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Preparation of chitin-based fluorescent hollow particles by Pickering emulsion polymerization using functional chitin nanofibers
BIOMAC-14006; No of Pages 7 International Journal of Biological Macromolecules xxx (xxxx) xxx
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Preparation of chitin-based fluorescent hollow particles by Pickering emulsion polymerization using functional chitin nanofibers Seiichiro Noguchi, Kazuya Yamamoto, Jun-ichi Kadokawa ⁎ Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan
a r t i c l e
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Article history: Received 28 October 2019 Received in revised form 19 November 2019 Accepted 27 November 2019 Available online xxxx Keywords: Chitin nanofiber Fluorescent hollow particle Pickering emulsion polymerization
a b s t r a c t This study investigated the preparation of chitin-based fluorescent hollow particles by Pickering emulsion polymerization of styrene using bifunctional chitin nanofibers (ChNFs) as stabilizer, giving CNF-based composite particles, followed by solubilizing out inner polystyrene. In addition to the introduction of anionic maleyl groups on ChNFs to improve dispersibility in aqueous ammonia, polymerizable methacryl groups were substituted on ChNFs as second functionalization to provide ability in copolymerization with styrene for stabilization of hollow structures. Consequently, after the formation of styrene-in-water Pickering emulsion using the bifunctional ChNFs as stabilizer, radical polymerization was conducted in the presence of potassium persulfate as an initiator to produce the composite particles. The hollow particles were then fabricated by solubilizing out inner polystyrene with toluene, which stably dispersed in water. Encapsulation of a fluorescent dye, pyrene, into the cavity of the hollow particles was achieved by hydrophobic interaction with polystyrene present on the inner walls, which could be released by treatment of the resulting fluorescent hollow particles with surfactant, oleyl alcohol, in water. The same Pickering emulsion polymerization system was also performed in the presence of a pyrene derivative having a polymerizable group to obtain fluorescent composite/hollow particles with pyrene moieties covalently bound to polystyrene. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Hollow particles contain interior hollow structure, which is usually covered by a solid shell. They have been applied in many fields such as drug delivery [1], catalysis [2], and medical imaging/diagnostics [3,4] due to the excellent features, such as light weight, high filling ratio, low coefficients of thermal expansion. We previously prepared chitin-based hollow particles by means of Pickering emulsion polymerization of styrene using anionic chitin nanofibers (ChNFs) as stabilizer, followed by solubilizing out inner polystyrene (Fig. 1a) [5]. Pickering emulsions are emulsions of any type, either oil-in water, water-in-oil, or even multiple, stabilized by solid particles or other types of solid materials, in place of surfactants in general emulsions [6,7]. In addition, Pickering emulsion polymerization using polymerizable hydrophobic substrates has been proven as a fascinating method to fabricate composite particles with well-defined morphology. For example, the preparation of composite particles has been conducted using biological molecules as stabilizer such as cellulose nanofibers [8,9]. Such emulsion polymerization products utilizing naturally abundant substrates with controlled fashions will allow significant advances for biomedical and environmentally benign applications [10,11]. ⁎ Corresponding author. E-mail address: [email protected] (J. Kadokawa).
Chitin is an abundant aminopolysaccharide composed of β(1→4)linked N-acetyl-D-glucosamine repeating units [12–14], which still remains an unutilized biomass resource, primarily because of its intractable bulk structure and insolubility in water and common organic solvents. An efficient approach for the functionalization of chitin is nanofibrillation, that is, the fabrication of nanocrystals and nanofibers [15–17]. Such chitin nanomaterials have been used as stabilizer in Pickering emulsion systems [18–30]. We have developed bottom-up approach to fabricate ChNFs, in which a dispersion of self-assembled ChNFs with ca. 20–60 nm in width was facilely obtained by regeneration from a chitin ion gel with an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), using methanol, followed by ultrasonication [31,32], based on the fact that AMIMBr formed the ion gel with chitin by heating the mixture [33]. Filtration of the resulting ChNF/methanol dispersion gave a film with highly entangled nanofiber morphology. The self-assembled ChNFs have been used for composition with other polymers to fabricate composite materials [34]. In the following study, carboxylate anions were additionally introduced on the surface of the self-assembled ChNF film by reaction of hydroxy groups with maleic anhydride [35]. The resulting anionic ChNF film showed better redispersibility than the original ChNF film under aqueous conditions, such as in aqueous ammonia. As abovementioned, we previously reported styrene-in-water Pickering emulsion polymerization of styrene using the anionic maleylated
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Fig. 1. (a) Pickering emulsion polymerization of styrene using anionic maleylated chitin nanofibers (ChNFs) as stabilizer to produce composite particles and conversion into hollow particles by solubilizing out inner polystyrene and (b) SEM image of spin-coated sample from aqueous dispersion of products after solubilizing out inner polystyrene.
ChNFs as stabilizer (ChNFs/styrene = 0.1 (w/w)) to fabricate ChNF/ polystyrene composite particles (Fig. 1a) [5]. ChNFs contributed to forming the sufficient emulsion system as stabilizer, owing to their amphiphilic property in the presence of anionic maleyl groups, in addition to their good dispersibility in aqueous media. By solubilizing out inner polystyrene from the resulting composite particles with toluene, ChNF-based hollow particles were successfully fabricated. However, when the produced hollow particles were attempted to be isolated from the toluene dispersion and re-dispersed in water, the SEM image of a spin-coated sample from the aqueous dispersion observed to not
remain the hollow morphology (Fig. 1b). This result indicated instability of the shells in the hollow structure against the above isolation and redispersion procedures, probably because which were made up by physical interaction of polystyrene among ChNFs. To improve stability of the ChNF-based hollow particles, in the present study, ChNFs were further functionalized by polymerizable methacryl groups in addition to maleyl groups to add ability in copolymerization with styrene during Pickering emulsion polymerization (Fig. 2). Consequently, the hollow particles, which were fabricated using such bifunctional ChNFs, were re-dispersed in water while
Fig. 2. Pickering emulsion polymerization of styrene or a mixture of styrene with (1-pyrene)methyl methacrylate (PMM) using bifunctional chitin nanofibers (ChNFs) as stabilizer to produce composite particles and conversion into hollow particles by solubilizing out inner polystyrene.
