Polyion Complex Vesicles with Solvated Phosphobetaine Shells Formed from Oppositely Charged Diblock Copolymers

Diblock copolymers consisting of a hydrophilic poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) block and either a cationic or anionic block were prepared from (3-(methacrylamido)propyl)trimethylammonium chloride (MAPTAC) or sodium 2-(acrylamido)-2-methylpropanesulfonate (AMPS). Polymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) radical polymerization using a PMPC macro-chain transfer agent. The degree of polymerization for PMPC, cationic PMAPTAC, and anionic PAMPS blocks was 20, 190, and 196, respectively. Combining two solutions of oppositely charged diblock copolymers, PMPC-b-PMAPTAC and PMPC-b-PAMPS, led to the spontaneous formation of polyion complex vesicles (PICsomes). The PICsomes were characterized using 1H NMR, static abd dynamic light scattering, transmittance electron microscopy (TEM), and atomic force microscopy. Maximum hydrodynamic radius (Rh) for the PICsome was observed at a neutral charge balance of the cationic and anionic diblock copolymers. The Rh value and aggregation number (Nagg) of PICsomes in 0.1 M NaCl was 78.0 nm and 7770, respectively. A spherical hollow vesicle structure was observed in TEM images. The hydrodynamic size of the PICsomes increased with concentration of the diblock copolymer solutions before mixing. Thus, the size of the PICsomes can be controlled by selecting an appropriate preparation method.


Introduction
Polymer vesicles prepared by self-association of block copolymers, which are of great interest because of their potential application in fields such as materials science and biochemistry. Usually, polymer vesicles are prepared by self-assembly of amphiphilic block copolymers by the solvent switch method [1,2] or the organic-solvent free method [3]. For the solvent switch method, an amphiphilic diblock copolymer is dissolved in an organic solvent that can be mixed with water, such as dimethylsulfoxide (DMSO), N,N-diethylformamide (DMF), tetrahydrofuran (THF), or 1,4-dioxane, to prepare a homogeneous polymer solution, followed by the gradual addition water to the organic solvent solution. The hydrophilic block chains become solvated to form the vesicle shell, which

Preparation of PMPC
The PMPC macro-chain transfer agent (PMPC macro-CTA) was prepared according to a method modified from previously reports [18]. MPC (6.03 g, 20.4 mmol) was dissolved in a mixture of methanol and water (38.8 mL, 7/5, v/v), followed by addition of CPD (423 mg, 1.38 mmol) and V-501 (48.0 mg, 0.171 mmol) to the solution. The solution was degassed by purging with argon gas for 0.5 h. Polymerization was performed at 70 °C for 6 h. The reaction mixture was dialyzed against pure water for two days. PMPC was obtained by freeze-drying (6.05 g, 93.8%). The number-average molecular weight (Mn(NMR)), degree of polymerization (DP) estimated from 1 H NMR, and molecular weight distribution (Mw/Mn) estimated from gel-permeation chromatography (GPC) were 6.21 × 10 3 g/mol, 20, and 1.03, respectively.

Preparation of Polyion Complex Vesicles (PICsomes)
The P 20 M 190 and P 20 A 196 were dissolved separately in NaCl aqueous solutions, and the solutions were left standing overnight at room temperature to achieve complete dissolution. A P 20 M 190 solution was added dropwise to a P 20 A 196 solution over a period of 5 min at room temperature with stirring to prepare the PIC vesicles (PICsomes), and the mixture was left standing for at least 1 h prior to measurement. The mixing ratio of the block copolymers was represented by the mole fraction of positively charged unit (f + = [MAPTAC]/([AMPS] + [MAPTAC])) and hence complete charge neutralization was achieved at f + = 0.5.

