pH-Responsive Polyion Complex Vesicle with Polyphosphobetaine

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pH-Responsive Polyion Complex Vesicle with Polyphosphobetaine Shells Yuki Ohara, Keita Nakai, Sana Ahmed, Kazuaki Matsumura, Kazuhiko Ishihara, and Shin-ichi Yusa Langmuir, Just Accepted Manuscript • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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A research paper for Langmuir pH-Responsive Polyion Complex Vesicle with Polyphosphobetaine Shells

Yuki Ohara†, Keita Nakai†, Sana Ahmed‡, Kazuaki Matsumura‡, Kazuhiko Ishihara§, Shin-ichi Yusa*,† †

Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo,

2167 Shosha, Himeji, Hyogo 671-2280, Japan. E-mail: [email protected] ‡

School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1

Asahidai, Nomi, Ishikawa 923-1292, Japan §

Department of Materials Engineering, School of Engineering, The University of Tokyo,

7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

Abstract: When a bioactive molecule is taken into cells by endocytosis, it is sometimes unable to escape from the lysosomes, resulting in inefficient drug release. We prepared pH-responsive polyion complex (PIC) vesicles that collapse under acidic conditions such as those inside a lysosome. Furthermore, under acidic conditions, cationic polymer was released from the PIC vesicles to break the lysosome membranes. Diblock copolymers (P20M167 and P20A190)

consisted

of

water-soluble

zwitterionic

poly(2-methacryloyloxyethyl

phosphorylcholine) (PMPC) block and cationic or anionic blocks were synthesized via reversible

addition-fragmentation

chain

transfer

(RAFT)

1

ACS Paragon Plus Environment

radical

polymerization.

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Poly(3-(methacrylamidopropyl) trimethylammonium chloride) (PMAPTAC) and poly(sodium 6-acrylamidohexanoate) (PAaH) were used as the cationic and anionic blocks, respectively. The pendant hexanoate groups in the PAaH block are ionized in basic water and in phosphate buffered saline (PBS), while the hexanoate groups are protonated in acidic water. In basic water PIC vesicles were formed from charge neutralized mixture of oppositely charged diblock copolymers. The interface of PIC vesicle and water exists biocompatible PMPC shells. Under acidic conditions, the PIC vesicles collapsed, because the charge balance shifted due to protonation of the PAaH block. After collapse of the PIC vesicles, P20A190 formed micelles composed of protonated PAaH core and PMPC shells, while P20M167 was released as unimers. PIC vesicles can encapsulate hydrophilic non-ionic guest molecules into their hollow core. Under acidic conditions, the PIC vesicles can release the guest molecules and P20M167. The cationic P20M167 can break the lysosome membrane to efficiently release the guest molecules from the lysosomes to the cytoplasm.

■ INTRODUCTION Advances in polymer synthesis have allowed the fabrication of nanometer-sized polymer aggregates with well-controlled structures. The tailor-made polymers can form 2


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aggregates with specific structures, e.g., micelles, worms, and vesicles by self-association. Recently,

controlled/living

radical

polymerization

methods

such

as

reversible

addition-fragmentation chain transfer (RAFT),1 and atom transfer radical polymerization (ATRP),2 were established to synthesize structure controlled polymers. These techniques can be used to prepare precise molecular conformations such as random, diblock, triblock, and star-shaped. These polymers can be used as building blocks to fabricate well-organized complex aggregates. Molecular interactions such as electrostatic, hydrogen bonding, and hydrophobic are generally used to form interpolymer aggregates.3–6 Water-soluble polyion complex micelles can be formed by mixing oppositely charged diblock copolymers, employing the electrostatic interactions between them.7,8 The diblock copolymers are consisted of a polyelectrolyte and non-ionic water-soluble poly(ethylene glycol) (PEG) blocks. The PIC micelle core is formed from oppositely charged polyelectrolytes, and the shells are formed from non-ionic hydrophilic PEG chains. PIC micelles can incorporate charged polymers such as DNA, RNA, and enzymes into their core via electrostatic interactions. One advantage of PIC micelles is that they can be prepared using water without organic solvents. Kataoka et al.9 have described the preparation of water-soluble PIC micelles by mixing anionic diblock copolymers (PEG-P(Asp)) composed of PEG with poly(α,β-aspartic acid); cationic diblock copolymers (PEG-P(Asp-AP)) composed

of

PEG

with

poly([5-aminopentyl]-α,β-aspartamide)

