7650B microscope operated at 80 kV, the TEM specimens were got by putting a drop of the nanoparticle ethanol suspension on a carbon-coated copper grid. The fluorescence data were obtained from the fluorescence spectrophotometer (FSP, model: C11367-11), which purchased from Hamamatsu (Japan). The UV-visible absorption spectrum was recorded on a spectrometer (TU-1810, Persee). The size distribution of OSA-Phe-OSA FPNs in water was determined using a zeta Plus particle size analyzer (ZetaPlus, Brookhaven Instruments, Holtsville, NY). Preparation of oxidized sodium alginate (OSA) The synthetic procedure of OSA with great water solubility derives from previous method with a slight modification.88 In brief, SA (2.0 g) was dissolved in 200 mL distilled water, then sodium periodate (2.5 g) was added into SA solution and stirring in the dark at room temperature for 6 h. The reaction was terminated by adding ethylene glycol (3 mL) and magnetically stirring for another 1 h. Afterwards, the oxidized solution was dialyzed with dialysis bag (MWCO: 3500 Da) for three days with frequent distilled water. Finally, the large amount of ethanol was added to dialyzed OSA solution to obtain white precipitate. The white powder could be achieved after filtrating and freeze drying. Thus-obtained pure OSA powder was storing in low temperature environment for further application and characterizations. Preparation of AIE-active OSA biopolymers (OSA-Phe-OSA) The specific synthetic route of OSA-Phe-OSA is shown in Fig. 1. The synthetic procedure of AIE dye doped biopolymers (OSA-Phe-OSA) by simple and facile Schiff based condensation method was described. The OSA (300 mg) was dissolved in distilled water (5 mL), then synthetic AIEgen PheNH2


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(100 mg) in THF (5 mL) was mixed with OSA solution. The mixture was magnetically stirred at room temperature for 12 h. After then, the THF solvent was removed from mixture solution by the rotary evaporate. The fluorescent OSA biopolymers were freeze drying to obtain red powders. Finally, thus-obtained red OSA-Phe-OSA polysaccharides were washed with THF to remove residual Phe-NH2 dyes for 5 times until eluant become clear. The dried OSA-Phe-OSA biopolymers can be obtained after freeze drying. On the other hand, in the procedure of removing THF from mixture solution, these fluorescent OSA biopolymers can self-assemble into nanoparticles due to their amphiphilic property. Therefore, the morphology of resulting OSA-Phe-OSA is spherical size with Phe as core and OSA as protected shells of polysaccharide nanoparticles. SA

OSA

Phe-NH2 OSA-Phe-OSA

Fig. 1 Synthetic route of OSA and AIE-active OSA-Phe-OSA polymers.

Cytotoxicity evaluation of fluorescent OSA-Phe-OSA nanoparticles Fluorescent nanoparticles possessed promising biomedical applications potential due to their remarkable advantages contained intense emission for convenient recognition, easy endocytosis by cells and prolong the cycling time in blood system. However, the ideal biomedical nanoprobes should have the great biocompatibility. To evaluate the biomedical application potential of synthetic OSA-Phe-OSA FPNs, the cell viability of OSA-Phe-OSA FPNs with desirable red fluorescence (fluorescence quantum yield is 7%) on L929 cells was calculated by cell counting kit-8 (CCK-8) assay.89-92 Cells were put into 96-well microplates at a density of 5 × 104 cells mL–1 in 160 µL of respective media containing 10% fetal bovine serum (FBS). After the cell attachment for 24 h, the OSA-Phe-OSA FPNs with different concentrations (20-100 µg mL–1) were incubated with cells for 24 h. After then, the residual FPNs existed the outside of cells were removed and cells were washed with



