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Cite this: J. Mater. Chem. B, 2022,
10, 7570

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Carboxy-functionalized pH responsive capsule
polymer particles fabricated by particulateinterfacial photocrosslinking†Published on 01 March 2022. Downloaded on 10/20/2024 4:06:31 AM.

Yukiya Kitayama* and Atsushi Harada*pH-responsive capsule particles show promise for various applications, such as self-healing materials,
micro/nanoreactors, and drug delivery systems. Herein, carboxy-functionalized capsule polymer
particles possessing neutral–alkaline pH responsive controlled release capability were newly fabricatedby interfacial photocrosslinking of spherical photoreactive polymer [poly(2-carboxyethyl acrylate-co-2cinnamoylethyl methacrylate): P(CEA-CEMA)] particles and a subsequent encapsulation process. Using
P(CEA-CEMA) particles, the shell-crosslinked hollow polymer particles were fabricated by the particulateinterfacial photocrosslinking procedure. Furthermore, the encapsulation of sulforhodamine B as a model
dye into the hollow particles was also performed. Under acidic pH conditions, encapsulated molecules
were stably retained in the P(CEA-CEMA) capsules with negligible release of sulforhodamine B. However,
the encapsulated sulforhodamine B was gradually or drastically released from the capsule particles
under neutral or basic conditions, respectively, indicating that the neutral–alkaline pH responsive
Received 30th December 2021,
Accepted 1st March 2022

controlled release from the capsules was successfully achieved by regulating the release kinetics. These
results demonstrate that the fabrication routes of hollow and capsule particles based on particulate

DOI: 10.1039/d1tb02866ainterfacial photocrosslinking can be successfully applied to carboxy-functionalized photoreactive
polymer particles, and the capsule polymer particles possessing pH-responsive release properties under

rsc.li/materials-b

neutral–basic conditions were successfully fabricated.

1. Introduction
Polymer capsules with controlled-release properties triggered by pHchanges are of great importance in a wide range of applications,such as self-healing materials,1–3 micro/nanoreactors,4–8 and drugdelivery systems.9–11 Hollow polymer particles have great potential
for the above applications because they can stably encapsulate
various functional molecules. Several fabrication routes ofhollow polymer particles have been widely developed to date.
The sacrificial template-assisted approach has been widely developed utilizing various approaches for shell-fabrication such as
layer-by-layer,12,13 seeded polymerization,14,15 surface-initiated living radical polymerization,16 and phenol–metal coordination.17,18
Self-assembly of block copolymer or colloidal particles (i.e., byformation of colloidsomes19–21 and polymer vesicles using block
Department of Applied Chemistry, Graduate School of Engineering, Osaka
Prefecture University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
E-mail: kitayama@chem.osakafu-u.ac.jp, harada@chem.osakafu-u.ac.jp† Electronic supplementary information (ESI) available: Materials, apparatus, 1HNMR and UV-vis spectra of P(CEA-CEMA)-50, fluorescence spectrum of P(CEACEMA)-50, photocrosslinking and pH-responsive properties of P(CEA-CEMA)-50
particles, and release kinetics of sulforhodamine B from the capsules. See DOI:
10.1039/d1tb02866a

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copolymer self-assembly,22–24 or polymerization-induced selfassembly which was pioneered by Armes and coworkers22,25,26)
is also a powerful approach for hollow/capsule fabrication.
Inverse miniemulsion periphery polymerization has beendeveloped as another fabrication route for hollow/capsules byZetterlund and coworkers.27,28 The self-assembled phaseseparated polymer method has been developed by Okubo,
Minami and coworkers, which is categorized as one of thepolymerization-induced self-assembly approaches in the(micro)suspension polymerization system.29–31
Recently, we developed a new fabrication route for shellcrosslinked hollow polymer particles based on the interfacial
photocrosslinking of polymer particles.32,33 In this approach,[2p + 2p] photoinduced dimerization between alkene groups ofdiﬀerent polymer chains occurs only at the interface of thepolymer particles (Scheme 1). After the photoirradiation, thenon-crosslinked polymers are removed from the particle core,resulting in the shell-crosslinked hollow polymer particles
being obtained. Furthermore, capsule polymer particles weresuccessfully fabricated by a posteriori encapsulation of various
functional molecules in the shell-crosslinked hollow polymer
particles utilizing the solvent exchange process (Scheme 2).
Furthermore, functionalization of the capsules, in particular,This journal is © The Royal Society of Chemistry 2022