Encapsulation of Texas Red-Labeled Dextran (Dex)
Dex (0.040 mg, 5.71 × 10 −10 mol) was dissolved in PBS buffer (4 mL), and P 20 M 190 (0.5 g/L) and P 20 A 196 (0.5 g/L) were dissolved in PBS buffer solutions containing Dex separately. The solutions were allowed to stand overnight at room temperature. The P 20 M 190 solution was added to the P 20 A 196 solution over a period of 5 min with stirring. The f + value was kept constant at 0.5. The solution (4 mL) was dialyzed using a polycarbonate membrane with 100-nm pore size (Harvard Apparatus, Holliston, MA, USA) against fresh PBS buffer (400 mL) for 18 h, changing the PBS buffer 3 times to remove the free Dex that was not incorporated into the hollow core of the PICsome. After dialysis, fluorescence emission of the PBS buffer in the dialyzer was measured. As a reference, fluorescence of the PBS buffer solution of Dex without PICsomes was also measured using a similar procedure. The weight of the Dex incorporated into the PICsomes was calculated using a calibration curve. The loading efficiency (LE) and loading capacity (LC) of Dex were calculated according to the following equations:

Measurements
The GPC measurements for the cationic polymer were obtained using a Jasco (Tokyo, Japan) RI-2031 Plus refractive index detector equipped with a Jasco PU-8020 pump and a Shodex (Tokyo, Japan) OHpak SB-804 HQ column (exclusion limit~10 7 ) working at 40 • C under a flow rate of 0.60 mL/min. A 0.30 M aq. Na 2 SO 4 solution containing 0.50 M acetic acid was used as the eluent. The values of M n (GPC) and M w /M n were calibrated using standard poly(2-vyniypyridine) samples. The GPC measurements for the anionic polymer were obtained using a Tosoh RI-8020 refractive index detector (Tosoh, Tokyo, Japan) equipped with a Shodex 7.0-µm bead size GF-7M HQ column (exclusion limit~10 7 ) working at 40 • C under a flow rate of 0.60 mL/min. A phosphate buffer (50 mM, pH 9.0) containing 10 vol % acetonitrile was used as the eluent. The values of M n (GPC) and M w /M n were determined using standard sodium poly(styrenesulfonate) samples. 1 H NMR spectra were obtained with a Bruker (Yokohma, Japan) DRX-500 spectrometer operating at 500.13 MHz with a deuterium lock. Light-scattering measurements were performed using an Otsuka Electronics Photal (Osaka, Japan) DLS-7000HL equipped with a multi-τ, digital time correlator (ALV-5000E). A helium-neon (He-Ne) laser (10.0 mW at 632.8 nm) was used as a light source. Sample solutions for light scattering measurements were filtered with a 0.45-µm membrane filter. From static light scattering (SLS) measurements, the weight-average molecular weight (M w ), z-average radius of gyration (R g ), and second virial coefficient (A 2 ) values were calculated by the relation: where R θ is the difference between the Rayleigh ratio of the solution and that of the solvent, K = 4π 2 n 2 (dn/dC p ) 2 /N A λ 4 with dn/dC p being the refractive index increment against C p , N A being Avogadro's number, and q the magnitude of the scattering vector. The q value was calculated from q = (4πn/λ)sin(θ/2), where n is the refractive index of the solvent, λ is the light source wavelength (=632.8 nm), and θ is the scattering angle. By measuring R θ for a set of C p and θ, values of M w , R g , and A 2 were estimated from Zimm plots. The known Rayleigh ratio of toluene was used for calibration of the instrument. Values of dn/dC p at 633 nm were determined using an Otsuka Electronics Photal (Osaka, Japan) DRM-3000 differential refractometer. In our dynamic light scattering (DLS) experiments, inverse Laplace transform (ILT) analysis was performed using the REPES algorithm [19][20][21] to obtain the relaxation time distribution, τA(τ). The relaxation rate (Γ = τ −1 ) is a function of θ [22]. The diffusion coefficient in the limit of zero angle (D) was calculated from D = (Γ/q 2 ) q→0 . The hydrodynamic radius (R h ) was provided by the Stokes-Einstein equation, R h = k B T/(6πηD), where k B is Boltzmann constant, T is absolute temperature, and η is solvent viscosity. The ζ-potential measurements were obtained using a Malvern (Worcestershire, UK) Zetasizer Nano-ZS ZEN3600 equipped with a He-Ne laser light source (4 mW at 632.8 nm). The ζ-potential was calculated from the electrophoretic mobility (µ) using the Smoluchowski