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phosphate buffer saline (PBS) five times. 10 µL of CCK-8 dye and 100 µL of Dulbecco’s modified eagle medium (DMEM) cell culture medium were added to each well and incubated for another 3 h. Afterward, plates were analyzed using a microplate reader (VictorШ, Perkin-Elmer). Measurements of formazan dye absorbance were carried out at 450 nm, with the reference wavelength at 620 nm. The values were proportional to the number of live cells. The percent reduction of CCK-8 dye was compared to controls, which represented 100% CCK-8 reduction. Three replicate wells were used per microplate, and the experiment was operated for three times. Cell survival was expressed as absorbance relative to that of untreated controls. Results are presented as mean ± standard deviation (SD). Cell imaging based OSA-Phe-OSA FPNs Biological imaging of L929 cells using OSA-Phe-OSA FPNs was conducted by the confocal laser scanning microscope (CLSM) Zeiss 710 3-channel (Zeiss, Germany), as the excitation wavelength of laser was set as 488 nm. L929 cells were cultured in supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U mL–1 penicillin, and 100 µg mL–1 of streptomycin. The environment of cell culture was controlled in a humidified condition of 95% air and 5% CO2 in RPMI1640 culture medium at 37 °C. Culture medium should be updated every day to maintain the exponential growth of the cells. On the day before treatment, cells were set in a glass bottom dish with a density of 1 × 105 cells per dish. On the day of treatment, the OSA-Phe-OSA FPNs (80 µg mL-1) was incubated with cells for 3 h at 37 °C. Afterward, the cells were washed three times with PBS to remove the OSA-Phe-OSA FPNs and then fixed with 4% paraformaldehyde for 10 min at room temperature. Result and discussion Characterization of OSA-Phe-OSA FPNs Fluorescent nanoparticles with unique AIE feature have promising application potentials for optical imaging and cancer diagnose and treatment. Comparing to conventional fluorescent diagnose system, such as inorganic quantum dots, organic dyes and fluorescent proteins, AIE-active FPNs possess a series of merits including great dispersibility in physiological solution, strong fluorescence, favourable photostability, excellent biocompatibility and stained ability for living cells. Since the first discovery of AIE phenomenon, bioimaging applications and cancer therapeutic system based on AIEgens have acquired many achievements. This work introduces that preparation of AIE-active polysaccharide nanoparticles by a facile method. Comparing with commercial biocompatible polymers such as PEG and BSA, polysaccharide SA is resourceful and low cost. More importantly, excellent cytocompatibility


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and abundant hydroxyl groups of OSA make synthetic OSA-Phe-OSA FPNs easy endocytosis by cells and convenient introduction of functional components such as targeted agents, proteins, anticancer drug, DNA and RNA by the reaction with abundant hydroxyl groups. To evidence the successful preparation of AIE-active polysaccharide nanoparticles, many characterizations such as TEM, 1H NMR, DLS, FT-IR, UV-Vis and FL spectra have been conducted. Furthermore, to evaluate the biomedical application potential of OSA-Phe-OSA FPNs, cells viability evaluation and long-term imaging effect have also been carried out. As shown in Fig. 2, the 1H NMR spectra of OSA and OSA-Phe-OSA samples were compared to evidence successful preparation of AIE-active biopolymers. Focusing on the OSA spectrum, the peak at 8.3 ppm was ascribed to the –CHO groups. The hydrogen atoms of sugar moiety were located between 3.4-4.5 ppm. The 1H NMR signals about abundant hydroxyl group of sugar moiety were found from 4.8 to 5.6 ppm. After conjugating with PheNH2, as shown the OSA-Phe-OSA 1H NMR spectrum, the peak at 8.3 ppm is disappeared, while a new peak at 7.8 ppm appears, which can be contributed to the aromatic ring. Because of the formation of amphiphilic nanoparticles, the OSA-Phe-OSA samples just greatly dispersed in D2O, leading to difficult detection of aromatic rings. Previous work have demonstrated that aromatic peaks were difficult detection in the samples of AIE-active polysaccharides.93 On the other hand, there are other new peaks at 1.7, 2.2 and 2.5 ppm. These peaks can be assigned to the alkyl chain of PheNH2. Therefore, successful conjugation of PheNH2 with OSA polysaccharides by the Schiff base reaction according to the 1H NMR results. Of course, other characterizations would synergistically evidence this conclusion.