(P(Asp-AP));

and

water-soluble PIC vesicles (PICsome) by mixing anionic PEG-P(Asp) and cationic 3


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homopolymer, P(Asp-AP). The PIC membrane of PICsome consists of a sandwich structure with PEG shell layers inside and outside. As the weight ratio of PEG chains in the shells drops below 10%, the PIC aggregates transform from micelles to vesicles. It is expected that stimuli-responsive polymer aggregates will be used as carriers for bioactive molecules, because stimuli-responsive dissociation by external stimuli such as pH, light, and temperature can allow local drug administration. Kabanov et al.10 reported that UV-responsive vesicles were prepared by mixing azobenzene-containing cationic surfactants with diblock copolymers composed of PEG and anionic polyacrylate blocks, employing electrostatic attractive interactions. The vesicles were irradiated with UV light to induce dissociation by photoisomerization of the azobenzene group from trans-to-cis. Kataoka et al.11 reported synthesis of water-soluble PIC micelles by mixing cationic polyethyleneimine with anionic diblock copolymers containing an ester bond between PEG and anionic oligodeoxynucleotide (ODN). The pH-responsive PIC micelles released ODN at pH 5.5, since the ester bond was cleaved under acidic conditions. In general, when extracellular components are taken into normal cells, they enter the lysosomes by endocytosis. The inside of the lysosome is acidic with a pH of 4.5 to 5.0. Some acid-responsive carrier systems were found to release encapsulated drugs. A diblock copolymer consisted of a permanent hydrophilic block and a pH-responsive hydrophobic block that becomes hydrophilic at acidic conditions forms pH-responsive polymer micelles in water.12,13 Because both blocks become hydrophilic under acidic conditions, encapsulated 4


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drugs are released from the polymer micelles. Drugs are attached to polymer chains via acid-labile bonds such as hydrazone bonds, which can be cleaved by acids.14,15 Micelles formed by diblock copolymers, in which hydrophobic and hydrophilic blocks are connected with acid-labile bonds, can release drugs due to destabilization of the micelle structure by acids. Zhu et al.16 reported synthesis of amphiphilic diblock copolymers consisted of hydrophilic PEG and a hydrophobic stearyl group (C18), which was connected to the PEG chain with an acid-labile bond, hydrazone. The amphiphilic block copolymers formed pH-responsive polymer micelles comprising C18 core and PEG shells in water. The polymer micelles could release encapsulated drugs under acidic conditions such as inside lysosomes, because the micelles were unstable with respect to cleavage of the hydrazone bond. As described above, various acid-responsive carriers for bioactive molecules have been proposed. In many cases, encapsulated drugs cannot be efficiently delivered into the cytoplasm, because the carrier remains inside the lysosome after entering a cell by endocytosis. In the current study, we focused on the acidity inside lysosomes and prepared pH-responsive PIC vesicles that can release drugs by dissociation under acidic conditions. To release drugs effectively from inside the lysosomes, we proposed a system whereby destruction of the lysosomal membranes is induced by cationic polymers released from PIC vesicles

after

their

collapse

under

acidic

conditions.17,18

We

used

poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC, P) as a biocompatible zwitterionic water-soluble block (Figure 1a).19 Hydrophilic PMPC shows protein antifouling property, 5


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inhibiting protein denaturation, and excellent biocompatibility because of the pendant hydrophilic phosphorylcholine group. Oppositely charged polymers (P20M167 and P20A190) consisted of PMPC and cationic poly(3-(methacrylamidopropyl) trimethylammonium chloride) (PMAPTAC, M), or anionic poly(sodium 6-acrylamidohexanoate) (PAaH, A) blocks, were synthesized via reversible addition-fragmentation chain transfer (RAFT) radical polymerization. Subscripts in the block copolymer symbols indicate the degree of polymerization (DP) of each block. The PMAPTAC block is always positively charged regardless of the pH. While at pH ≥ 7, the pendant hexanoate groups in the PAaH block have anionic charges, under acidic conditions, the pendant hexanoate groups are protonated to neutralize the charges (Figure 1b). Therefore, at pH ≥ 7, a stoichiometrically charge neutralized mixture of P20M167 and P20A190 formed PIC vesicles by electrostatic attractive interactions between cationic PMAPTAC and anionic PAaH blocks. The interface of PIC vesicle and water exists biocompatible PMPC shells. However, under acidic conditions, the PIC vesicles collapsed due to the charge imbalance between P20M167 and P20A190, because the anionic charge in PAaH was neutralized by protonation of the pendant hexanoate groups (Figure 1c). When the PIC vesicles collapsed, P20A190 formed core-shell micelles consisted of hydrophobic PAaH block core and hydrophilic PMPC shells. Cationic P20M167 unimers, which can decompose lysosomal membranes, were released from PIC vesicles under acidic conditions. Therefore, it is expected that pH-responsive PIC vesicles will allow efficient controlled release of drugs from lysosomes to cytoplasm (Figure 1d). 6