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Journal of Materials Chemistry B

Scheme 1 Schematic illustration of interfacial photocrosslinking of P(CEA-CEMA)-50 particles for the preparation of hollow particles. pH-responsive
release of P(CEA-CEMA)-50 capsule particles. P(CEA-CEMA): poly(2-carboxyethylacrylate-co-2-cinnamoylethyl methacrylate).

the controlled release of encapsulated molecules and materials,
is of great importance in various applications. Various stimuli
responsive capsules have also been successfully developed
through interfacial photocrosslinking by designing photoreactive monomers. For example, redox-responsive capsules
were developed by using the designed photoreactive monomer
possessing a disulfide linkage between the vinyl group and thecinnamoyl group.34 In the capsules, the polymer main chain
and crosslinking points (cinnamoyl dimer) were cleaved by theaddition of reducing agents, leading to the degradation ofpolymer capsules. The use of a coumarin-based photoreactive
monomer for the interfacial photocrosslinking approach led to

Yukiya Kitayama is assistantprofessor of the Graduate Schoolof Engineering at Osaka Prefecture
University. He received his PhD
under the supervision of Prof. Dr
Masayoshi Okubo from Kobe
University. From April to Octoberin 2012, he was a postdoctoral
fellow in Prof. Dr Craig J. Hawker
group in the University of California
at Santa Barbara. From November
2012 to January in 2020, he got
academic position as an assistantYukiya Kitayama
professor in Prof. Dr Toshifumi
Takeuchi group at Kobe University, Japan. In February 2020, he won
Leading Initiative for Excellent Young Researchers (LEADER) programfrom Japan Society for the Promotion of Science, and he moved to thecurrent position in Osaka Prefecture University. His research interests
are in polymer chemistry, colloid chemistry, molecular recognition, anddrug delivery.

This journal is © The Royal Society of Chemistry 2022

the fabrication of photodegradable capsule particles.35 As
another approach, stimuli-responsive capsules were fabricatedusing a simple copolymer of stimuli-responsive comonomers withphotoreactive monomers. Recently, we have successfully fabricated acidic pH-responsive polymer capsules by the interfacial
photocrosslinking of photoreactive spherical polymer particles
possessing tertiary amine groups as pH-responsive groups.36,37
An advantage of the fabrication route using a copolymer of pH
responsive and photoreactive monomers is the high simplicity ofprocedures and starting materials. However, only tertiary aminefunctionalized capsules possessing acidic pH-responsiveness havebeen reported to date, but the versatility of this fabrication routeof pH responsive capsules is unclear.

Scheme 2 Schematic illustration of solvent exchange procedures
applied to P(CEA-CEMA)-50 particles for the preparation of capsuleparticles. P(CEA-CEMA): poly(2-carboxyethylacrylate-co-2-cinnamoylethyl
methacrylate).

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In this study, we fabricated carboxy-functionalized
pH-responsive polymer capsules possessing controlled releasecapability in neutral–basic conditions to expand the varietyof pH-responsive polymer capsules that can be prepared by
the interfacial photocrosslinking of photoreactive random
copolymer particles. Using the carboxy-functionalized capsules,
the controlled release of the encapsulated molecules should beachieved at neutral–alkaline conditions. Through this research,
we demonstrate the high versatility of the interfacial photocrosslinking approach using a simple random copolymer for
creating a wide range of pH-responsive polymer capsules.