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remaining the hollow structure, owing to stabilization of the shells by covalent linkages between the two components in the shells. Furthermore, fluorescent hollow particles incorporating pyrene moieties were also fabricated both physically by encapsulation in the cavity by hydrophobic interaction and chemically by copolymerization with a polymerizable pyrene monomer during Pickering emulsion polymerization. Release of pyrene physically present in the hollow particles was also attempted using surfactant. 2. Experimental section 2.1. Materials and methods Chitin powder from crab shell was purchased from Wako Pure Chemicals, Tokyo, Japan. An ionic liquid, AMIMBr, was prepared by reaction of 1-methylimidazole with 3-bromo-1-propene according to the method adapted from the literature procedure [36]. Other reagents and solvents were available commercially and used without further purification. A maleylated ChNF film was prepared by substitution at hydroxy groups on ChNFs with maleic anhydride (30 equiv. with total hydroxy groups) under melted condition (m.p. of maleic anhydride = ca. 53 °C) in the presence of perchloric acid according to our previous study [35]. The degree of substitution (DS) value of the maleyl group was calculated from the integrated ratio of the CH= signal to the H1 signals in the 1H NMR spectrum (DCl/D2O) to be 23.0% with total hydroxy groups. (1-Pyrene)methyl methacrylate (PMM) was prepared by reaction of 1-pyrenemethanol with methacryloyl chloride in the presence of triethylamine in THF according to the literature procedure [37]. The structure was evaluated by the 1H NMR spectrum (CDCl3); δ 1.97 (s, CH3, 3H), 5.56, 6.15 (2 s, CH2=, 2H), 5.89 (s, CH2O, 2H), 7.99–8.19 (m, aromatics, 9H). 1H NMR spectra were recorded on JEOL ECA 600 and JEOL ECX400 spectrometers. The SEM images were obtained using Hitachi S\\4100H electron microscope. The average particle diameters were calculated on the basis of 50 objects for each SEM image. Fluorescence spectra were recorded on a FP-6300Q3 spectrometer. 2.2. Preparation of maleylated/methacrylated (bifunctional) ChNF film A mixture of chitin (0.110 g, 0.59 mmol) with AMIMBr (1.00 g, 4.92 mmol) was allowed to stand at room temperature for 24 h and subsequently heated with stirring at 100 °C for 24 h to obtain a chitin ion gel (10 wt%). The gel was then soaked in methanol (40 mL) at room temperature for 48 h, followed by ultrasonication (Branson 1510, 42 kHz, 70 W) for 10 min to yield a dispersion of self-assembled ChNFs in methanol. The dispersion was subjected to filtration to isolate the ChNFs, which were then dried at 60 °C at 3 h under reduced pressure to obtain a self-assembled ChNF film. The resulting ChNF film (0.101 g, 0.490 mmol) and N,N-dimethyl-4aminopyridine (DMAP, 0.003 g, 0.025 mmol) were mixed with methacrylic anhydride (1.5 g, 9.73 mmol). After the mixture was heated at 70 °C for 3 h with stirring, maleic anhydride (1.5 g, 15.3 mmol) was added and additionally heated at that temperature for 3 h. After the mixture was left standing at room temperature for 30 min, methanol (40 mL) was added to disperse the product, followed by ultrasonication. The bifunctionalized product was isolated by filtration and dried at 60 °C for 3 h under reduced pressure to obtain a maleylated/ methacrylated ChNF film (ca. 0.1 g). The DS value of the maleyl and methacryl groups were calculated from the integrated ratio of the CH = and CH3– signals to the H1 signals in the 1H NMR spectrum (DCl/D2O) to be ca. 10.0–11.5 and 6.6–8.0% with total hydroxy groups, respectively.
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for 12 h, followed by ultrasonication for 4 h to give a dispersion. After styrene (1.1 mL, 9.6 mmol) was added to the dispersion, the mixture was ultrasonicated for 10 min to give a Pickering emulsion. After nitrogen gas was bubbled into the emulsion for 5 min on an ice-water bath, an aqueous solution (1.0 mL) of potassium persulfate (9.2 mg, 0.034 mmol, 0.35 mol% with styrene) was added. The resulting emulsified mixture was then kept at 70 °C for 24 h with stirring under nitrogen atmosphere. The reaction mixture was centrifugated for 45 min (3500 rpm) and the residual product was lyophilized to give the composite particles (0.552 g). For SEM measurement, a mixture of the product (1.0 mg) with water (10 mL) was ultrasonicated for 1 h to give a redispersion. 2.4. Preparation of ChNF/polystyrene composite particles with pyrene moieties A mixture of the maleylated/methacrylated ChNF film (0.101 g) with 1.0 mol/L aqueous ammonia (10 mL) was stirred at room temperature for 12 h, followed by ultrasonication for 4 h to give a dispersion. After styrene (1.1 mL, 9.6 mmol), which dissolved PMM (4.2 mg, 0.014 mmol) was added to the dispersion, the mixture was ultrasonicated for 10 min to give a Pickering emulsion. After nitrogen gas was bubbled into the emulsion for 5 min on an ice-water bath, an aqueous solution (1.0 mL) of potassium persulfate (9.2 mg, 0.034 mmol, 0.35 mol% with styrene) was added. The resulting emulsified mixture was then kept at 70 °C for 24 h with stirring under nitrogen atmosphere. The reaction mixture was centrifugated for 45 min (3500 rpm) and the residual product was lyophilized to give the composite particles (0.626 g). For SEM measurement, a mixture of the product (1.0 mg) with water (10 mL) was ultrasonicated for 1 h to give a redispersion. 2.5. Preparation of hollow particles The procedures for immersion of the resulting composite particles (5.0 mg) in toluene (20 mL) for 10 min and decantation of toluene were repeated 6 times. The resulting material was finally immersed in toluene (20 mL) to give a dispersion of the hollow particles, which were isolated by filtration and dried at 60 °C for 2 h. The residual product (1.5 mg) was immersed in water (10 mL) to give a aqueous dispersion, which was subjected to SEM measurement. 