relation, ζ = ηµ/ε (κa >> 1), where η is viscosity, ε is the dielectric constant of the medium, and κ and a are the Debye-Hückel parameter and particle radius, respectively [23]. Transmission electron microscopy (TEM) observations were performed using a Jeol JEM-2100 instrument at an accelerating voltage of 200 kV. Samples for TEM were prepared by placing one drop of the aqueous solution on a copper grid coated with thin films of Formvar. Excess water was blotted using filter paper. The samples were stained by sodium phosphotungstate and dried under vacuum for one day. Atomic force microscope (AFM) observations were performed with a JPK Nano Wizard 3 (JPK Instruments, Berlin, Germany) microscope. The sample of PICsome was applied onto a freshly cleaved mica surface. Excess water was blotted using filter paper and the sample dried for 10 min at 25 • C. Measurements were obtained in tapping mode using the Olympus (Tokyo, Japan) OMCLAC 160 TN-W2 silicon AFM probes (nominal spring constant, k = 42 N/m, resonance frequency ca. 300 kHz, tip radius < 10 nm). Height and size information were extracted using JPK data processing software (Version 5.1.8, JPK Instruments, Berlin, Germany). Fluorescence emission spectra were recorded on a Hitachi (Tokyo, Japan) F-2500 fluorescence spectrophotometer. Fluorescence spectra of Dex were measured with excitation at 550 nm. Excitation and emission slit widths were maintained at 10 nm.

Results and Discussion
To obtain oppositely charged diblock copolymers (P 20 M 190 and P 20 A 196 ), block copolymerization was conducted using PMPC macro-CTA with DP = 20 via RAFT radical polymerization. The conversions of MAPTAC and AMPS were estimated from 1 H NMR measurements after polymerization reached 93.0% and 95.0%, respectively. The molecular characteristics of PMPC, P 20 M 190 , and P 20 A 196 are summarized in Table 1. The theoretical number-average molecular weight (M n (theo)) was calculated using:    Figure 2c shows the 1 H NMR spectrum for the polyion complex vesicle (PICsome) composed of P 20 M 190 and P 20 A 196 with f + = 0.5 in D 2 O containing 0.1 M NaCl. The intensity of resonance peaks associated with the PMAPTAC and PAMPS blocks was weak compared with those associated with the PMPC block. These observations suggest that the motion of the PMAPTAC and PAMPS blocks was highly restricted due to formation of the PIC core. The mobility of PMPC chains may be higher than that of the PMAPTAC and PAMPS chains because the PMPC chains form shells surrounding the PIC.
Aggregates formed by electrostatic interactions sometimes depend on the mixing pathway [24][25][26]. We studied PICsome size dependence on the mixing pathway. A standard method is that a P 20 M 190 solution was added dropwise to a P 20 A 196 solution over a period of 5 min at room temperature with stirring. The P 20 A 196 solution was added to the P 20 M 190 solution, and the P 20 M 190 solution was added to the P 20 A 196 solution immediately. These two additional methods had no effect on the size of PICsome with f + = 0.5.  If the polymer main chain forms completely planar zigzag structure, the distance between one carbon to the next carbon is about 0.25 nm [27]. Hence, we can calculate the end-to-end distance of fully expanded polymer chains. The end-to-end distance of fully extended P20M190 and P20A196 chains were calculated as 52.5 and 54.0 nm, respectively. The Rh of 78.0 nm found for the PICsome was larger than those expected from the fully extended length of the P20M190 and P20A196 chains. These observations indicate that the shape of the PICsome is not a simple core-shell spherical micelle. Large compound aggregates or vesicles should be formed by mixing P20M190 and P20A196. Relaxation rates (Γ) measured at different θ plotted against the square of the scattering vector (q 2 ) are shown in Figure 3b. A line passing through the origin suggests that all of the relaxation modes were virtually diffusive [28].   