2. Results and discussion
For creation of neutral–alkaline responsive capsule polymer
particles by interfacial photocrosslinking, functional polymers

Paperpossessing photoreactivity and carboxy groups as pH responsive groups were prepared. In this study, 2-cinnamoylethyl
methacrylate (CEMA), which was selected as a photoreactive
monomer, was synthesized by the conjugation of cinnamoyl
chloride and 2-hydroxyethyl methacrylate in the presence oftriethylamine. CEMA possesses UV-absorbing properties with amaximum absorbance at l = 280 nm, and the cinnamoyl group
undergoes a [2p + 2p] photoinduced dimerization reaction byphotoirradiation.38,39 2-Carboxyethyl acrylate (CEA), which
was selected as an alkali-pH responsive monomer, has a
pH-responsive carboxylic acid group, which means that the
carboxylic acid is deprotonated under neutral–alkaline pH
conditions, leading to increased water solubility.
Conventional radical polymerization of CEA and CEMA
was performed using 2,2 0 -azobis(isobutyronitrile) (AIBN) in a
mixture of tetrahydrofuran (THF) and dimethylformamide (DMF).
The number-average molecular weight of P(CEA-CEMA)-50 was

Fig. 1 (a) UV-Vis spectra and (b) absorbance changes at l = 280 nm of P(CEA-CEMA)-50 thin film prepared on a quartz substrate after various
photoirradiation periods. (c) pH titration curve, (d) diﬀerential of the pH curve, and (e) degree of protonation under various pH conditions of P(CEACEMA)-50 in water. P(CEA-CEMA): poly(2-carboxyethylacrylate-co-2-cinnamoylethyl methacrylate).

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estimated to be approximately 5800 Da (polydispersity index:
2.2) by gel permeation chromatography (GPC) after methylationof the carboxy groups of P(CEA-CEMA)-50. The CEMA
molar content of the obtained polymer was estimated to beapproximately 53 mol% by 1H-NMR spectroscopy, indicatingthat CEA and CEMA were copolymerized by conventional
radical polymerization in a solution polymerization system(Fig. S1, ESI†). Furthermore, the cinnamoyl group was clearlyobserved in the 1H-NMR spectrum, indicating that side
reactions with the cinnamoyl group did not occur during thepolymerization. This conclusion is also supported by the UV-vis
spectrum of the P(CEA-CEMA)-50 solution, which indicates thatthe obtained P(CEA-CEMA)-50 absorbs UV radiation at a
maximum of l = 280 nm (Fig. S2, ESI†). A polymer thin film
was prepared on a quartz substrate to investigate the photoreactivity of P(CEA-CEMA)-50 in the polymer film state. According
to the UV-vis spectra, the absorbance of the cinnamoyl
groups in P(CEA-CEMA)-50 gradually decreased with increasing

Journal of Materials Chemistry B
photoirradiation periods (Fig. 1). This phenomenon is a resultof the [2p + 2p] photodimerization reaction in the polymer thinfilm state. It is worth noting that the absorbance at l = 280 nm
derived from free cinnamoyl groups did not completely disappear
even after suﬃcient photoirradiation time. This result is reasonable because the cinnamoyl groups can dimerize only when two
diﬀerent cinnamoyl groups are placed close together in thepolymer thin film. The degree of photodimerization of cinnamoyl
groups in the film is roughly according to the A/A0 values shownin Fig. 1b because the maximum absorbance wavelength ofthe cinnamoyl group is shifted to a shorter wavelength after
dimerization. Furthermore, the pH-responsive properties ofP(CEA-CEMA)-50 in aqueous media were investigated by acid–base titration. The apparent pKa derived from the carboxy
groups in P(CEA-CEMA)-50 was evaluated using an acid–basetitration experiment, and the value was determined to be approximately 6.8, indicating that P(CEA-CEMA)-50 is pH-responsive(Fig. 1).

Fig. 2 Optical micrographs (a–c) and particle size distributions (d–f) of P(CEA-CEMA)-50 particles (a) before and (b) after interfacial photocrosslinking,and (c) after subsequent removal of the non-crosslinked polymer. P(CEA-CEMA): poly(2-carboxyethylacrylate-co-2-cinnamoylethyl methacrylate).