2.6. Separation of copolymer from hollow particles The abovementioned hollow particles (5.0 mg) were treated with 1.0 mol/L aqueous NaOH (30 mL) at 80 °C for 4 h. After pH value was adjusted to 3 by adding 1.0 mol/L aqueous HCl and the resulting mixture was stirred at room temperature for 1 h, the insoluble material was isolated by filtration and dried at 60 °C for 4 h under reduced pressure. The fraction soluble in CDCl3 was then analyzed by the 1H NMR measurement. 2.7. Encapsulation of pyrene in hollow particles Pyrene (35 mg) was added in the aqueous dispersion of hollow particles (3.5 mg/10 mL), and the mixture was stirred at room temperature for 24 h. After the hollow particles were isolated by filtration and washed with acetone, the products were re-dispersed in water (10 mL) for fluorescent measurement. 2.8. Release of pyrene from hollow particles
2.3. Preparation of ChNF/polystyrene composite particles A mixture of the maleylated/methacrylated ChNF film (0.096 g) with 1.0 mol/L aqueous ammonia (10 mL) was stirred at room temperature
An aqueous dispersion (10 mL) of pyrene-encapsulated hollow particles (3.5 mg) containing oleyl alcohol (1.0 mL) was stirred at room temperature for 2 days. After the hollow particles were isolated by
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filtration, washed with acetone, and dried at 60 °C for 2 h, the products were re-dispersed in water (10 mL) for fluorescent measurement. 3. Results and discussion 3.1. Preparation of stable hollow particles using maleylated/methacrylated ChNFs To improve stability of the shells in the hollow particles, we attempted to comprise covalent linkages between the two components, i.e., ChNFs and polystyrene, by radical copolymerization for their construction. In addition to anionic maleyl groups for improving dispersibility in aqueous ammonia, accordingly, polymerizable methacryl groups were introduced on the surface of ChNFs using methacrylic anhydride. The reaction of methacrylic anhydride with hydroxy groups on ChNFs was first performed in the presence of DMAP to give methacrylated ChNFs, which were then substituted with maleyl groups by addition of maleic anhydride to the reaction mixture to produce the maleylated/methacrylated (bifunctional) ChNFs. The 1H NMR spectrum of the sample hydrolyzed and solubilized from the product in DCl/D2O (Fig. S1a) supported the introduction of both the maleyl and methacryl groups, in which the CH= signals c and d and the CH2=C(CH3)C=O signals a and b from the former and latter groups were detected at δ 6.43/6.56 (methines assignable to maleyl group and fumaryl group, a little present produced by isomerization under acidic condition, respectively) and at δ 1.63 (methyl) and 5.33/5.88 (methylene), respectively. The values of degrees of substitution (DSs) with total hydroxy groups by both the reactions at 70 °C for 3 h were calculated from the integrated ratios of the CH= and CH3– signals to the H1 (anomeric protons) signals to be ca. 10.0–11.5 and 6.6–8.0%, respectively. The SEM image of the produced bifunctional ChNF film in
Fig. 3b shows highly-entangled nanofiber morphology similar to that observed for the original ChNF film before functionalization in Fig. 3a, suggesting that functionalization has not affected nanostructure of the film. The resulting bifunctional ChNF films were then dispersed in 1.0 mol/L aqueous ammonia (10 mL) by stirring the mixture at room temperature for 12 h, followed by ultrasonication for 4 h according to the procedure reported by us [35]. When styrene (1.1 mL, ChNFs/styrene = 0.1 (w/w)) was mixed with the obtained dispersions and the mixtures were ultrasonicated for homogenization, styrene-in-water emulsion was efficiently formed. Pickering emulsion polymerization of styrene was then performed by the same procedure as that using the maleylated ChNFs reported in our previous literature [5]. After nitrogen gas was bubbled into the emulsion on an ice-water bath, radical polymerization of styrene was carried out in the presence of potassium persulfate (0.35 mol% with styrene) at 70 °C for 24 h with stirring under nitrogen atmosphere (Fig. 2). The product was isolated by centrifugation and lyophilized to obtain ChNF/polystyrene composite particles. As the product was redispersed well in water by ultrasonication, the SEM image of a spincoated sample from the re-dispersion has been taken, which clearly observes the uniform particle morphology with an average diameter of 365 nm (Fig. 3c). The composite particles were then converted into ChNF-based hollow particles by solubilizing out inner polystyrene from the composite particles through repetitions of immersion in toluene for 10 min and decantation 6 times. The SEM image of a spin-coated sample from the dispersion of the resulting material in toluene shows clear hollow morphology (Fig. 3d). Furthermore, the SEM image also shows the presence of holes in shells as seen in previous studies on the fabrication of hollow particles [38,39]. The holes were probably generated by reaching out the inner polystyrene with toluene during the above the solubilizing
Fig. 3. SEM images of (a) ChNF film, (b) maleylated/methacylated (bifunctional) ChNF film, (c–e) spin-coated samples from aqueous dispersion of composite particles, toluene dispersion of hollow particles, and aqueous dispersion of hollow particles by Pickering emulsion polymerization of styrene using bifunctional ChNFs, and (f–h) spin-coated samples from aqueous dispersion of composite particles, toluene dispersion of hollow particles, and aqueous dispersion of hollow particles by Pickering emulsion copolymerization of styrene with PMM using bifunctional ChNFs.