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Figure 1. (a) Chemical structures of diblock copolymers (P20M167 and P20A190). (b) pH-responsive behavior of the pendant hexanoate group in P20A190. (c) Schematic illustration of encapsulation and release guest molecules by pH-responsive polyion complex (PIC) vesicles composed of P20M167 and P20A190. (d) Conceptual illustration of incorporation of drug-encapsulating PIC vesicle into a cell by endocytosis, and controlled release of the drugs from lysosome to cytoplasm.

■ RESULTS AND DISCUSSION GPC measurements of PMPC20, P20A190, and P20M167 were performed to determine Mn(GPC) and Mw/Mn (Figure S2). The GPC curves were unimodal with narrow Mw/Mn values 7


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below 1.38 (Table 1), suggesting that the chemical structures of all the polymers were controlled.

Figure 2. 1H NMR spectra measured in D2O of (a) P20M167, (b) P20A190, and (c) after mixing P20M167 and P20A190 at pH 12.0.

1

H NMR spectrum of the PMPC20 macro-chain transfer agent (macro-CTA) was measured in

D2O (Figure S3). The degree of polymerization (DP(NMR)) and number-average molecular weight (Mn(NMR)) of PMPC20 were determined by comparing the area integrated intensity ratio between peaks attributed to the terminal phenyl protons at 7.5–7.9 ppm and the pendant 8


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methylene protons at 3.7 ppm (Table 1). The 1H NMR spectra for P20M167 and P20A190 were measured in D2O at pH 12.0 (Figure 2). The DP of the PMAPTAC block was determined by comparing the peaks attributed to the PMPC pendant methylene protons at 3.7 ppm and the PMAPTAC pendant methyl protons at 3.4 ppm (Figure 2a). The DP of the PAaH block was determined by comparing the peaks attributed to the pendant methylene protons in the PMPC block at 3.7 ppm and in the PAaH block at 3.1 ppm (Figure 2b).

Table 1. Number-average molecular weight (Mn), degree of polymerization (DP), and molecular weight distribution (Mw/Mn) of PMPC20, P20M167, and P20A190. Mn(theo)

Mn(NMR)

× 104

× 104

(g/mol)

(g/mol)

PMPC20

0. 618

0. 618

20

20

0.421

1.03

P20M167

4.58

4.30

180a

167a

3.84

1.06

P20A190

4.14

4.14

190b

190b

5.33

1.38

samples

a

Mn(GPC) DP(theo)

DP(NMR)

× 104

Mw/Mn

(g/mol)

DP of the PMAPTAC block. bDP of the PAaH block.

The theoretical value of number-average molecular weight (Mn(theo)) was determined using equation (1):

9


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M n theo  

M 0 p M + M CTA 0 100 m CTA

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(1)

where [M]0 and [CTA]0 are the initial molar concentration of monomer and CTA, respectively, p is the percent conversion after polymerization, Mm and MCTA are the molecular weight of the monomer and CTA, respectively. The values of Mn(theo) for P20M167 and P20A190 were close to Mn(NMR), indicating that the polymerizations proceeded according to the controlled mechanism (Table 1). 1