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Solvent evaporation was carried out to prepare parent
P(CEA-CEMA)-50 particles.40,41 In the solvent evaporation
process, the organic solvent for dissolving P(CEA-CEMA)-50
must not be dissolved in aqueous media, and thus the organic
solvent for the solvent evaporation method for P(CEA-CEMA)-50
was first investigated (Table S1, ESI†). P(CEA-CEMA)-50 was notdissolved in hexane, ethyl acetate, methanol, ethanol, andtoluene. The poor solubility in these solvents might be causedby the high hydrogen bonding capabilities of the polymer
chains. Acetone, THF, DMF, dimethylacetamide, and dimethyl
sulfoxide (DMSO) can dissolve P(CEA-CEMA)-50, indicating thatthese solvents can work as a good solvent; however thesesolvents are not suitable for the solvent evaporation process
due to their high miscibility with water. The polymer was
dissolved in chloroform and methyl ethyl ketone. Due to thelimited water miscibility of these solvents, these solvents are
suitable for the solvent evaporation method for P(CEA-CEMA)50. In this study, chloroform was selected as a solvent because
it has lower boiling point and water solubility than methyl ethyl
ketone (water solubility at 25 1C: 7.95 mg mL1 forchloroform,42 223 mg mL1 for methyl ethyl ketone;43 boilingpoint: 61.12 1C for chloroform,44 79.59 1C for methyl ethyl
ketone44). P(CEA-CEMA)-50 was first dissolved in chloroform,

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and the polymer solution was homogenized in an acidic aqueous
medium (pH 4.0) dissolving poly(vinyl alcohol) (PVA) using ahomogenizer, where acidic water is a poor solvent of P(CEACEMA)-50. After homogenization, chloroform was slowly evaporated from the dispersion, leading to the formation of spherical
P(CEA-CEMA)-50 particles (Fig. 2). Then, the interfacial photocrosslinking reaction of P(CEA-CEMA)-50 particles was carried out
by photoirradiation (l = 254 nm) to prepare the hollow polymer
particles. The spherical particle morphology was maintained evenafter photoirradiation (Fig. 2). Non-photoirradiated P(CEA-CEMA)50 particles dissolved in THF, whereas a part of photoirradiated
P(CEA-CEMA)-50 particles remained even after THF washing.
The removal ratio of P(CEA-CEMA)-50 from the photoirradiatedparticles after 6 h of photoirradiation was estimated to be approximately 50% by UV-vis analysis (Fig. S3, ESI†). These results
confirm that photocrosslinking proceeded in the particle state.
After the washing process, hollow P(CEA-CEMA)-50 particles weresuccessfully obtained while maintaining a particle size similar tothat before polymer removal, indicating that photoirradiation led to
the formation of a crosslinked shell region at the particle interfaceof dispersed P(CEA-CEMA)-50 particles (Fig. 2). The selective crosslinking of the polymers at the interfacial region of the particles waswell according to our previous studies.32,36,37 We previously clarified

Fig. 3 Time lapse images of NaOH added aqueous dispersion of P(CEA-CEMA)-50 particles (a) before and (b) after interfacial photocrosslinking. P(CEACEMA): poly(2-carboxyethylacrylate-co-2-cinnamoylethyl methacrylate).

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disappeared after addition of a small quantity of NaOH solid,
when the aqueous medium became alkaline (good solvent forthe polymer) from acidic conditions (poor solvent for thepolymer) whereas, after photoirradiation, the P(CEA-CEMA)-50
particles were not dissolved and the swollen polymer particles
remained even after addition of a small quantity of solid NaOH.
To further clarify the successful preparation of hollow polymer
particles by interfacial photocrosslinking of parent polymer
particles, fluorescein-labeled P(CEA-CEMA)-50 was preparedby copolymerization with a small portion of fluorescein acrylamide. The fluorescence derived from the fluorescein-labeled

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that the non-reacted cinnamoyl groups in the polymer shell shieldcore region from the photoirradiation, where the cinnamoyl group
has a high absorption coeﬃcient. The photocrosslinking degree ofthe cinnamoyl group of P(CEA-CEMA)-50 in the crosslinked shell
region was evaluated to be approximately 32% by 1H-NMR afterhydrolysis of photoirradiated P(CEA-CEMA)-50 particles (after
removal of non-crosslinked polymers) (Fig. S4, ESI†).
To evaluate the eﬀect of the photoirradiation process on theparticle crosslinking reaction, in situ pH-switching experiments
were carried out (Fig. 3). Before photoirradiation, P(CEACEMA)-50 particles dispersed in acidic solution (pH 4.0)