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procedures. The presence of the holes in shells on the hollow particles has probably induced the encapsulation-release of a hydrophobic dye in the present study (vide infra). From the weight of the products obtained by evaporating toluene from the dispersions at 100 °C for 2 h, ca. 30 wt% polystyrenes were calculated to be remained. After the hollow particles were isolated by filtration, they were re-dispersed in water. The SEM image of a spin-coated sample from the aqueous dispersion shows morphology of the hollow particles (Fig. 3e), which are completely different from that resulted by the same procedure using the maleylated (monofunctional) ChNFs as shown in Fig. 1b. These observations strongly suggest improvement of stability of the shells in the hollow particles, probably owing to the formation of covalent linkages between ChNFs and polystyrene by radical copolymerization of methacryl groups on ChNFs with the closely present styrene during the production of composite particles. To confirm the occurrence of the copolymerization, the ester linkages in the polymer chains on ChNFs were cleaved by alkaline treatment of the hollow particles with aqueous NaOH. Then, aqueous HCl was added to the mixture for conversion of sodium methacrylate units into methacrylic acid units to make the separated copolymer insoluble in aqueous media, which was isolated by filtration and dried. The 1H NMR spectrum of the soluble fraction in the isolated product in CDCl3 (Fig. S1b) shows a signal a at 0.87 ppm assignable to methyl group of the methacrylic acid units besides large signals b and c ascribed to polystyrene, strongly supporting the occurrence of copolymerization of styrene with methacryl groups on ChNFs. As walls inside the shells of the hollow particles were considered to be hydrophobic, owing to the presence of polystyrene covalently linked on ChNFs, encapsulation of a hydrophobic fluorescent dye, pyrene, through the holes in shells was investigated. The encapsulation experiment was carried out by stirring a mixture of pyrene with the aqueous dispersion of the hollow particles at room temperature for 24 h. After filtration of the mixture, the isolated product was washed with acetone to remove the dye absorbed on the surfaces of the particles. The fluorescence spectrum of the re-dispersion of the resulting particles in water excited at 345 nm exhibits an emission maximum at 378 nm due to pyrene (Fig. 4b), indicating the presence of the dye in the products. On the other hand, when the same experiment has been conducted using the composite particles, where the cavity is filled by polystyrene, the fluorescence spectrum does not mostly show peaks by emission of pyrene (Fig. 4a). These results strongly support that pyrene has been encapsulated in the cavity of the hollow particles through the holes, probably by its hydrophobic interaction with polystyrene present at
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inner walls of the shell. Indeed, the aqueous dispersion of the resulting hollow particles observed blue florescent emission by UV-light irradiation at 365 nm (Fig. S2b), while such emission was not detected by the same light irradiation to the aqueous dispersion of the composite particles after treatment with pyrene (Fig. S2a). Release of pyrene through holes from the resulting hollow particles was investigated by treatment with surfactant, as it potentially interacts with hydrophobic substrates [40]. After the aqueous dispersion of the pyrene-encapsulated hollow particles was treated with oleyl alcohol at room temperature for 2 days in water, the product was isolated by filtration and washed with acetone. The fluorescence spectrum of the aqueous re-dispersion of the obtained hollow particles excited at 345 nm does not observe an emission peak from pyrene (Fig. 4c). Indeed, UV-light irradiation to the resulting dispersion at 365 nm did not induce fluorescent emission as shown in Fig. S2c. On the other hand, when the same procedure for the pyrene-encapsulated hollow particles was carried out without oleyl alcohol, the fluorescence spectrum excited at 345 nm exhibits an emission peak from pyrene (Fig. 4d). These results strongly suggest that release of pyrene successfully happens by treatment with surfactant, oleyl alcohol. However, intensity of the emission peak from pyrene after the control experiment in Fig. 4d slightly decreased compared with that of the pyreneencapsulated hollow particles in Fig. 4b, indicating that a little occurrence of spontaneous release of pyrene in aqueous dispersion without oleyl alcohol. This result has motivated us to fabricate other composite/hollow particles stably carrying fluorescent moieties through the formation of covalent linkages as described in the following section. 3.2. Preparation of fluorescent composite and hollow particles with pyrene moieties An attempt was made to incorporate fluorescent (pyrene) moieties into composite/hollow by covalent linkages with polystyrene using PMM having a polymerizable methacryl group. Pickering emulsion copolymerization of styrene with PMM using the bifunctional ChNFs as stabilizer was carried out by the same procedure as described above to produce composite particles (Fig. 2). The SEM image of the spincoated sample shows uniform particle morphology with an average diameter of 372 nm (Fig. 3f), which is similar as that for the abovementioned composite particles without pyrene moieties (Fig. 3c). The resulting composite particles were then converted into the corresponding hollow particles by solubilizing out inner polystyrene with toluene. The SEM images of the spin-coated samples from a
Fig. 4. Fluorescence spectra of aqueous dispersions after encapsulation experiments of (a) composite particles and (b) hollow particles and aqueous dispersions (c) after treatment of pyrene-encapsulated hollow particles with oleyl alcohol and (d) after control experiment without oleyl alcohol.
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Fig. 5. Fluorescence spectra of aqueous dispersions of (a) composite and (b) hollow particles by Pickering emulsion copolymerization of styrene with PMM and (c) aqueous dispersion after encapsulation experiment of hollow particles.
toluene dispersion and an aqueous re-dispersion, obtained according to the procedure in Fig. 2, exhibit hollow morphologies (Fig. 3g and h, respectively), indicating that the produced hollow particles are stable during the re-dispersion procedure in water. The fluorescence spectra of the aqueous dispersions of the resulting composite and hollow particles have been measured by excitation at 345 nm, which observe emission maxima at 378 nm due to pyrene (Fig. 5a and b, respectively). Intensity of the emission peak in Fig. 5b is lower than that in Fig. 5a, because of solubilizing out inner fluorescent copolymer during the procedure for conversion into the hollow particles. Both the aqueous dispersions observe blue florescent emission by UV-light irradiation at 365 nm (Figs. S2d and e). After encapsulation of pyrene into the hollow particles were carried out by the same procedure as above, intensity of the emission peak increased from that before encapsulation (Fig. 5b and c). This result indicates the presence of pyrene moieties in the hollow particles both by covalently linking and by hydrophobic interaction with polystyrene in the cavity. 4. Conclusion In this study, we investigated the preparation of chitin-based fluorescent hollow particles by Pickering emulsion polymerization of styrene using the bifunctional ChNFs as stabilizer, followed by solubilizing out inner polystyrene with toluene. The resulting hollow particles were stable by dispersing in water owing to the presence of methacrylate units on ChNFs covalently bound to styrene units. The fluorescent dye, pyrene, was then incorporated into the hollow particle both physically by encapsulation through hydrophobic interaction and chemically by using PMM during Pickering emulsion copolymerization. Release of pyrene from the former hollow particles was achieved by treatment with oleyl alcohol in water. The present fluorescent hollow particles have a potential to be practically employed as photofunctional materials. Furthermore, the encapsulation and release approaches conducted in this study will imply the present hollow particles in application as carriers for drug delivery system in the future. CRediT authorship contribution statement Seiichiro Noguchi: Investigation, Data curation. Kazuya Yamamoto: Data curation, Writing - original draft. Jun-ichi Kadokawa: Conceptualization, Methodology, Writing - review & editing.
