Thermo-responsive and Biocompatible Diblock Copolymers Prepared via Reversible Addition-fragmentation Chain Transfer (raft) Radical Polymerization

Poly(2-(methacryloyloxy)ethyl phosphorylcholine)-b-poly(N,N-diethyl acrylamide) (PMPC m-PDEA n) was synthesized via reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization. Below, the critical aggregation temperature (CAT) the diblock copolymer dissolved in water as a unimer with a hydrodynamic radius (R h) of ca. 5 nm. Above the CAT the diblock copolymers formed polymer micelles composed of a PDEA core and biocompatible PMPC shells, due to hydrophobic self-aggregation of the thermo-responsive PDEA block. A fluorescence probe study showed that small hydrophobic small guest molecules could be incorporated into the core of the polymer micelle above the CAT. The incorporated guest molecules were released from the core into the bulk aqueous phase when the temperature decreased to values below the CAT because of micelle dissociation.


Introduction
Phospholipids are a component of lipid bilayers in cell membranes; thus, they are of interest in the biochemical and biomedical fields as unique substrates.The phosphorylcholine group of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) is a component of cell membranes [1,2].MPC can be polymerized with other vinyl monomers, and the properties and functions of MPC polymers can be controlled by changing comonomers.Polymers with water-soluble MPC units show excellent biocompatibility and antithrombogenicity.
In water, many N-substituted polyacrylamides show phase separation upon heating [3], and the temperature at which phase transition occurs is called the lower critical solution temperature (LCST).The most extensively studied polymer is poly(N-isopropylacrylamide) (PNIPAM) [4].Below the LCST, the PNIPAM chains are dissolved as extended coils, while above the LCST they collapse into compact globules in a coil-to-globule transition [5].Poly(N,N-diethylacrylamide) (PDEA) is also a thermo-responsive polymer similar to PNIPAM.The LCST of PDEA is reported to be between 25 and 35 °C, depending on molecular weight, concentration, and external additives [6].The LCST is mainly governed by the balance between hydrogen bonds and hydrophobic interactions among alkyl side chains [7].Therefore, the LCST is accompanied by sharp changes in the degree of hydration, chain mobility, hydrophobicity, and so on.
Lowe et al. [8] reported the synthesis of diblock copolymers (PMPC m -PDEA n ) composed of hydrophilic PMPC and thermo-responsive PDEA blocks and investigated their thermo-responsive association behavior in water using NMR and dynamic light scattering (DLS) measurements.We were interested in the detailed thermo-responsive association behavior of PMPC m -PDEA n and the potential for controlled release of guest molecules, such as drugs, in the cores of polymer micelles.We focused on diblock copolymers based on a hydrophilic shell-forming PMPC block and a thermo-responsive core-forming PDEA block (Figure 1).PMPC m -PDEA n can be molecularly dissolved in water below the LCST for the PDEA block, as the PDEA block is hydrophilic under these conditions.However, when the temperature is elevated above LCST, the PDEA block becomes hydrophobic because of the breaking of hydrogen bonding interactions between the pendant amide bond in the PDEA block and water molecules, leading to the formation of micelles with a dehydrated PDEA core and hydrated PMPC shells.These polymer micelles can incorporate in their hydrophobic core hydrophobic guest molecules, such as pyrene and adriamycin hydrochloride (ADR).

Preparation of Poly(2-(methacryloyloxy)ethyl phosphorylcholine)-Based Chain Transfer Agent (PMPC 64 )
A poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC)-based chain transfer agent was prepared using a modified method reported previously [11].MPC (50.2 g, 170 mmol) was dissolved in 290 mL of water, and then CPD (696 mg, 2.31 mmol) and V-501 (80.7 mg, 0.288 mmol) were added to the solution.The mixture was deoxygenated by purging with Ar gas for 30 min.Polymerization was carried out at 70 °C for 2 h.After cooling the reaction mixture with an ice bath, the solution was dialyzed against pure water for a week.PMPC 64 was recovered by freeze-drying (19.1 g, 38.0% conversion).The number-average molecular weight (M n (NMR)) and degree of polymerization (DP) for PMPC 64 were 1.89 × 10 4 and 64, respectively, as estimated from 1 H nuclear magnetic resonance (NMR) terminal group analysis.The molecular weight distribution (M w /M n ), estimated from gel-permeation chromatography (GPC), was 1.19.

Preparation of Diblock Copolymers
A typical procedure for block copolymerization is as follows.PMPC 64 (2.00 g, M n (NMR) = 1.89 × 10 4 , M w /M n = 1.19),DEA (2.58 g, 20.3 mmol), and V-501 (5.92 mg, 0.0210 mmol) were dissolved in 32.0 mL of methanol.The solution was placed in a glass ampoule and outgassed on a vacuum line by six freeze-pump-thaw cycles, and then the ampoule was vacuum-sealed.Polymerization was carried out at 70 °C for 24 h.The reaction mixture was poured into a large excess of diethyl ether to precipitate the resulting polymer.The polymer (PMPC 64 -PDEA 66 ) was purified by reprecipitation from methanol into a large excess of diethyl ether (2.64 g, 57.6% conversion).The M n (NMR) and M w /M n values for PMPC 64 -PDEA 66 were 2.82 × 10 4 , M w /M n = 1.21, respectively.
To investigate the relationship between polymerization time and conversion, the monomer conversion was determined by 1 H NMR spectroscopy.Predetermined amounts of DEA, PMPC 64 , and V-501 were dissolved in methanol.The solution was transferred to an NMR tube containing an NMR lock tube of D 2 O and degassed by purging with Ar gas for 30 min.The cap was sealed and the solution was heated at 70 °C in a preheated oil bath for varying lengths of reaction time.Polymerization was terminated by rapid cooling with an ice bath.The monomer conversion estimated from 1 H NMR was monitored as a function of reaction time.