Journal of Materials Chemistry B

Fig. 4 Confocal laser scanning micrographs (z-stack images) of fluorescein-labeled P(CEA-CEMA)-50 particles (a) before and (b) after interfacial
photocrosslinking, and (c) after removal of the non-crosslinked polymer. Interval of Z-stack images is 1 mm. Left: Bright image. Right: Fluorescence
image. Line analysis of the internal fluorescence intensity of fluorescein-labeled P(CEA-CEMA)-50 particles (d) before and (e) after interfacial
photocrosslinking, and (f) after removal of the non-crosslinked polymer. (g) SEM image of photoirradiated P(CEA-CEMA)-50 particles after removal of
the non-crosslinked polymer. P(CEA-CEMA): poly(2-carboxyethylacrylate-co-2-cinnamoylethyl methacrylate).

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the photoirradiation (without washing), the homogeneous internal
morphology was maintained for P(CEA-CEMA)-50 particles; i.e. thefluorescence intensity derived from the polymers was observed
from the internal region of the particles (Fig. 4b and e). However,the fluorescence derived from fluorescein-labeled polymers was
only observed at the particle shell region with negligible fluorescence intensity in the internal core region of the particles after the

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P(CEA-CEMA)-50 was confirmed by fluorescence spectra (Fig.
S4, ESI†). From the z-stack images obtained using confocal laser
scanning microscopy (CLSM), the fluorescence derived fromfluorescein-labeled P(CEA-CEMA)-50 was observed in the internal
region (Fig. 4a). From the line analysis of the fluorescence image,polymer particles prepared using fluorescein-labeled P(CEA-CEMA)50 had homogeneous internal fluorescence intensity (Fig. 4d). After

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Fig. 5 (a) CLSM images (z-stack images) of P(CEA-CEMA)-50 capsule particles containing sulforhodamine B. Interval of z-stack images is 1 mm. Top:
Bright image. Bottom: Fluorescence image. (b) Zeta potential of P(CEA-CEMA)-50 hollow particles at various pH conditions. Time-lapse CLSM images ofP(CEA-CEMA)-50 capsule particles containing sulforhodamine B after addition of NaOH granules: (c) sulforhodamine B channel, (d) fluorescein channel,
and (e) bright field. (f) Average particle size of polymer capsules before (left) and after (right) NaOH addition. P(CEA-CEMA): poly(2-carboxyethylacrylateco-2-cinnamoylethyl methacrylate).

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washing process of the photoirradiated polymer particles toremove the non-crosslinked polymers (Fig. 4c and f). Furthermore, the internal hollow morphology can be directly observedin the washed P(CEA-CEMA)-50 by scanning electron microscopy (Fig. 4g). These results strongly suggest that the interfacial region of the P(CEA-CEMA)-50 particles was selectively
crosslinked and non-crosslinked polymers were successfully
removed from the photoirradiated P(CEA-CEMA)-50 particles,
resulting in hollow polymer particles being prepared by theinterfacial photocrosslinking of spherical polymer particles.
Encapsulation by the P(CEA-CEMA)-50 hollow particles was
carried out using a solvent exchange procedure.34 Shellcrosslinked P(CEA-CEMA)-50 hollow particles were allowed toswell through incubation in a THF/DMSO mixture containing
sulforhodamine B, a model fluorescent molecule. The swelling
allowed the diﬀusion of sulforhodamine B into the capsules.
Subsequently, the solvent was exchanged for an acidic aqueous
solution, a poor solvent, which shrunk the polymer shells,
leading to the physical encapsulation of the molecules diﬀused
into hollow particles. After the solvent exchange process, thez-stack image of the P(CEA-CEMA)-50 capsule particles wasobserved by CLSM. Fluorescence derived from sulforhodamine
B was observed in the core region of the P(CEA-CEMA)-50
capsule particles, indicating that sulforhodamine B was
successfully encapsulated in P(CEA-CEMA)-50 hollow particles
by solvent exchange (Fig. 5).
The pH-responsive properties of the P(CEA-CEMA)-50 hollow
polymer particles were investigated from the change in the zeta
potential of the hollow polymer particles with pHs. Under
increasing pH conditions, the zeta potential of P(CEA-CEMA)50 hollow particles gradually decreased (Fig. 5). As discussed
above, P(CEA-CEMA)-50 has a pH-responsive carboxy group,and the apparent pKa of P(CEA-CEMA)-50 existed at pH 6.8.