H NMR measurements of P20A190 have been performed in D2O at pH 12.0 and 3.0

(Figure S4). The peaks attributed to the PMPC and PAaH blocks were observed at pH 12.0, which suggests that P20A190 dissolves in the solution as unimers. Alternatively, at pH 3.0, the peaks attributed to the PAaH block disappeared, and only the peaks of the PMPC block were observed. It is known that NMR peaks broaden and disappear as the molecular motion of the protons decreases.20,21 Under acidic conditions, P20A190 formed core-shell micelles う with hydrophobic protonated PAaH core and hydrophilic PMPC shells. We measured the Rh and light scattering intensity (LSI) for P20A190 as a function of pH to confirm the formation of micelles under acidic conditions (Figure S5). Aqueous P20A190 was prepared at Cp = 5.0 g/L at pH 12.0. A small amount of HCl aqueous solution was added to the solution to change the pH. Above pH 5.5, P20A190 dissolved in aqueous solutions as unimers because of electrostatic repulsion of the anionic PAaH blocks. Whereas, below pH 5.5, P20A190 formed polymer micelles with PAaH core and PMPC shells, because the pendant 10


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hexanoate groups in the PAaH block were protonated and therefore hydrophobic. The pKa for P20A190 was 5.3, estimated by a pH titration method (Figure S6). When the pH was above the pKa, P20A190 dissolved in the aqueous solutions as unimers. On the other hand, when the pH was below the pKa, P20A190 formed micelles because of hydrophobic interactions of the protonated PAaH blocks.

Figure 3. Hydrodynamic radius (Rh) distributions for (a) P20M167, (b) P20A190 at Cp = 5.0 g/L, and (c) P20M167/P20A190 PIC vesicles with f+ = 0.5 at pH 12.0 and Cp = 0.1 g/L.

Table 2. Dynamic Light Scattering (DLS) Data for P20M167, P20A190, and P20M167/P20A190 at pH 12.0 samples

Rh (nm)

LSI (Mcps)

PDI

P20M167

5.1

0.27

0.171

P20A190

6.8

0.34

0.204

P20M167/P20A190a

66.3

31.6

0.058

11


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a

Stoichiometrically charge neutralized mixture at f+ = 0.5.

Figure 3 shows Rh distributions for P20M167, P20A190, and a charge neutralized mixture of the oppositely charged diblock copolymers (P20M167/P20A190), which formed PIC aggregates at pH 12.0. The Rh distributions were unimodal for P20M167 and P20A190 with Rh of 5.1 and 6.8 nm, respectively (Table 2). These small Rh values suggested that the diblock copolymers dissolve as unimers in aqueous solutions. The Rh of P20M167/P20A190 increased to 66.3 nm, which indicated that PIC aggregates were formed due to electrostatic interactions. The expanded polymer chain lengths of P20M167 and P20A190 were calculated as 46.8 and 52.5 nm, respectively, using computer simulation. The Rh of P20M167/P20A190 was larger than the expanded end-to-end chain distance, which suggests that the PIC aggregates were not conventional core-shell spherical micelles but may be large compound micelles,22,23 worm-like micelles,24,25 or vesicles. The LSIs for P20M167 and P20A190 were 0.27 and 0.34 Mcps, respectively. The LSI for P20M167/P20A190 increased to 31.6 Mcps because of the formation of large aggregates. To confirm the structure of the stoichiometrically charge neutralized mixture of P20M167 and P20A190, we performed TEM with P20M167/P20A190 (Figure 4a). The vesicle structures were confirmed by TEM. The average radius of the vesicles was 63.6 nm, determined from TEM; this is close to the Rh (= 66.3 nm) determined by DLS.

12


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Figure 4 TEM images for P20M167/P20A190 with f+ = 0.5 at Cp = 0.1 g/L in 0.01 M aqueous NaCl at (a) pH 12.0 and (b) 3.0.

Figure 5 (a) Hydrodynamic radius (Rh, ○) and light scattering intensity (LSI, 13


) of

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P20M167/P20A190 as a function of f+ (=[MAPTAC]/([MAPTAC]+[AaH])) in 0.01 M aqueous NaCl at 25°C. (b) Zeta-potential as a function of f+ in 0.01 M aqueous NaCl. The total concentrations of the diblock copolymers were fixed at 0.1 g/L.

The Rh, LSI, and zeta-potential for the P20M167/P20A190 mixture were plotted as a function of f+ at pH 12.0 (Figure 5). At f+ = 0.5, Rh and LSI for the P20M167/P20A190 mixture reached their maximum values, and the zeta-potential was almost zero. When opposite charges in P20M167 and P20A190 were stoichiometrically charge neutralized by mixing, the size of the PIC aggregates reached a maximum. The charge balance is the most important factor in preparing PIC vesicles with a maximum size. If the f+ value for the PIC aggregates deviated from 0.5, the Rh and LSI decreased. with collapse of the vesicle structure.