If the polymer main chain forms completely planar zigzag structure, the distance between one carbon to the next carbon is about 0.25 nm [27]. Hence, we can calculate the end-to-end distance of fully expanded polymer chains. The end-to-end distance of fully extended P 20 M 190 and P 20 A 196 chains were calculated as 52.5 and 54.0 nm, respectively. The R h of 78.0 nm found for the PICsome was larger than those expected from the fully extended length of the P 20 M 190 and P 20 A 196 chains. These observations indicate that the shape of the PICsome is not a simple core-shell spherical micelle. Large compound aggregates or vesicles should be formed by mixing P 20 M 190 and P 20 A 196 . Relaxation rates (Γ) measured at different θ plotted against the square of the scattering vector (q 2 ) are shown in Figure 3b. A line passing through the origin suggests that all of the relaxation modes were virtually diffusive [28].  If the polymer main chain forms completely planar zigzag structure, the distance between one carbon to the next carbon is about 0.25 nm [27]. Hence, we can calculate the end-to-end distance of fully expanded polymer chains. The end-to-end distance of fully extended P20M190 and P20A196 chains were calculated as 52.5 and 54.0 nm, respectively. The Rh of 78.0 nm found for the PICsome was larger than those expected from the fully extended length of the P20M190 and P20A196 chains. These observations indicate that the shape of the PICsome is not a simple core-shell spherical micelle. Large compound aggregates or vesicles should be formed by mixing P20M190 and P20A196. Relaxation rates (Γ) measured at different θ plotted against the square of the scattering vector (q 2 ) are shown in Figure 3b. A line passing through the origin suggests that all of the relaxation modes were virtually diffusive [28].  To confirm the stability of the PICsome size, R h values were measured at various standing times. The R h values were nearly constant and independent of time until 150 h, suggesting that the structure of the PICsome does not change with time (data not shown). Scattering intensities of the PICsomes were also independent of time.
To further characterize the PICsomes, SLS measurements were performed for θ from 30 to 130 • with a 20 • increment. The refractive index increment (dn/dC p ) for P 20 M 190 , P 20 A 196 , and PICsome in 0.1 M NaCl were determined individually. Values for M w (SLS), R g , and A 2 were estimated from Zimm plots. Aggregation number (N agg ) for PICsomes (i.e., number of PMPC shell chains per one PICsome) was calculated by dividing M w (SLS) with that of unimers. The structure of the PICsome was also characterized by combining DLS and SLS to determine the R g /R h ratio. The density (d) of P 20 M 190 , P 20 A 196 , and PICsome can be calculated by: ratio is a structure-sensitive parameter that provides information about the density distribution of the particles and thereby about particle morphology [29,30]. The R g /R h ratio equals 0.775 for a homogeneous hard sphere, 1.0 for a thin hard spherical shell (e.g., vesicle), and increases significantly for a less dense structure and for a polydisperse solution because large molecules of a broad distribution will contribute more to R g than to R h , provided that internal modes of motion are absent [31]. The large R g /R h ratios (>4) for P 20 M 190 and P 20 A 196 suggest that the polymer chains were expanded due to electrostatic repulsions in the pendant ions. The R g /R h ratio for a polymeric vesicle may be less than or greater than 1.0, depending on the thickness and density of the wall [32]. The R g /R h ratio of the PICsome was 1.12, which is close to unity, indicates that the PICsome was a vesicle [33]. The A 2 value for the PICsome was less than those for P 20   The structure of the PICsome was confirmed by TEM observations, which showed incomplete spherical hollow vesicle structures (Figure 4). The vesicle structures may shrink during the drying process done prior to TEM observation. The PICsome diameter determined from the TEM images was 171 nm, which is close to the value obtained from the light scattering data. The AFM height image of the PICsome confirmed that the PICsome formed spherical structures that were slightly flattened due to the adsorption and drying process ( Figure 5). The height of the PICsome observed in the AFM image was ca. 100 nm. image of the PICsome confirmed that the PICsome formed spherical structures that were slightly flattened due to the adsorption and drying process ( Figure 5). The height of the PICsome observed in the AFM image was ca. 100 nm.   