Journal of Materials Chemistry B
Notably, the zeta potential behavior was in good agreement withthe water solubility of the non-photoirradiated P(CEA-CEMA)-50
particles. That is, non-photoirradiated P(CEA-CEMA)-50 particles
were partially dissolved at pH 6.8, whereas they were almost
completely dissolved at pH 7.4–11, according to the transmittance results (Fig. S5, ESI†). Therefore, the carboxy groups at thesurface of the P(CEA-CEMA)-50 hollow particles were deprotonated under neutral and alkaline conditions in the aqueous
medium.
pH-switching experiments were then carried out to investigate
the pH-responsive release properties of the P(CEA-CEMA)-50
capsule particles. Here, a small quantity of solid NaOH
was added to the aqueous dispersion of fluorescein-labeled
P(CEA-CEMA)-50 polymer capsules containing sulforhodamine
B. As fluorescein is a pH-dependent fluorescent molecule,the fluorescence intensity drastically increased when the pHchanged from acidic to neutral/basic. Sulforhodamine B, in
contrast, is not pH-dependent in the range of pH 3.0–10.0.45
Just after the addition of NaOH, the fluorescence intensity
derived from fluorescein was negligible and the fluorescencederived from sulforhodamine B was observed from the particles. After several seconds, the fluorescence intensity derived
from fluorescein increased and the fluorescence derived from
sulforhodamine B rapidly disappeared from the particle coreregion (Fig. 5c–e and Fig. S6, ESI†). However, the fluorescencederived from sulforhodamine B slowly decreased at the particle
shell layer, possibly due to non-specific p–p stacking or hydrophobic interactions between the polymer and dyes. Notably, theaverage particle size increased after the addition of NaOH,
indicating that the P(CEA-CEMA)-50 capsule particles swelled
in alkaline conditions (Fig. 5f).
The pH-responsive release capability of P(CEA-CEMA)-50
capsule particles containing sulforhodamine B was investigated.

Fig. 6 pH responsive release properties of P(CEA-CEMA)-50 capsule particles containing sulforhodamine B. (a) Release of sulforhodamine B from
P(CEA-CEMA)-50 capsule particles after diﬀerent incubation times under diﬀerent pH conditions. (b–f) Bright field (left) and fluorescence field (right)
images obtained from CLSM of P(CEA-CEMA)-50 particles after incubation in various aqueous media under different pH conditions. CLSM: confocal laser
scanning microscopy. P(CEA-CEMA): poly(2-carboxyethylacrylate-co-2-cinnamoylethyl methacrylate).

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The P(CEA-CEMA)-50 capsule particles were incubated in various aqueous media at diﬀerent pH conditions. After several
incubation periods, the absorbance of the released sulforhodamine B in the aqueous media was measured by UV-vis
spectroscopy. In acidic aqueous media (pH 4.0 and 5.5), theabsorbance did not increase even after 24 h incubation,
indicating that the P(CEA-CEMA)-50 capsule particles rigidly
retained sulforhodamine B under acidic conditions (Fig. 6). In
reality, fluorescence derived from sulforhodamine B was clearlyobserved from the capsule particles in the CLSM image. At
neutral pH (6.8), the absorbance derived from sulforhodamine
B gradually increased with incubation time. Furthermore,
the absorbance drastically increased in the initial stage ofincubation in alkaline aqueous media (pH 8.5 and 10). From
the absorbance derived from sulforhodamine B, the loading
eﬃciency and the loading amount into the capsules wereapproximately 0.15% and 34 mg/1 mg-particles. These results
indicate that whereas neutral and alkaline conditions
stimulated the continuous and burst release of encapsulated
sulforhodamine B, respectively. From CLSM images, thefluorescence derived from sulforhodamine B was absent fromthe capsule particles incubated at pH 6.8, 8.5, and 10. theP(CEA-CEMA)-50 capsule particles were successfully endowedwith pH-responsive controlled-release properties triggered byneutral–alkaline pH conditions.