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Fig. 2 Picture shows the 1H NMR spectra of PheNH2 (A), OSA (B) and OSA-Phe-OSA (C). Comparing to 1H NMR spectrum of OSA sample, the spectrum of OSA-Phe-OSA shows new peaks at the position of 7.8 and 1.7 ppm, which was respectively ascribed to aromatic rings and alkyl chain existed in PheNH2 AIEgen, suggesting successful connection between OSA and PheNH2 with Schiff base bonds.

FT-IR characterization technique was also used to confirm successful reaction between OSA and PheNH2 by the formation of Schiff base bonds (Fig. 3). According to FT-IR spectra of three samples (SA, OSA and OSA-Phe-OSA), we also can confirm prosperous syntheses of AIE-active polysaccharides. The broad peak at 3340 cm-1 was –OH groups because of the existence of abundant –OH in most polysaccharides. After reacting with PheNH2, some new peaks were first appeared in the spectrum of OSA-Phe-OSA. For instance, the peak at 2185 cm-1 was the stretching vibration of –CN, which is consistent with –CN group of PheNH2 dye according to its IR spectrum (Fig. S1). According to previous work in our group, the structure of PheNH2 contained –CN group, while SA and OSA samples don’t contain this group. In addition, the position ranging from 650 to 780 cm-1 appears some new peaks, which were ascribed to the out-of-plane blending vibration of aromatic rings. Therefore, we can confirm that successful preparation of OSA-Phe-OSA. Furthermore, a new peak at 1548 cm-1 was contributed to stretching vibration of C=N bond. Combining analysis of 1H NMR and FT-IR spectra, we can demonstrate successful preparation AIE-active OSA-Phe-OSA biopolymers via a facile Schiff base condensation reaction at room temperature.

Fig. 3 FT-IR spectra of SA, OSA and OSA-Phe-OSA samples. Some characteristic peaks at 2185 cm-1


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and 1548 cm-1 were respectively ascribed to –CN and C=N groups, and peaks ranging from 650 cm-1 to 780 cm-1 were contributed to the out-of-plane blending vibration of aromatic rings, demonstrating successful preparation of AIE-active fluorescent OSA biopolymers.

Beside 1H NMR and FT-IR measurements, X-ray photoelectron spectroscopy (XPS) was also used to evidence that successful preparation of OSA-Phe-OSA fluorescent natural polymers with novel AIE feature. As is well known, the polysaccharide OSA is majorly constructed by four elements of H, C, O, Na, and ionic OSA possess great water solubility and biocompatibility. After conjugating with PheNH2 AIEgen, this biocompatible OSA was endowed with desirable luminescence, and other new elements were thereby introduced into OSA polysaccharide. As shown in Fig. 4, except four elements (H, C, O, Na), other two new elements of S and N could be discovered in the OSA-Phe-OSA samples (Fig. 4A). Respectively, Fig. 4B-F shows clear peaks of S 2p, C 1s, N 1s, O 1s and Na 1s signal, which respectively locate at 169.13, 287.41, 400.02, 533.01 and 1062.53 eV. The appearance of new elements (S, N) could demonstrate that PheNH2 fluorophore was linked with OSA polysaccharide. On the other hand, Table 1 shows that element content of C, N, O, S and Na in fluorescent OSA-Phe-OSA samples. The element content of S and N is 0.39% and 0.94%, while Na content is 1.48%. According to the content data of S and Na elements, we can calculate the content of PheNH2 dye in OSA-Phe-OSA polysaccharide is about 26.4%, while polysaccharide chain content in FPNs is about 73.6%.

Fig. 4 XPS spectra of OSA-Phe-OSA fluorescent natural polymers: (A) survey signal, (B) S 2p signal, (C) C 1s signal, (D) N 1s signal, (E) O 1s signal and (F) Na 1s signal.