14


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Figure 6. (a) Hydrodynamic radius (Rh, ○), light scattering intensity (LSI,

), and (b)

zeta-potential (●) for P20M167/P20A190 as a function of pH in 0.01 M aqueous NaCl.

The Rh, LSI, and zeta-potential for P20M167/P20A190 PIC vesicles at f+ = 0.5 were plotted as a function of the solution pH (Figure 6). The aqueous PIC vesicles were formed at pH 11.6, and then pH was decreased using aqueous HCl. At 11.6 ≥ pH ≥ 6.5, the Rh, LSI, and zeta-potential were about 60 nm, 30 Mcps, and −2 mV, respectively, and were constant and independent of the pH. In this pH region, the oppositely charged diblock copolymers formed PIC vesicles due to electrostatic interactions. At 6.5 > pH ≥ 5.5, the Rh increased from 60 to 70 nm, and the LSI decreased from 30 to 25 Mcps, which suggested that PIC vesicles may be swollen without collapsing. The decrease in the LSI suggests a decline in the density of the

15


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PIC vesicles. In this pH region, some pendant hexanoate groups in the PAaH block were protonated to decrease negative charges. Therefore, the charge balance deviated, and repulsion of excess cationic charges in the PMAPTAC block caused swelling of the PIC vesicle. The zeta potential increased from −2 to 5.5 as the pH decreased from 6.5 to 5.5 due to excess cationic charges in the PMAPTAC block. At 5.5 > pH ≥ 4.0, the Rh decreased from 70 to 32 nm, and the LSI also decreased from 25 to 2.8 Mcps. These observations suggested that the PIC vesicles collapsed, because the charge balance deviated from f+ = 0.5 because of complete protonation of the PAaH pendant hexanoate anions. The zeta-potential increased from 5.5 to 16 mV as the pH decreased from 5.5 to 4.0, which indicated cationic P20M167 may be released from PIC vesicles simultaneously with the collapse. These observations suggested that PIC vesicles collapsed below pH 5.5. The Rh (=32.4 nm) for P20M167/P20A190 below pH 4.0 was larger than those of unimers (Rh = 5.1–6.8 nm). P20A190 formed polymer micelles below pH 4.0 due to hydrophobic interactions of the protonated PAaH blocks, because the Rh (= 32.4 nm) was close to that of the P20A190 micelles (Figure S5). The charges of the P20A190 micelles should be neutral, because the surface was coated by the shell of PMPC chains. The zeta-potential was 16 mV below pH 4.0, which indicated that the P20M167 polymer chains may exist as unimers. The P20A167 micelles were also confirmed via TEM at pH 3.0 (Figure 4b). The average radius of the P20A167 micelles was 33.7 nm, which is similar to the Rh (= 32.4 nm) determined by DLS. 16


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A stoichiometrically charge neutralized mixture of P20M167 and P20A190 PBS solutions at pH 7.3 formed PIC vesicles with Rh = 64.5 nm (Figure S7). When the pH was adjusted to 3.1 by adding excess aqueous HCl, the Rh decreased to 38.0 nm. These observations indicated that the formation and pH-responsive collapse of PIC vesicles can be controlled in PBS.

Table 3. Dynamic and Static Light Scattering Results for P20M167/P20A190 in 0.01 M aqueous Sodium Chloride at pH 12.0 and 3.0 A2 × 10−4

Mw(SLS) Rg pH

7

× 10

Rh

Nagg (nm)

(nm)

Rg/Rh

(mol mL/g2)

(g/mol) 12.0

29.8

5,850

71.3

66.3

1.07

0.0382

3.0

1.61

282

34.0

32.4

1.05

2.18

To investigate pH induced structural changes in the P20M167/P20A190 mixture in more detail, we measured SLS at pH 12.0 and 3.0. The weight-average molecular weight (Mw(SLS)) was estimated from extrapolation to zero of both the scattering angle (θ) and Cp in the Zimm plot (Figure S8). Table 3 shows the light scattering results of P20M167/P20A190 at pH 12.0 and 3.0. The aggregation number (Nagg) values were estimated from ratio of Mw(SLS) to the average Mw values of P20M167 and P20A190 calculated using Mn(NMR) and Mw/Mn. The 17