3. Conclusions
In this study, P(CEA-CEMA)-50 capsule particles capable ofneutral–alkaline pH responsive controlled release were successfully prepared by the interfacial photocrosslinking of sphericalpolymer particles. P(CEA-CEMA)-50 prepared by conventional
radical copolymerization of pH-responsive and photoreactive
monomers exhibited [2p + 2p] photoreactive and pH-responsive
properties. P(CEA-CEMA)-50 hollow particles were successfully
prepared by interfacial photocrosslinking, and the subsequent
encapsulation procedure allowed sulforhodamine B to be
trapped inside the capsule. Under acidic conditions, the polymer capsules rigidly retained sulforhodamine B in the internal
space, whereas neutral and alkaline conditions stimulated thecontinuous and burst release of encapsulated sulforhodamine
B, respectively, from the capsule particles. The alkali-pHresponsive capsule particles can be applied for research in avariety of fields, such as mitochondria-targeted drug delivery
systems, after minor adjustments to optimize the response to
pH conditions. Through this research, we have successfully
demonstrated the high versatility of the fabrication route of
pH-responsive capsules utilizing interfacial photocrosslinkingof simple random copolymer particles prepared from pHresponsive and photoreactive monomers. Utilizing theapproach, various pH responsive capsules possessing different
pH-responsive groups such as imidazole, primary amine,secondary amine, phosphate, and pyridine groups will becreated by a simple procedure based on the interfacial
photocrosslinking.

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4. Experimental section
4.1. Synthesis of carboxy-functionalized photoreactive
polymers
Functional polymers possessing pH-responsive and photoreactive groups were synthesized as follows. For the preparationof P(CEA-CEMA)-50, CEA (1.5 mmol), CEMA (1.5 mmol), and theinitiator, AIBN (12.4 mg), were dissolved in a mixture of THF(10 mL) and DMSO (2 mL) in a 15 mL Schlenk tube. To prepare
fluorescein-labeled P(CEA-CEMA)-50, a small quantity of fluorescein acrylamide (30 mmol) was added to the prepolymerization
solution, where fluorescein acrylamide was synthesized as previously reported.46 After N2/vacuum cycles, conventional radical
polymerization was performed at 60 1C for 16 h. The polymer
was precipitated in water, and the precipitated polymer was
further washed with methanol to remove the remaining monomers and initiators. The obtained polymer was dried in vacuo.
Notably, the number after the polymer represents the initialmolar ratio of CEMA in the monomers. For GPC analysis,
carboxy groups of P(CEA-CEMA)-50 were methylated by trimethylsilyldiazomethane as the reported procedure.47–49 In
brief, P(CEA-CEMA)-50 (20 mg) was dissolved in THF (5 mL),and a hexane solution of trimethylsilyldiazomethane (10%,0.1 mL) was added to the polymer solution. After overnight
reaction at room temperature, excess acetic acid was added toquench the trimethylsilyldiazomethane.
4.2.