Figure 6a shows the Rh and light scattering intensity values for PICsomes in 0.1 M NaCl as a function of f + . Total polymer concentration was kept constant at 0.5 g/L. An increase in Rh indicates an increase in the size of the PICsome. The maximum Rh value was observed at f + = 0.5. In general, scattering intensity depends on molecular weight of the particles. Therefore, an increase in scattering intensity indicates an increase in Nagg for the PICsome, which suggests that stoichiometric charge neutralization in the mixture of the two oppositely charged P20M190 and P20A196 leads to formation of PICsomes with maximum size and aggregation number. Plots of Rh (and scattering intensity) vs. f + were asymmetric (i.e., the Rh and scattering intensities for PIC aggregates with f + = 0.6 and 0.8 were larger than those with f + = 0.4) [34]. To confirm the structure of PIC aggregates with f + = 0.4, 0.6, and 0.8, TEM images were obtained (Figure 7). Results showed that PIC aggregates with f + = 0.4 were micelle-like spherical particles without a hollow core. In contrast, PIC aggregates with f + = 0.6 and 0.8 clearly possessed hollow core vesicle structures. The PIC aggregates composed of P20M190 and P20A196 with excess PMAPTAC blocks tended to form vesicles, presumably because the pendant quaternary amino groups surrounded by three methyl groups in the PMAPTAC blocks were more hydrophobic compared to the pendant sulfonate groups in the PAMPS blocks. When f + is larger than 0.5, excess PMAPTAC blocks existed in the aggregate, dehydration of PIC aggregates was promoted, and solubility was less than that at f + < 0.5. For aggregates formed from conventional amphiphilic diblock copolymers in water, the greater the hydrophobicity of the aggregate, the more likely diblock copolymers are to form vesicles rather than spherical core-shell micelles [35]. Therefore, PIC aggregates with f + ≥ 0.5 tend to form vesicles. image of the PICsome confirmed that the PICsome formed spherical structures that were slightly flattened due to the adsorption and drying process ( Figure 5). The height of the PICsome observed in the AFM image was ca. 100 nm.   Figure 6a shows the Rh and light scattering intensity values for PICsomes in 0.1 M NaCl as a function of f + . Total polymer concentration was kept constant at 0.5 g/L. An increase in Rh indicates an increase in the size of the PICsome. The maximum Rh value was observed at f + = 0.5. In general, scattering intensity depends on molecular weight of the particles. Therefore, an increase in scattering intensity indicates an increase in Nagg for the PICsome, which suggests that stoichiometric charge neutralization in the mixture of the two oppositely charged P20M190 and P20A196 leads to formation of PICsomes with maximum size and aggregation number. Plots of Rh (and scattering intensity) vs. f + were asymmetric (i.e., the Rh and scattering intensities for PIC aggregates with f + = 0.6 and 0.8 were larger than those with f + = 0.4) [34]. To confirm the structure of PIC aggregates with f + = 0.4, 0.6, and 0.8, TEM images were obtained (Figure 7). Results showed that PIC aggregates with f + = 0.4 were micelle-like spherical particles without a hollow core. In contrast, PIC aggregates with f + = 0.6 and 0.8 clearly possessed hollow core vesicle structures. The PIC aggregates composed of P20M190 and P20A196 with excess PMAPTAC blocks tended to form vesicles, presumably because the pendant quaternary amino groups surrounded by three methyl groups in the PMAPTAC blocks were more hydrophobic compared to the pendant sulfonate groups in the PAMPS blocks. When f + is larger than 0.5, excess PMAPTAC blocks existed in the aggregate, dehydration of PIC aggregates was promoted, and solubility was less than that at f + < 0.5. For aggregates formed from conventional amphiphilic diblock copolymers in water, the greater the hydrophobicity of the aggregate, the more likely diblock copolymers are to form vesicles rather than spherical core-shell micelles [35]. Therefore, PIC aggregates with f + ≥ 0.5 tend to form vesicles.  