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Nagg values at pH 12.0 and 3.0 were 5,850 and 282, respectively. These observations indicated that P20M167/P20A190 formed PIC vesicles with a large Nagg under basic conditions, and PIC vesicles collapsed due to the non-stoichiometric charge balance under acidic conditions. At pH 3, Nagg was not one, even after disintegration of PIC vesicles suggested the formation of P20A190 micelles because of hydrophobic interactions of the protonated PAaH blocks. The Nagg was 282 at pH 3, which was estimated using the average Mw(SLS) of the mixture of P20A190 micelles and P20M167 unimers. Therefore, the actual Nagg of the P20A190 micelles may be above 282. The Rg values estimated from SLS at pH 12.0 and 3.0 were 71.3 and 34.0 nm, respectively. The ratio of Rg to Rh (Rg/Rh) indicates the morphology of aggregates in solution.26,27 When Rg/Rh is close to 1, the aggregate is spherical. The Rg/Rh ratios at pH 12.0 and 3.0 were 1.07 and 1.05, respectively, which indicated that both the PIC vesicles and P20A190 micelles were spherical. These results are consistent with TEM observations at pH 12.0 and 3.0 (Figure 4). The P20M167 unimers were not observed with TEM at pH 3.0 due to their small size. The A2 values at pH 12.0 and 3.0 were 3.82 × 10−6 and 2.18 × 10−4 mol mL/g2, respectively. The smaller A2 value suggests reduced solubility in solvents.28,29 Thus, the solubility of PIC vesicles at pH 12.0 was less than those of P20A190 micelles and P20M167 unimers at pH 3.0. The PIC vesicles were covered with short PMPC shells with DP = 20. The dispersion stability of the PIC vesicles may be low due to the thin hydrated PMPC shells. The solubility of P20M167/P20A190 at pH 3.0 increased compared with that at pH 12.0, because 18


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P20A190 formed polymer micelles with PMPC shells and P20M167 formed unimers. Density (d) of the PIC vesicles can be estimated using Mw(SLS), Rg, and the following equation: d

3M w SLS 4N A Rg

(2)

where NA is Avogadro’s number. The d value for the PIC vesicle was 0.326 g/cm3, which was close to d (=0.376 g/cm3) of the PIC vesicles formed by oppositely charged diblock copolymers comprising polyelectrolyte and PMPC blocks.30 Separately P20M167 and P20A190 were dissolved in aqueous solutions containing fluorescence-labelled guest molecules, TD70 at pH 12.0, and then the aqueous solutions were mixed to prepare TD70-encapsulating PIC vesicles. The solution was dialyzed against water at pH 12.0 to remove unencapsulated TD70. A 50 nm dialysis membrane was used. Although TD70 can permeate the pores, the PIC vesicles were larger than the pore size. As a control experiment, a TD70 solution without PIC vesicles was dialyzed against water at pH 12.0, and fluorescence spectra were measured. Fluorescence from TD70 was not observed in the control solution after dialysis for 72 h. Fluorescence from encapsulated TD70 from the aqueous solution containing PIC vesicles was observed after dialysis for 72 h (Figure S9). PIC vesicles can encapsulate water-soluble non-ionic TD70 guest molecules into the interior of their hollow core. The weight of TD70 encapsulated into PIC vesicles was estimated from the calibration curve (Figure S10). The loading capacity (LC) and loading efficiency (LE) estimated from 19


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equations (S1) and (S2) were 8.63% and 21.4%, respectively.

Figure 7. Release profiles of Texas red-labelled dextran (TD70) encapsulated PIC vesicles in water at pH 3.0 (○) and 12.0 (△).

The aqueous TD70-encapsulating PIC vesicles at pH 12.0 were divided into two equal portions. One portion was adjusted to pH 3.0 and was dialyzed against aqueous solution at pH 3.0 using a 50 nm dialysis membrane. For the other portion, the pH was maintained at 12.0, and this was dialyzed against aqueous solution at pH 12.0 in the same manner. From fluorescence intensities of the aqueous solutions inside the dialysis bag at varying times, the release rate of encapsulated TD70 from PIC vesicles was monitored (Figure 7). At pH 3.0 the release rate was faster than that at pH 12.0. After 24 h, the release amounts of TD70 at pH 12.0 and 3.0 were 22.4% and 67.4%, respectively. A significant difference was observed in the release rate of TD70 from PIC vesicles in response to the pH change. Water-soluble non-ionic guest molecules can be encapsulated into PIC vesicles at pH 12.0. The encapsulated guest 20


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molecules can be released at pH 3.0 due to collapse of the PIC vesicles.