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Preparation of chitin-based fluorescent hollow particles by Pickering emulsion polymerization using functional chitin nanofibers Seiichiro Noguchi, Kazuya Yamamoto, Jun-ichi Kadokawa ⁎ Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan
a r t i c l e
i n f o
Article history: Received 28 October 2019 Received in revised form 19 November 2019 Accepted 27 November 2019 Available online xxxx Keywords: Chitin nanofiber Fluorescent hollow particle Pickering emulsion polymerization
a b s t r a c t This study investigated the preparation of chitin-based fluorescent hollow particles by Pickering emulsion polymerization of styrene using bifunctional chitin nanofibers (ChNFs) as stabilizer, giving CNF-based composite particles, followed by solubilizing out inner polystyrene. In addition to the introduction of anionic maleyl groups on ChNFs to improve dispersibility in aqueous ammonia, polymerizable methacryl groups were substituted on ChNFs as second functionalization to provide ability in copolymerization with styrene for stabilization of hollow structures. Consequently, after the formation of styrene-in-water Pickering emulsion using the bifunctional ChNFs as stabilizer, radical polymerization was conducted in the presence of potassium persulfate as an initiator to produce the composite particles. The hollow particles were then fabricated by solubilizing out inner polystyrene with toluene, which stably dispersed in water. Encapsulation of a fluorescent dye, pyrene, into the cavity of the hollow particles was achieved by hydrophobic interaction with polystyrene present on the inner walls, which could be released by treatment of the resulting fluorescent hollow particles with surfactant, oleyl alcohol, in water. The same Pickering emulsion polymerization system was also performed in the presence of a pyrene derivative having a polymerizable group to obtain fluorescent composite/hollow particles with pyrene moieties covalently bound to polystyrene. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Hollow particles contain interior hollow structure, which is usually covered by a solid shell. They have been applied in many fields such as drug delivery [1], catalysis [2], and medical imaging/diagnostics [3,4] due to the excellent features, such as light weight, high filling ratio, low coefficients of thermal expansion. We previously prepared chitin-based hollow particles by means of Pickering emulsion polymerization of styrene using anionic chitin nanofibers (ChNFs) as stabilizer, followed by solubilizing out inner polystyrene (Fig. 1a) [5]. Pickering emulsions are emulsions of any type, either oil-in water, water-in-oil, or even multiple, stabilized by solid particles or other types of solid materials, in place of surfactants in general emulsions [6,7]. In addition, Pickering emulsion polymerization using polymerizable hydrophobic substrates has been proven as a fascinating method to fabricate composite particles with well-defined morphology. For example, the preparation of composite particles has been conducted using biological molecules as stabilizer such as cellulose nanofibers [8,9]. Such emulsion polymerization products utilizing naturally abundant substrates with controlled fashions will allow significant advances for biomedical and environmentally benign applications [10,11]. ⁎ Corresponding author. E-mail address: [email protected] (J. Kadokawa).
Chitin is an abundant aminopolysaccharide composed of β(1→4)linked N-acetyl-D-glucosamine repeating units [12–14], which still remains an unutilized biomass resource, primarily because of its intractable bulk structure and insolubility in water and common organic solvents. An efficient approach for the functionalization of chitin is nanofibrillation, that is, the fabrication of nanocrystals and nanofibers [15–17]. Such chitin nanomaterials have been used as stabilizer in Pickering emulsion systems [18–30]. We have developed bottom-up approach to fabricate ChNFs, in which a dispersion of self-assembled ChNFs with ca. 20–60 nm in width was facilely obtained by regeneration from a chitin ion gel with an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), using methanol, followed by ultrasonication [31,32], based on the fact that AMIMBr formed the ion gel with chitin by heating the mixture [33]. Filtration of the resulting ChNF/methanol dispersion gave a film with highly entangled nanofiber morphology. The self-assembled ChNFs have been used for composition with other polymers to fabricate composite materials [34]. In the following study, carboxylate anions were additionally introduced on the surface of the self-assembled ChNF film by reaction of hydroxy groups with maleic anhydride [35]. The resulting anionic ChNF film showed better redispersibility than the original ChNF film under aqueous conditions, such as in aqueous ammonia. As abovementioned, we previously reported styrene-in-water Pickering emulsion polymerization of styrene using the anionic maleylated
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Fig. 1. (a) Pickering emulsion polymerization of styrene using anionic maleylated chitin nanofibers (ChNFs) as stabilizer to produce composite particles and conversion into hollow particles by solubilizing out inner polystyrene and (b) SEM image of spin-coated sample from aqueous dispersion of products after solubilizing out inner polystyrene.
ChNFs as stabilizer (ChNFs/styrene = 0.1 (w/w)) to fabricate ChNF/ polystyrene composite particles (Fig. 1a) [5]. ChNFs contributed to forming the sufficient emulsion system as stabilizer, owing to their amphiphilic property in the presence of anionic maleyl groups, in addition to their good dispersibility in aqueous media. By solubilizing out inner polystyrene from the resulting composite particles with toluene, ChNF-based hollow particles were successfully fabricated. However, when the produced hollow particles were attempted to be isolated from the toluene dispersion and re-dispersed in water, the SEM image of a spin-coated sample from the aqueous dispersion observed to not
remain the hollow morphology (Fig. 1b). This result indicated instability of the shells in the hollow structure against the above isolation and redispersion procedures, probably because which were made up by physical interaction of polystyrene among ChNFs. To improve stability of the ChNF-based hollow particles, in the present study, ChNFs were further functionalized by polymerizable methacryl groups in addition to maleyl groups to add ability in copolymerization with styrene during Pickering emulsion polymerization (Fig. 2). Consequently, the hollow particles, which were fabricated using such bifunctional ChNFs, were re-dispersed in water while
Fig. 2. Pickering emulsion polymerization of styrene or a mixture of styrene with (1-pyrene)methyl methacrylate (PMM) using bifunctional chitin nanofibers (ChNFs) as stabilizer to produce composite particles and conversion into hollow particles by solubilizing out inner polystyrene.