Figure 6a shows the R h and light scattering intensity values for PICsomes in 0.1 M NaCl as a function of f + . Total polymer concentration was kept constant at 0.5 g/L. An increase in R h indicates an increase in the size of the PICsome. The maximum R h value was observed at f + = 0.5. In general, scattering intensity depends on molecular weight of the particles. Therefore, an increase in scattering intensity indicates an increase in N agg for the PICsome, which suggests that stoichiometric charge neutralization in the mixture of the two oppositely charged P 20 M 190 and P 20 A 196 leads to formation of PICsomes with maximum size and aggregation number. Plots of R h (and scattering intensity) vs. f + were asymmetric (i.e., the R h and scattering intensities for PIC aggregates with f + = 0.6 and 0.8 were larger than those with f + = 0.4) [34]. To confirm the structure of PIC aggregates with f + = 0.4, 0.6, and 0.8, TEM images were obtained (Figure 7). Results showed that PIC aggregates with f + = 0.4 were micelle-like spherical particles without a hollow core. In contrast, PIC aggregates with f + = 0.6 and 0.8 clearly possessed hollow core vesicle structures. The PIC aggregates composed of P 20 M 190 and P 20 A 196 with excess PMAPTAC blocks tended to form vesicles, presumably because the pendant quaternary amino groups surrounded by three methyl groups in the PMAPTAC blocks were more hydrophobic compared to the pendant sulfonate groups in the PAMPS blocks. When f + is larger than 0.5, excess PMAPTAC blocks existed in the aggregate, dehydration of PIC aggregates was promoted, and solubility was less than that at f + < 0.5. For aggregates formed from conventional amphiphilic diblock copolymers in water, the greater the hydrophobicity of the aggregate, the more likely diblock copolymers are to form vesicles rather than spherical core-shell micelles [35]. Therefore, PIC aggregates with f + ≥ 0.5 tend to form vesicles.  To confirm PICsome neutralization at f + = 0.5, the ζ-potential was measured as a function of f + (Figure 6b). At f + = 0, the aqueous solution of P20A196 has a negative ζ-potential value of −29 mV because the PAMPS block has pendant anionic sulfonate groups. At f + = 1, the aqueous solution of P20M190 has a positive ζ-potential value of +27 mV because the PMAPTAC block has pendant cationic quaternary amino groups. The ζ-potential was zero at f + = 0.5 because the charges of the PAMPS and PMAPTAC blocks were neutralized. The PICsome was composed of a PIC core and PMPC shells. The pendant phosphorylcholine groups in the PMPC shells contain anionic phosphate and cationic quaternary amine. However, the ζ-potential of PMPC homopolymer was zero (data not shown) because of neutralization of the anion and cation pair within a single polymer chain. Therefore, the ζ-potential of PICsome was zero at f + = 0.5.  To confirm PICsome neutralization at f + = 0.5, the ζ-potential was measured as a function of f + (Figure 6b). At f + = 0, the aqueous solution of P20A196 has a negative ζ-potential value of −29 mV because the PAMPS block has pendant anionic sulfonate groups. At f + = 1, the aqueous solution of P20M190 has a positive ζ-potential value of +27 mV because the PMAPTAC block has pendant cationic quaternary amino groups. The ζ-potential was zero at f + = 0.5 because the charges of the PAMPS and PMAPTAC blocks were neutralized. The PICsome was composed of a PIC core and PMPC shells. The pendant phosphorylcholine groups in the PMPC shells contain anionic phosphate and cationic quaternary amine. However, the ζ-potential of PMPC homopolymer was zero (data not shown) because of neutralization of the anion and cation pair within a single polymer chain. To confirm PICsome neutralization at f + = 0.5, the ζ-potential was measured as a function of f + (Figure 6b). At f + = 0, the aqueous solution of P 20 A 196 has a negative ζ-potential value of −29 mV because the PAMPS block has pendant anionic sulfonate groups. At f + = 1, the aqueous solution of P 20 M 190 has a positive ζ-potential value of +27 mV because the PMAPTAC block has pendant cationic quaternary amino groups. The ζ-potential was zero at f + = 0.5 because the charges of the PAMPS and PMAPTAC blocks were neutralized. The PICsome was composed of a PIC core and PMPC shells. The pendant phosphorylcholine groups in the PMPC shells contain anionic phosphate and cationic quaternary amine. However, the ζ-potential of PMPC homopolymer was zero (data not shown) because of neutralization of the anion and cation pair within a single polymer chain. Therefore, the ζ-potential of PICsome was zero at f + = 0.5.