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Table 1 Elemental signal peak and Quantification Name

Start BE

Peak BE

End BE

Atomic %

S 2p

175.33

169.13

157.53

0.39

C 1s

298.33

287.41

279.53

57.38

N 1s

410.33

400.02

392.53

0.94

O 1s

545.33

533.01

525.53

39.80

Na 1s

1079.33

1071.87

1062.53

1.48

Fluorophore PheNH2 with unique AIE feature has strong stained ability. When the PheNH2 connects with biocompatible OSA by the formation of Schiff base bonds, resulting in OSA possesses strong emission property under UV irradiation. On the other hand, when OSA conjugated with PheNH2 could endow the final polymers great water dispersity. As shown in Fig. 5A, the inset image shows clear red water solution of OSA-Phe-OSA fluorescent polymers and the background of word “Phe” can be directly observed. It demonstrated the excellent water dispersibility of OSA-Phe-OSA FPNs. UV-Vis spectrum of OSA-Phe-OSA water solution is used to analyze the structure of OSA-Phe-OSA FPNs. The absorption spectrum of pure OSA in water demonstrates that no obvious peak ranging from 300 to 800 nm. After conjugating with PheNH2, two obvious characteristic peaks respectively locate at 325 nm and 431 nm, suggesting that abundant π-π* conjugated systems existed in OSA-Phe-OSA samples. According to the chemical structure of PheNH2 from Scheme 1, this AIEgen is constructed by many π-π* conjugated systems. Herein, we can conclude successful preparation of AIE-active polysaccharides. On the other hand, due to fluorophore with AIE feature exists in OSA polymers, resulting in strong emission of OSA-Phe-OSA polymers. According to Fig. 5B, strong red fluorescence of OSA-Phe-OSA aqueous solution can be noticed after exposure under UV lamp shown in inset of Fig. 5B. FL spectrum of OSA-Phe-OSA solution shows the maximum emission wavelength is 607 nm, while optimal excitation wavelength locates at 495 nm. On the basis of optical pictures and spectra, these OSA-Phe-OSA polymers have great dispersibility in aqueous solution and strong red fluorescence, which primarily shows their promising potential in biomedical applications. Meanwhile, the unique AIE property of OSA-Phe-OSA polymers shows in Fig. S2, which is opposite to pure


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synthetic AIEgen (PheNH2). This reason is that original PheNH2 dye has great solubility in THF solvent, while aggregates in water. However, OSA-Phe-OSA polymers possess bad solubility in any organic solvents and great dispersity in aqueous solution.

Fig. 5 UV-Vis and fluorescent optical spectra of OSA-Phe-OSA polymer in aqueous solution. (A) UV-Vis spectrum of OSA-Phe-OSA in distilled water, the inset using word “Phe” as background shows fluorescent polymers dispersed in water; (B) FL spectrum of OSA-Phe-OSA polymer in distilled water, the inset show strong fluorescence of OSA-Phe-OSA in water by irradiating under UV lamp.

Fluorescent nanoparticles should have excellent stability in human body. To evaluate the stability of fluorescent OSA-Phe-OSA polymers, the relationship of fluorescent strength vs irradiating time and physiological condition should be measured. Firstly, the change of fluorescence intensity of OSA-Phe-OSA polymers in aqueous solution after irradiating by UV lamp for 24 h should be determined. As shown in Fig. 6A, the fluorescence intensity of OSA-Phe-OSA aqueous solution is 2280 a.u., while the fluorescence intensity of polymer solution reduced to 1670 a.u. After irradiating with UV lamp for 24 h. the reduced fluorescence intensity is calculated about 26.8%, demonstrating better photostability of OSA-Phe-OSA polymers. On the other hand, the pH environment of extracellular compartments and intracellular organelles is different. Usually, the pH value in normal cells environment is about 7.35-7.45, while physiological environment of cancer cells and lysosome is acidic. To evaluate the influence of different pH physiological environment for OSA-Phe-OSA FPNs, the FL spectra of OSA-Phe-OSA polymers in different physiological conditions with pH 7.4, 5.5 and 4.5 was investigated. As shown in Fig. 6B, the similar excitation and emission wavelengths of OSA-Phe-OSA solution could be observed. However, the fluorescence intensity is enhanced when fluorescent polysaccharides solution dispersed in acidic environment. This phenomenon suggests that OSA-Phe-OSA can still keep excellent fluorescence in acidic conditions. On the other hand, to evaluate



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their stability in acid and alkaline condition, the change of fluorescence intensity in different pH environment is shown in Fig. S4, the fluorescence strength is stable in alkaline condition, while reduced fluorescence intensity in strong acid environment. Even so, the fluorescence strength still keep desirable in pH 4-6 condition, Which make them promising potential in various biomedical applications such as cancer trace, anticancer drug delivery and cancer therapy.