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remaining the hollow structure, owing to stabilization of the shells by covalent linkages between the two components in the shells. Furthermore, fluorescent hollow particles incorporating pyrene moieties were also fabricated both physically by encapsulation in the cavity by hydrophobic interaction and chemically by copolymerization with a polymerizable pyrene monomer during Pickering emulsion polymerization. Release of pyrene physically present in the hollow particles was also attempted using surfactant. 2. Experimental section 2.1. Materials and methods Chitin powder from crab shell was purchased from Wako Pure Chemicals, Tokyo, Japan. An ionic liquid, AMIMBr, was prepared by reaction of 1-methylimidazole with 3-bromo-1-propene according to the method adapted from the literature procedure [36]. Other reagents and solvents were available commercially and used without further purification. A maleylated ChNF film was prepared by substitution at hydroxy groups on ChNFs with maleic anhydride (30 equiv. with total hydroxy groups) under melted condition (m.p. of maleic anhydride = ca. 53 °C) in the presence of perchloric acid according to our previous study [35]. The degree of substitution (DS) value of the maleyl group was calculated from the integrated ratio of the CH= signal to the H1 signals in the 1H NMR spectrum (DCl/D2O) to be 23.0% with total hydroxy groups. (1-Pyrene)methyl methacrylate (PMM) was prepared by reaction of 1-pyrenemethanol with methacryloyl chloride in the presence of triethylamine in THF according to the literature procedure [37]. The structure was evaluated by the 1H NMR spectrum (CDCl3); δ 1.97 (s, CH3, 3H), 5.56, 6.15 (2 s, CH2=, 2H), 5.89 (s, CH2O, 2H), 7.99–8.19 (m, aromatics, 9H). 1H NMR spectra were recorded on JEOL ECA 600 and JEOL ECX400 spectrometers. The SEM images were obtained using Hitachi S\\4100H electron microscope. The average particle diameters were calculated on the basis of 50 objects for each SEM image. Fluorescence spectra were recorded on a FP-6300Q3 spectrometer. 2.2. Preparation of maleylated/methacrylated (bifunctional) ChNF film A mixture of chitin (0.110 g, 0.59 mmol) with AMIMBr (1.00 g, 4.92 mmol) was allowed to stand at room temperature for 24 h and subsequently heated with stirring at 100 °C for 24 h to obtain a chitin ion gel (10 wt%). The gel was then soaked in methanol (40 mL) at room temperature for 48 h, followed by ultrasonication (Branson 1510, 42 kHz, 70 W) for 10 min to yield a dispersion of self-assembled ChNFs in methanol. The dispersion was subjected to filtration to isolate the ChNFs, which were then dried at 60 °C at 3 h under reduced pressure to obtain a self-assembled ChNF film. The resulting ChNF film (0.101 g, 0.490 mmol) and N,N-dimethyl-4aminopyridine (DMAP, 0.003 g, 0.025 mmol) were mixed with methacrylic anhydride (1.5 g, 9.73 mmol). After the mixture was heated at 70 °C for 3 h with stirring, maleic anhydride (1.5 g, 15.3 mmol) was added and additionally heated at that temperature for 3 h. After the mixture was left standing at room temperature for 30 min, methanol (40 mL) was added to disperse the product, followed by ultrasonication. The bifunctionalized product was isolated by filtration and dried at 60 °C for 3 h under reduced pressure to obtain a maleylated/ methacrylated ChNF film (ca. 0.1 g). The DS value of the maleyl and methacryl groups were calculated from the integrated ratio of the CH = and CH3– signals to the H1 signals in the 1H NMR spectrum (DCl/D2O) to be ca. 10.0–11.5 and 6.6–8.0% with total hydroxy groups, respectively.
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for 12 h, followed by ultrasonication for 4 h to give a dispersion. After styrene (1.1 mL, 9.6 mmol) was added to the dispersion, the mixture was ultrasonicated for 10 min to give a Pickering emulsion. After nitrogen gas was bubbled into the emulsion for 5 min on an ice-water bath, an aqueous solution (1.0 mL) of potassium persulfate (9.2 mg, 0.034 mmol, 0.35 mol% with styrene) was added. The resulting emulsified mixture was then kept at 70 °C for 24 h with stirring under nitrogen atmosphere. The reaction mixture was centrifugated for 45 min (3500 rpm) and the residual product was lyophilized to give the composite particles (0.552 g). For SEM measurement, a mixture of the product (1.0 mg) with water (10 mL) was ultrasonicated for 1 h to give a redispersion. 2.4. Preparation of ChNF/polystyrene composite particles with pyrene moieties A mixture of the maleylated/methacrylated ChNF film (0.101 g) with 1.0 mol/L aqueous ammonia (10 mL) was stirred at room temperature for 12 h, followed by ultrasonication for 4 h to give a dispersion. After styrene (1.1 mL, 9.6 mmol), which dissolved PMM (4.2 mg, 0.014 mmol) was added to the dispersion, the mixture was ultrasonicated for 10 min to give a Pickering emulsion. After nitrogen gas was bubbled into the emulsion for 5 min on an ice-water bath, an aqueous solution (1.0 mL) of potassium persulfate (9.2 mg, 0.034 mmol, 0.35 mol% with styrene) was added. The resulting emulsified mixture was then kept at 70 °C for 24 h with stirring under nitrogen atmosphere. The reaction mixture was centrifugated for 45 min (3500 rpm) and the residual product was lyophilized to give the composite particles (0.626 g). For SEM measurement, a mixture of the product (1.0 mg) with water (10 mL) was ultrasonicated for 1 h to give a redispersion. 2.5. Preparation of hollow particles The procedures for immersion of the resulting composite particles (5.0 mg) in toluene (20 mL) for 10 min and decantation of toluene were repeated 6 times. The resulting material was finally immersed in toluene (20 mL) to give a dispersion of the hollow particles, which were isolated by filtration and dried at 60 °C for 2 h. The residual product (1.5 mg) was immersed in water (10 mL) to give a aqueous dispersion, which was subjected to SEM measurement. 