To confirm that PICsomes with f + = 0.5 are at equilibrium or in a kinetically frozen state, excess P 20 A 196 was added to the aqueous PICsome solution with f + = 0.5 to change the f + value. A kinetically frozen state means that the polymer chains cannot break free from the aggregate. The size of a PICsome with f + = 0.5 in the kinetically frozen state should not be affected by the addition of excess P 20 A 196 . The size of a PICsome in the equilibrium state may decrease upon addition of excess P 20 A 196 . Figure 8 shows R h distributions for PICsomes with f + = 0.5 and PIC aggregates with f + = 0.4 and 0.2 formed by the addition of P 20 A 196 to the PICsome with f + = 0.5. The R h value of the PICsome with f + = 0.5 was 78.0 nm at C p = 0.5 g/L. When a P 20 A 196 solution at C p = 0.5 g/L was added to the PICsome solution, which changed the f + to 0.4 and 0.2, the R h values of the PIC aggregates decreased to 49 and 7.9 nm, respectively. This observation suggested that PICsomes formed by mixing oppositely charged diblock copolymers existed in an equilibrium state in water. Thus, small pairs of the oppositely charged diblock copolymers may dissociate from and associate with the PICsome [36]. NaCl concentrations in the aqueous solution are very important for stability of PICsomes because they were formed by electrostatic interactions. We measured the R h values of PICsomes in various NaCl concentrations. When NaCl concentration was 0.5 M, the R h value was 77.8 nm, which is close to the value (R h = 78.0 nm) in 0.1 M NaCl aqueous solutions. Therefore, at least below 0.5 M of NaCl concentration, PICsomes were stable.   The relation between PICsome size and Cp in 0.1 M NaCl is shown in Figure 9. The sample solutions were prepared by two different methods. The first method involved preparing separate aqueous P20M190 and P20A196 solutions with a target Cp from 0.001 to 1 g/L before mixing a pair of two oppositely charged diblock copolymers. Then, the two aqueous P20M190 and P20A196 solutions with the same Cp were mixed to form a PICsome solution (Figure 9a). The second method involved mixing pairs of oppositely charged diblock copolymer solutions at Cp = 1, 0.5, and 0.01 g/L to form PICsomes. Subsequently, the aqueous PICsome solutions were diluted with 0.1 M NaCl to adjust Cp to the target value (Figure 9b). These PICsome solutions prepared via these two Cp adjustment methods were measured using DLS to determine Rh. The Rh values for PICsomes depended on the value of Cp of the P20M190 and P20A196 aqueous solutions before mixing to form the PICsome. When each P20M190 and P20A196 solution was prepared at Cp = 1 g/L, the Rh value for the PICsome was ca. 100 nm. In contrast, when each aqueous P20M190 and P20A196 solution was prepared at Cp = 0.01 g/L, the Rh value for the PICsome was ca. 38 nm. The size of the PICsomes could be controlled by adjusting the Cp values of oppositely charged diblock copolymer solutions before mixing. When 0.1 M NaCl PICsome solutions were diluted with 0.1 M NaCl, the Rh values for the PICsome remained nearly constant, independent of Cp. These findings suggest that the structure of the PICsome, once prepared, is stable against dilution. In general, sonication and extrusion techniques are used to control the size of vesicles [37]. However, the easily adjustable size of the stable PICsome system The relation between PICsome size and C p in 0.1 M NaCl is shown in Figure 9. The sample solutions were prepared by two different methods. The first method involved preparing separate aqueous P 20 M 190 and P 20 A 196 solutions with a target C p from 0.001 to 1 g/L before mixing a pair of two oppositely charged diblock copolymers. Then, the two aqueous P 20 M 190 and P 20 A 196 solutions with the same C p were mixed to form a PICsome solution (Figure 9a). The second method involved mixing pairs of oppositely charged diblock copolymer solutions at C p = 1, 0.5, and 0.01 g/L to form PICsomes. Subsequently, the aqueous PICsome solutions were diluted with 0.1 M NaCl to adjust C p to the target value (Figure 9b). These PICsome solutions prepared via these two C p adjustment methods were measured using DLS to determine R h . The R h values for PICsomes depended on the value of C p of the P 20 M 190 and P 20 A 196 aqueous solutions before mixing to form the PICsome. When each P 20 M 190 and P 20 A 196 solution was prepared at C p = 1 g/L, the R h value for the PICsome was ca. 100 nm. In contrast, when each aqueous P 20 M 190 and P 20 A 196 solution was prepared at C p = 0.01 g/L, the R h value for the PICsome was ca. 38 nm. The size of the PICsomes could be controlled