Viability (%)

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Concentration (g/mL)

Figure 8. Cytotoxicity of PIC vesicle. L929 cells were incubated for 48 h with various PIC vesicle concentrations, followed with MTT assay. Data were described as ratio of untreated cells. Results are expressed using means ± SD.

The result of the cytotoxicity assay was given in Figure 8. Even 150 μg/mL did not show any reduction of viability compared with 0 μg/mL. It showed clearly that PIC vesicle has no cytotoxicity in the concentration range used for cell applications.

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10 µm

Figure 9. Fluorescence microscopic observation of TD70-encapsulating PIC vesicles after incubating for 24 h in L929 cells. The late endosomes were labelled with Lysotracker green, and nuclei were labelled with Hoechst blue prior to observation.

To establish an effective therapeutic response, it is important for materials to release their therapeutic cargoes into the cytoplasm of the cells without approaching the lysosome organelles. Lysosome organelles have been known to degrade the materials due to the presence of acidic enzymes, which are responsible for loss of activity of the therapeutic cargoes. We examined the endosomal escape ability of TD70-encapsulating PIC vesicles after incubating for 24 h in L929 cells. The late endosome was labelled by Lysotracker green, and the nucleus was labelled by Hoechst blue prior to observation (Figure 9). The yellow region indicates overlap with the green color from Lysotracker and the red color from Texas red in TD70. This indicates that large amounts of TD70-encapsulating PIC vesicles were trapped in lysosomes. However, we observed separation of red fluorescence from the green signal in L929 cells. This indicates that TD70 was efficiently released from late endosomes. A possible

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mechanism for the release of TD70-encapsulating PIC vesicles could be related to the pH-responsive nature of nanocapsules. This result further validates the release of TD70 from PIC vesicles.

■ CONCLUSIONS Cationic and anionic diblock copolymers (P20M167 and P20A190) with narrow Mw/Mn were synthesized via RAFT polymerization. The pendant hexanoate groups in the PAaH block of P20A190 were ionized and protonated under basic and acidic conditions, respectively. A stoichiometrically charge neutralized mixture of P20M167 and P20A190 under basic conditions formed PIC vesicles due to electrostatic attractive interactions of the cationic PMAPTAC block and the anionic PAaH block. Under acidic conditions, the PIC vesicles collapsed to P20M167 unimers and P20A190 polymer micelles. We confirmed that hydrophilic non-ionic guest molecules can be encapsulated into PIC vesicles under neutral and basic conditions. The encapsulated guest molecules can be released from PIC vesicles under acidic conditions. It is expected that PIC vesicles are biocompatible, because their surfaces are covered with PMPC shells. When pH-responsive PIC vesicles were taken into a cell by endocytosis, destruction of lysosomal membranes was induced by cationic P20M167 unimers released by the collapse of PIC vesicles in the acidic environment inside lysosomes. Therefore, it is expected that PIC vesicles can achieve effective drug administration into cytoplasm. ■ ASSOCIATED CONTENT 23


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Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental details, materials, preparation of PMPC20, P20M167, P20A190, and PIC vesicle, characterization of the polymers and PIC vesicle, and fluorescence of TD70 (PDF)

■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID S. Yusa: 0000-0002-2838-5200, K. Matsumura: 0000-0001-9484-3073 Notes The author declares no competing financial interest.

■ ACKNOWLEDGMENTS This work was financially supported by a Grant-in-Aid for Scientific Research (17H03071 and 16K14008) from the Japan Society for the Promotion of Science (JSPS), JSPS Bilateral Joint Research Projects, and the Cooperative Research Program of “Network Joint Research Center for Materials and Devices (20174031).”

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Graphical Abstract pH-Responsive Polyion Complex Vesicle with Polyphosphobetaine Shells

Yuki Ohara†, Keita Nakai†, Sana Ahmed‡, Kazuaki Matsumura‡, Kazuhiko Ishihara§, Shin-ichi Yusa*,†

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pH-Responsive Polyion Complex Vesicle with Polyphosphobetaine

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