2.6. Separation of copolymer from hollow particles The abovementioned hollow particles (5.0 mg) were treated with 1.0 mol/L aqueous NaOH (30 mL) at 80 °C for 4 h. After pH value was adjusted to 3 by adding 1.0 mol/L aqueous HCl and the resulting mixture was stirred at room temperature for 1 h, the insoluble material was isolated by filtration and dried at 60 °C for 4 h under reduced pressure. The fraction soluble in CDCl3 was then analyzed by the 1H NMR measurement. 2.7. Encapsulation of pyrene in hollow particles Pyrene (35 mg) was added in the aqueous dispersion of hollow particles (3.5 mg/10 mL), and the mixture was stirred at room temperature for 24 h. After the hollow particles were isolated by filtration and washed with acetone, the products were re-dispersed in water (10 mL) for fluorescent measurement. 2.8. Release of pyrene from hollow particles
2.3. Preparation of ChNF/polystyrene composite particles A mixture of the maleylated/methacrylated ChNF film (0.096 g) with 1.0 mol/L aqueous ammonia (10 mL) was stirred at room temperature
An aqueous dispersion (10 mL) of pyrene-encapsulated hollow particles (3.5 mg) containing oleyl alcohol (1.0 mL) was stirred at room temperature for 2 days. After the hollow particles were isolated by
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filtration, washed with acetone, and dried at 60 °C for 2 h, the products were re-dispersed in water (10 mL) for fluorescent measurement. 3. Results and discussion 3.1. Preparation of stable hollow particles using maleylated/methacrylated ChNFs To improve stability of the shells in the hollow particles, we attempted to comprise covalent linkages between the two components, i.e., ChNFs and polystyrene, by radical copolymerization for their construction. In addition to anionic maleyl groups for improving dispersibility in aqueous ammonia, accordingly, polymerizable methacryl groups were introduced on the surface of ChNFs using methacrylic anhydride. The reaction of methacrylic anhydride with hydroxy groups on ChNFs was first performed in the presence of DMAP to give methacrylated ChNFs, which were then substituted with maleyl groups by addition of maleic anhydride to the reaction mixture to produce the maleylated/methacrylated (bifunctional) ChNFs. The 1H NMR spectrum of the sample hydrolyzed and solubilized from the product in DCl/D2O (Fig. S1a) supported the introduction of both the maleyl and methacryl groups, in which the CH= signals c and d and the CH2=C(CH3)C=O signals a and b from the former and latter groups were detected at δ 6.43/6.56 (methines assignable to maleyl group and fumaryl group, a little present produced by isomerization under acidic condition, respectively) and at δ 1.63 (methyl) and 5.33/5.88 (methylene), respectively. The values of degrees of substitution (DSs) with total hydroxy groups by both the reactions at 70 °C for 3 h were calculated from the integrated ratios of the CH= and CH3– signals to the H1 (anomeric protons) signals to be ca. 10.0–11.5 and 6.6–8.0%, respectively. The SEM image of the produced bifunctional ChNF film in
Fig. 3b shows highly-entangled nanofiber morphology similar to that observed for the original ChNF film before functionalization in Fig. 3a, suggesting that functionalization has not affected nanostructure of the film. The resulting bifunctional ChNF films were then dispersed in 1.0 mol/L aqueous ammonia (10 mL) by stirring the mixture at room temperature for 12 h, followed by ultrasonication for 4 h according to the procedure reported by us [35]. When styrene (1.1 mL, ChNFs/styrene = 0.1 (w/w)) was mixed with the obtained dispersions and the mixtures were ultrasonicated for homogenization, styrene-in-water emulsion was efficiently formed. Pickering emulsion polymerization of styrene was then performed by the same procedure as that using the maleylated ChNFs reported in our previous literature [5]. After nitrogen gas was bubbled into the emulsion on an ice-water bath, radical polymerization of styrene was carried out in the presence of potassium persulfate (0.35 mol% with styrene) at 70 °C for 24 h with stirring under nitrogen atmosphere (Fig. 2). The product was isolated by centrifugation and lyophilized to obtain ChNF/polystyrene composite particles. As the product was redispersed well in water by ultrasonication, the SEM image of a spincoated sample from the re-dispersion has been taken, which clearly observes the uniform particle morphology with an average diameter of 365 nm (Fig. 3c). The composite particles were then converted into ChNF-based hollow particles by solubilizing out inner polystyrene from the composite particles through repetitions of immersion in toluene for 10 min and decantation 6 times. The SEM image of a spin-coated sample from the dispersion of the resulting material in toluene shows clear hollow morphology (Fig. 3d). Furthermore, the SEM image also shows the presence of holes in shells as seen in previous studies on the fabrication of hollow particles [38,39]. The holes were probably generated by reaching out the inner polystyrene with toluene during the above the solubilizing
Fig. 3. SEM images of (a) ChNF film, (b) maleylated/methacylated (bifunctional) ChNF film, (c–e) spin-coated samples from aqueous dispersion of composite particles, toluene dispersion of hollow particles, and aqueous dispersion of hollow particles by Pickering emulsion polymerization of styrene using bifunctional ChNFs, and (f–h) spin-coated samples from aqueous dispersion of composite particles, toluene dispersion of hollow particles, and aqueous dispersion of hollow particles by Pickering emulsion copolymerization of styrene with PMM using bifunctional ChNFs.