Preparation of polymer particles

The solvent evaporation method36 was used to prepare polymer
particles possessing pH-responsive and photoreactive polymers.
P(CEA-CEMA)-50 (50 mg) was dissolved in chloroform (1 mL),and each solution was homogenized in phthalic acid buﬀer (pH4.0) containing PVA (25 mL, 0.0067 wt%) using a homogenizer at
12 000 rpm for 1 min. The chloroform in the polymer droplets
was slowly evaporated at room temperature with gentle stirring.
The average sizes of more than 100 particles in the optical
micrographs were estimated using ImageJ software.50
4.3. Interfacial photocrosslinking for hollow particle
fabrication
Interfacial photocrosslinking was performed as follows: P(CEACEMA)-50 particles (solid content: 2 mg mL1) dispersed inphthalic acid buﬀer (pH 4.0, 3 mL) containing PVA (0.0067 wt%)
were separately placed in 6 mL vials. Each dispersion was
photoirradiated (l = 254 nm, 2 mW cm2, GL 15, Toshiba,
Tokyo, Japan) for diﬀerent lengths of time, with gentle stirring
at room temperature. Each dispersion was then washed fivetimes with THF by centrifugation to remove the non-crosslinked
polymers. After centrifugation, the solvent was replaced withfresh THF/DMSO (9/1, v/v, B1 mL), followed by 10 mM phthalicacid buﬀer (pH 4.0, 1 mL) containing PVA (0.0067 wt%). Before
SEM observation, the P(CEA-CEMA)-50 hollow particles dispersed in the acidic aqueous solution was freeze-dried, and thedried particles were used for SEM observation after platinum
coating.

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4.4.

Encapsulation of sulforhodamine B

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Hollow P(CEA-CEMA)-50 particles were prepared from thecorresponding seed particles (2 mg mL1, 3 mL) using theabove procedure (photoirradiation time: 6 h). The hollow
particles were then dispersed in THF (0.9 mL) and subsequently
mixed with DMSO (0.1 mL) containing sulforhodamine B (final
concentration: 1 mg mL1). After 3 h of incubation, theparticles were separated by centrifugation, and the solvent
was replaced with phthalic acid buﬀer (pH 4.0, 1 mL). The
particles were then washed with 10 mM phthalic acid buﬀer(pH 4.0, 1 mL) five times by centrifugation to remove the nonencapsulated sulforhodamine B. Finally, the washed capsuleparticles were dispersed in fresh 10 mM phthalic acid buﬀer(pH 4.0, 1 mL).
4.5.

Determination of pH-responsive release properties

Hollow P(CEA-CEMA)-50 particles were prepared by the above
procedure using seed P(CEA-CEMA)-50 particles (solid content:
2 mg mL1; 3 mL  15). The hollow P(CEA-CEMA)-50 particles
were then dispersed in THF (40.5 mL) before mixing with DMSO
(4.5 mL) containing sulforhodamine B (final concentration:
1 mg mL1). After 3 h of incubation, the particles were separated
by centrifugation, and the solvent was replaced with 10 mM
phthalic acid buﬀer (pH 4.0, 45 mL). The particles were thenwashed five times with 10 mM phthalic acid buﬀer (pH 4.0) toremove non-loaded free sulforhodamine B. The washed capsuleparticles were then dispersed in 10 mM phthalic acid buﬀer(pH 4.0, 45 mL). A solvent of capsule particle dispersion (1 mL)
prepared by the above-described procedure was then changed tothe aqueous solution with diﬀerent pH conditions (final pH: 4.0,
5.5, 6.8, 8.5, and 10.0) by centrifugation. After various incubation
periods, the supernatant of each capsule particle dispersion wascorrected by centrifugation. The UV-vis spectra of the supernatants (l = 565 nm) were recorded to estimate the pH-dependent
controlled-release capabilities of the capsule polymers.

Abbreviations
AIBN, 2,2 0 -azobis(isobutyronitrile); CEMA, 2-cinnamoylethyl
methacrylate; CLSM, confocal laser scanning microscopy; DMF,
dimethylformamide; GPC, gel permeation chromatography;
P(CEA-CEMA), poly(2-carboxyethyl acrylate-co-2-cinnamoylethyl
methacrylate); TEA, trimethylamine; THF, tetrahydrofuran.

Conflicts of interest
The authors declare no competing financial interest.

Acknowledgements
This work was supported by JSPS KAKENHI (Grant number
21H02004 for Y. K.) and partially supported by the KOSE
Cosmetology Research Foundation, Takahashi Industrial andEconomic Research Foundation, and the Leading Initiative for
Excellent Young Researchers, MEXT, Japan (Y. K.).

This journal is © The Royal Society of Chemistry 2022

Journal of Materials Chemistry B

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