Fig. 6 FL spectra of OSA-Phe-OSA dispersed in aqueous solution. (A) Fluorescence intensity value vs time under irradiating by UV lamp for 24 h; (B) Different pH environment (4.5, 5.5 and 7.4).

As is well-known, OSA has great hydrophilic property, while PheNH2 is hydrophobic fluorescent dye. When they are connected by a facile Schiff base reaction, thus-prepared OSA-Phe-OSA polymers have significant amphiphilic property. When they dispersed in aqueous solution, hydrophobic PheNH2 was first aggregated into core, while hydrophilic OSA was coated on the surface of hydrophobic core and directly extended in water. As shown in Fig. 7A, TEM images are used to prove that self-assembly of OSA-Phe-OSA into nanoparticles in aqueous solution. Many spherical nanoparticles can be directly observed from Fig. 7A, the diameter of these nanoparticle is about 200-300 nm. Dynamic light scattering (DLS) technique is also used to estimate the hydrodynamic size of OSA-Phe-OSA FPNs. As we can see from Fig. 7B, the major distribution of particle size of OSA-Phe-OSA ranges from 100 to 400 nm, and the compact district is 160 nm to 300 nm, which is closed to TEM result. Therefore, combining TEM and DLS results, we think the particle size of these OSA-Phe-OSA FPNs is about 250 nm. The smaller particles size of OSA-Phe-OSA FPNs makes them easy endocytosis by living cells for various biomedical applications. Furthermore, in order to determine the stability of OSA-Phe-OSA in aqueous solution, their critical micelles concentration (CMC) value is detected (Fig. S2). Adopting light scattering method to detect CMC value of OSA-Phe-OSA FPNs in aqueous solution, through detecting the fluorescence intensity of OSA-Phe-OSA polymers with different concentrations, the CMC


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value is calculated to be 0.067 mg/mL. The low CMC value demonstrates that FPNs with unique AIE feature can stably exist in aqueous solution. On the other hand, the inset of Fig. S2 reveals clear “Tyndall effect” of OSA-Phe-OSA FPNs, which can also prove successful formation of fluorescent nanoparticles via self-assembly.

A

B

PDI = 0.29

200 nm

Fig. 7 (A) TEM image of OSA-Phe-OSA dispersed in aqueous solution; (B) Histogram shows particle size distribution of OSA-Phe-OSA. Combing with this two characterization techniques, we think the particles size of OSA-Phe-OSA is about 250 nm.

3.2 Cytotoxicity evaluation of OSA-Phe-OSA FPNs The great water dispersibility, strong fluorescence, great photostability, stable structure with low CMC value and smaller size make the OSA-Phe-OSA FPNs promising biomedical applications. To research their biomedical application potential, the biocompatibility of OSA-Phe-OSA FPNs should be first investigated. As shown in Fig. 8, different concentrations of OSA-Phe-OSA FPNs are used to culture L929 cells. When the concentration of FPNs is 20 µg mL–1, the cell viability is 97%. When the culture concentration of FPNs was increased to 60 µg mL–1, the cell viability value still kept above 93 %. When concentration of FPNs was 100 µg mL–1, the survival cells are still closed to 90%. According to the results of cells viability, we can confirm synthetic OSA-Phe-OSA FPNs possess excellent biocompatibility, which makes them have promising biomedical applications.



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Fig. 8 Cytotoxicity evaluation of OSA-Phe-OSA FPNs for L929 cells. The cells were incubated with 20-100 µg mL–1 of OSA-Phe-OSA FPNs for 24 h.