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procedures. The presence of the holes in shells on the hollow particles has probably induced the encapsulation-release of a hydrophobic dye in the present study (vide infra). From the weight of the products obtained by evaporating toluene from the dispersions at 100 °C for 2 h, ca. 30 wt% polystyrenes were calculated to be remained. After the hollow particles were isolated by filtration, they were re-dispersed in water. The SEM image of a spin-coated sample from the aqueous dispersion shows morphology of the hollow particles (Fig. 3e), which are completely different from that resulted by the same procedure using the maleylated (monofunctional) ChNFs as shown in Fig. 1b. These observations strongly suggest improvement of stability of the shells in the hollow particles, probably owing to the formation of covalent linkages between ChNFs and polystyrene by radical copolymerization of methacryl groups on ChNFs with the closely present styrene during the production of composite particles. To confirm the occurrence of the copolymerization, the ester linkages in the polymer chains on ChNFs were cleaved by alkaline treatment of the hollow particles with aqueous NaOH. Then, aqueous HCl was added to the mixture for conversion of sodium methacrylate units into methacrylic acid units to make the separated copolymer insoluble in aqueous media, which was isolated by filtration and dried. The 1H NMR spectrum of the soluble fraction in the isolated product in CDCl3 (Fig. S1b) shows a signal a at 0.87 ppm assignable to methyl group of the methacrylic acid units besides large signals b and c ascribed to polystyrene, strongly supporting the occurrence of copolymerization of styrene with methacryl groups on ChNFs. As walls inside the shells of the hollow particles were considered to be hydrophobic, owing to the presence of polystyrene covalently linked on ChNFs, encapsulation of a hydrophobic fluorescent dye, pyrene, through the holes in shells was investigated. The encapsulation experiment was carried out by stirring a mixture of pyrene with the aqueous dispersion of the hollow particles at room temperature for 24 h. After filtration of the mixture, the isolated product was washed with acetone to remove the dye absorbed on the surfaces of the particles. The fluorescence spectrum of the re-dispersion of the resulting particles in water excited at 345 nm exhibits an emission maximum at 378 nm due to pyrene (Fig. 4b), indicating the presence of the dye in the products. On the other hand, when the same experiment has been conducted using the composite particles, where the cavity is filled by polystyrene, the fluorescence spectrum does not mostly show peaks by emission of pyrene (Fig. 4a). These results strongly support that pyrene has been encapsulated in the cavity of the hollow particles through the holes, probably by its hydrophobic interaction with polystyrene present at
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inner walls of the shell. Indeed, the aqueous dispersion of the resulting hollow particles observed blue florescent emission by UV-light irradiation at 365 nm (Fig. S2b), while such emission was not detected by the same light irradiation to the aqueous dispersion of the composite particles after treatment with pyrene (Fig. S2a). Release of pyrene through holes from the resulting hollow particles was investigated by treatment with surfactant, as it potentially interacts with hydrophobic substrates [40]. After the aqueous dispersion of the pyrene-encapsulated hollow particles was treated with oleyl alcohol at room temperature for 2 days in water, the product was isolated by filtration and washed with acetone. The fluorescence spectrum of the aqueous re-dispersion of the obtained hollow particles excited at 345 nm does not observe an emission peak from pyrene (Fig. 4c). Indeed, UV-light irradiation to the resulting dispersion at 365 nm did not induce fluorescent emission as shown in Fig. S2c. On the other hand, when the same procedure for the pyrene-encapsulated hollow particles was carried out without oleyl alcohol, the fluorescence spectrum excited at 345 nm exhibits an emission peak from pyrene (Fig. 4d). These results strongly suggest that release of pyrene successfully happens by treatment with surfactant, oleyl alcohol. However, intensity of the emission peak from pyrene after the control experiment in Fig. 4d slightly decreased compared with that of the pyreneencapsulated hollow particles in Fig. 4b, indicating that a little occurrence of spontaneous release of pyrene in aqueous dispersion without oleyl alcohol. This result has motivated us to fabricate other composite/hollow particles stably carrying fluorescent moieties through the formation of covalent linkages as described in the following section. 3.2. Preparation of fluorescent composite and hollow particles with pyrene moieties An attempt was made to incorporate fluorescent (pyrene) moieties into composite/hollow by covalent linkages with polystyrene using PMM having a polymerizable methacryl group. Pickering emulsion copolymerization of styrene with PMM using the bifunctional ChNFs as stabilizer was carried out by the same procedure as described above to produce composite particles (Fig. 2). The SEM image of the spincoated sample shows uniform particle morphology with an average diameter of 372 nm (Fig. 3f), which is similar as that for the abovementioned composite particles without pyrene moieties (Fig. 3c). The resulting composite particles were then converted into the corresponding hollow particles by solubilizing out inner polystyrene with toluene. The SEM images of the spin-coated samples from a
Fig. 4. Fluorescence spectra of aqueous dispersions after encapsulation experiments of (a) composite particles and (b) hollow particles and aqueous dispersions (c) after treatment of pyrene-encapsulated hollow particles with oleyl alcohol and (d) after control experiment without oleyl alcohol.
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Fig. 5. Fluorescence spectra of aqueous dispersions of (a) composite and (b) hollow particles by Pickering emulsion copolymerization of styrene with PMM and (c) aqueous dispersion after encapsulation experiment of hollow particles.
toluene dispersion and an aqueous re-dispersion, obtained according to the procedure in Fig. 2, exhibit hollow morphologies (Fig. 3g and h, respectively), indicating that the produced hollow particles are stable during the re-dispersion procedure in water. The fluorescence spectra of the aqueous dispersions of the resulting composite and hollow particles have been measured by excitation at 345 nm, which observe emission maxima at 378 nm due to pyrene (Fig. 5a and b, respectively). Intensity of the emission peak in Fig. 5b is lower than that in Fig. 5a, because of solubilizing out inner fluorescent copolymer during the procedure for conversion into the hollow particles. Both the aqueous dispersions observe blue florescent emission by UV-light irradiation at 365 nm (Figs. S2d and e). After encapsulation of pyrene into the hollow particles were carried out by the same procedure as above, intensity of the emission peak increased from that before encapsulation (Fig. 5b and c). This result indicates the presence of pyrene moieties in the hollow particles both by covalently linking and by hydrophobic interaction with polystyrene in the cavity. 4. Conclusion In this study, we investigated the preparation of chitin-based fluorescent hollow particles by Pickering emulsion polymerization of styrene using the bifunctional ChNFs as stabilizer, followed by solubilizing out inner polystyrene with toluene. The resulting hollow particles were stable by dispersing in water owing to the presence of methacrylate units on ChNFs covalently bound to styrene units. The fluorescent dye, pyrene, was then incorporated into the hollow particle both physically by encapsulation through hydrophobic interaction and chemically by using PMM during Pickering emulsion copolymerization. Release of pyrene from the former hollow particles was achieved by treatment with oleyl alcohol in water. The present fluorescent hollow particles have a potential to be practically employed as photofunctional materials. Furthermore, the encapsulation and release approaches conducted in this study will imply the present hollow particles in application as carriers for drug delivery system in the future. CRediT authorship contribution statement Seiichiro Noguchi: Investigation, Data curation. Kazuya Yamamoto: Data curation, Writing - original draft. Jun-ichi Kadokawa: Conceptualization, Methodology, Writing - review & editing.
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