by adjusting the C p values of oppositely charged diblock copolymer solutions before mixing. When 0.1 M NaCl PICsome solutions were diluted with 0.1 M NaCl, the R h values for the PICsome remained nearly constant, independent of C p . These findings suggest that the structure of the PICsome, once prepared, is stable against dilution. In general, sonication and extrusion techniques are used to control the size of vesicles [37]. However, the easily adjustable size of the stable PICsome system described here indicates that the size of vesicles and polymersomes can be easily controlled by adjusting the C p before mixing a pair of oppositely charged diblock copolymers. To confirm the ability to incorporate hydrophilic guest molecules into the interior aqueous phase of PICsomes, fluorescence experiments were performed using Texas red-labeled Dex as a fluorescence probe. The hydrophilic Dex molecule contains no charged groups. The P20M190 and P20A196 were dissolved in Dex-containing PBS buffer solutions, and then these solutions were mixed to form PICsomes. The Dex molecules that could not be incorporated into the PICsomes were removed by dialysis against fresh PBS buffer for 18 h. Fluorescence spectra were obtained for the solution inside the dialyzer after dialysis ( Figure 10). Fluorescence emission with a maximum wavelength at 610 nm for Dex was observed, which indicates that the Dex molecules were incorporated into the hollow core of PICsome. In contrast, a blank solution in the absence of PICsomes produced no fluorescence from Dex because the small Dex molecules were removed when using a dialysis membrane with a pore size of 100 nm. These results demonstrate that PICsomes can incorporate Dex guest molecules into the hollow core. The weight of the Dex incorporated into the PICsomes was calculated using a calibration curve, and was 0.00315 mg. The LE and LC values determined using the encapsulated Dex weight were 78.8% and 1.58%, respectively. To confirm the ability to incorporate hydrophilic guest molecules into the interior aqueous phase of PICsomes, fluorescence experiments were performed using Texas red-labeled Dex as a fluorescence probe. The hydrophilic Dex molecule contains no charged groups. The P 20 M 190 and P 20 A 196 were dissolved in Dex-containing PBS buffer solutions, and then these solutions were mixed to form PICsomes. The Dex molecules that could not be incorporated into the PICsomes were removed by dialysis against fresh PBS buffer for 18 h. Fluorescence spectra were obtained for the solution inside the dialyzer after dialysis ( Figure 10). Fluorescence emission with a maximum wavelength at 610 nm for Dex was observed, which indicates that the Dex molecules were incorporated into the hollow core of PICsome. In contrast, a blank solution in the absence of PICsomes produced no fluorescence from Dex because the small Dex molecules were removed when using a dialysis membrane with a pore size of 100 nm. These results demonstrate that PICsomes can incorporate Dex guest molecules into the hollow core. The weight of the Dex incorporated into the PICsomes was calculated using a calibration curve, and was 0.00315 mg. The LE and LC values determined using the encapsulated Dex weight were 78.8% and 1.58%, respectively.
PICsomes produced no fluorescence from Dex because the small Dex molecules were removed when using a dialysis membrane with a pore size of 100 nm. These results demonstrate that PICsomes can incorporate Dex guest molecules into the hollow core. The weight of the Dex incorporated into the PICsomes was calculated using a calibration curve, and was 0.00315 mg. The LE and LC values determined using the encapsulated Dex weight were 78.8% and 1.58%, respectively.

Conclusions
A pair of oppositely charged diblock copolymers with well-controlled structures, P20M190 and P20A196, were prepared via RAFT using PMPC macro-CTA. Polyion complex vesicles (PICsomes) were formed by stoichiometric charge neutralization of a mixture of aqueous P20M190 and P20A196

Conclusions
A pair of oppositely charged diblock copolymers with well-controlled structures, P 20 M 190 and P 20 A 196 , were prepared via RAFT using PMPC macro-CTA. Polyion complex vesicles (PICsomes) were formed by stoichiometric charge neutralization of a mixture of aqueous P 20 M 190 and P 20 A 196 solutions. The surface of the PICsomes was covered with biocompatible PMPC shell chains. These PICsomes could incorporate water-soluble guest molecules without charge groups inside the interior aqueous phase, which indicates that these PICsomes may be useful as a molecular carrier of several bioactive compounds.