Bioimaging of OSA-Phe-OSA FPNs On the basis of cytotoxicity evaluation result of OSA-Phe-OSA FPNs, the ability of OSA-Phe-OSA FPNs for biological imaging was investigated by using CLSM. According to the result of cell viability, we know ultra-low cytotoxicity of synthetic FPNs when concentration of FPNs reaches 80 µg mL–1. Therefore, we choose this concentration to evaluate their bioimaging ability. As shown in Fig. 9, we can observe almost all of L929 cells ingest effectively OSA-Phe-OSA FPNs, and the cellular morphology keep normal after culture with OSA-Phe-OSA FPNs during the cell imaging procedure, further indicating the good biocompatibility of OSA-Phe-OSA FPNs. On the other hand, the weak fluorescence can be observed in cell nuclei, demonstrating these FPNs are majorly uptake into cytoplasm instead of cell nuclei. This phenomenon is decided by direct endocytosis process of living cells. Most importantly, strong fluorescence can be observed after cells uptake these FPNs for 3 h, evidencing strong stained performance of OSA-Phe-OSA FPNs for living cells. Considered many carboxyl groups have been introduced in the OSA-Phe-OSA FPNs. These AIE-active FPNs should be also promising for drug delivery applications owing to the coordination interaction between carboxyl groups and cisplatin. It has been demonstrated that the carboxyl groups could effectively carry cisplatin and controlled release the anticancer agent through a pH-dependent behavior.94 Therefore, these AIE-active FPNs could also be utilized for controlled delivery of cisplatin with self-report capability.


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Fig. 9 CLSM images of OSA-Phe-OSA FPNs in L929 cells for 3 h. (A) excited with 488 laser, (B) bright field and (C) merged image of A and B. Scale bar = 20 µm.

Conclusion In conclusion, AIE-active FPNs based on PheNH2 with far-red emission as the core and OSA as the shells have been prepared by a facile Schiff base condensation reaction in this work. Thanks to encapsulating the AIEgen into nanoparticles, the final OSA-Phe-OSA FPNs possess strong fluorescence and great photostability. On the other hand, the introduction of OSA to serve as protected coating of FPNs endows them desirable water dispersibility and biocompatibility. In addition, low CMC value and suitable size make these AIE-active FPNs can be stably existed in physiological environment and easily endocytosed by living cells. From the evaluation of cytotoxicity and biological imaging of OSA-Phe-OSA FPNs for living cells, the excellent biocompatibility and stained performance make them have promising potential in various biomedical applications. Comparing with other fluorescent nanoparticles with short emission wavelength, these biocompatible FPNs with red fluorescence are more suitable for the applications of biological optical imaging, targeted tracer, cancer diagnose and treatment due to reduced damage for normal cells. Moreover, comparing to previous commercial biocompatible polymers, modified SA polysaccharides (OSA) are resourceful, convenient preparation and compatible for human tissue and cells, which can reduce cost for the preparation of FPNs. More importantly, abundant hydroxyl groups still retain on the surface of AIE-active FPNs, which are convenient for further conjugation of multifunctional components. We therefore expect these OSA-Phe-OSA FPNs show huge potential for various biomedical applications.

Acknowledgements



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This research was supported by the National Natural Science Foundation of China (Nos. 51363016, 21474057, 21564006, 21561022, 21644014), Natural Science Foundation of Jiangxi Province in China (Nos. 20161BAB203072, 20161BAB213066). References 1.

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Supporting Information



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The FT-IR spectrum of PheNH2, AIE feature, CMC and effect of pH on the fluorescence properties of OSA-Phe-OSA were provided in the supporting information.


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μm


B

C

AIE-active fluorescent polymeric nanoparticles (FPNs) are prepared by the formation of Schiff base bonds between the AIEgens with red emission and abundant oxidized sodium alginate with many hydrophilic groups such as hydroxyl and carboxyl. Thus-obtained FPNs possess low toxicity, great biocompatibility and stained performance for living cells, which make them wide application potentials in the biomedical field.

20 μm


20 μ

Novel Strategy toward AIE-Active Fluorescent Polymeric Nanoparticles

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