Measurements
GPC measurements were performed with a GPC system composed of a Tosoh DP-8020 pump, a Tosoh RI-8021 refractive index detector, and Shodex 7.0 μm bead size GF-7M HQ column (molecular weight range 10 7 -10 2 ) using a phosphate buffer (pH 9) containing 20 vol% acetonitrile as an eluent at a flow rate of 0.6 mL/min at 25 °C.Sample solutions were filtered with a 0.2-μm pore size membrane filter before measurements.The M n (GPC) and M w /M n for the polymers were calibrated with standard sodium poly(styrenesulfonate) samples. 1 H NMR spectra were obtained with a Bruker DRX-500 spectrometer.The sample solutions of the block copolymers at a polymer concentration (C p ) of 5.0 g/L for 1 H NMR measurements were prepared in D 2 O containing 0.1 M NaCl.The temperature was changed from 24 °C to 60 °C with a heating or cooling rate of 1 °C/min.Percent transmittance (T%) for 0.1 M NaCl aqueous solutions of diblock copolymer was measured with a U-3000 spectrophotometer (Hitachi, Tokyo, Japan) with a 1.0 cm path length quartz cell at various temperatures.The temperature was changed from 20 °C to 60 °C with a heating or cooling rate of 0.5 °C/min.
Static light scattering (SLS) and dynamic light scattering (DLS) measurements were performed at 25 °C with an Otsuka Electronics Photal DLS-7000HL light scattering spectrometer equipped with a 5000E multi-τ digital time correlator (ALV, Langen, Germany).Sample solutions for SLS and DLS measurements were filtered with a 0.2-μm pore size membrane filter.For SLS measurements, a He-Ne laser (10 mW at 632.8 nm) was used as a light source.The weight-average molecular weight (M w ), z-average radius of gyration (R g ), and second virial coefficient (A 2 ) values were estimated from the relation: where R θ is the Rayleigh ratio, K = 4π 2 n 2 (dn/dC p ) 2 /N A λ 4 , with n being the refractive index of the solvent, dn/dC p being the refractive index increment against C p , N A being Avogadro's number, and λ being the wavelength (= 632.8 nm), while q = (4πn/λ)sin(θ/2) with θ being 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.Toluene was used for calibration of the instrument.Values of dn/dC p were determined with an Otsuka Electronics Photal DRM-1020 differential refractometer at a wavelength of 632.8 nm.For DLS measurements, an He-Ne laser (10 mW at 632.8 nm) was used as a light source.The hydrodynamic radius (R h ) was calculated using the Einstein-Stokes relation R h = k B T/6πηD, where k B is Boltzmann's constant, T is the absolute temperature, and η is the solvent viscosity.Excitation emission spectra for pyrene were recorded on a Hitachi F-2500 fluorescence spectrophotometer.A pyrene-saturated aqueous stock solution was prepared as previously [12].Sample solutions were prepared by mixing aliquots of stock solutions of the polymer and pyrene (2.0 × 10 −7 M) in 0.1 M NaCl aqueous solutions.Excitation spectra were monitored at 390 nm.Excitation and emission slit widths were maintained at 2.5 and 20 nm, respectively.

Release Experiments
The release of ADR from the polymer micelles and blank experiments were performed according to a modified literature procedure [13].The release was monitored by a V-530 UV/VIS spectrophotometer (Jasco, Tokyo, Japan) equipped with a magnetic stirrer using a 1.0 cm path length quartz cell as a diffusion cell contained in a small glass cell separated from the surrounding solution by a dialysis membrane with a molecular weight cutoff of 15,000.First, 0.3 mL of a 0.1 M NaCl aqueous solution of ADR (0.06 mM) with and without PMPC 64 -PDEA 66 (C p = 50 g/L) was put inside a small glass cell.The temperature of the solution was maintained at 25 and 50 °C.The small glass cell, capped with a dialysis membrane with an o-ring, was immersed into 3.0 mL of 0.1 M NaCl with same the temperature as that of the blank and polymer solutions in the quartz cell.The cumulative amount of ADR released from the small glass cell was monitored by absorption at 480 nm.

Results and Discussion
We prepared PMPC 64 via RAFT using CPD as a water-soluble CTA.The M n (NMR) value, calculated from 1 H NMR peak area intensities of pendant methylene protons and terminal phenyl protons (Table 1), was 1.89 × 10 4 .The M n (GPC) and M w /M n values were 8.62 × 10 3 and 1.19, estimated from GPC.A notable observation was the marked deviation of M n (GPC) from M n (NMR).To verify the true molecular weight of PMPC 64 , SLS measurements were performed.The M w value of 2.04 × 10 4 determined by SLS was in fair agreement with M n (NMR), using M w /M n = 1.19.It should be mentioned that M n (GPC) values estimated by GPC are only apparent values, probably because sodium poly(styrenesulfonate) was used as a standard for molecular weight calibration, compared to PMPC 64 , which has a bulky phosphorylcholine side chain.To prepare the thermo-responsive diblock copolymer, DEA was polymerized via RAFT radical polymerization in methanol using the PMPC 64 macro-chain transfer agent.Figure 2 shows the pseudo-first-order kinetics plot for the polymerization of DEA in the presence of PMPC 64 at 70 °C under Ar in methanol containing a sealed tube of D 2 O to lock the NMR frequency.Monomer consumption was monitored by 1 H NMR spectroscopy as a function of polymerization time.There was an induction period of ca. 30 min, which may have been due to a slow rate of formation of the radical fragment [14].A monomer conversion of 83% was reached within 5 h.The downward curvature was observed, which indicates a decrease in the concentration of propagating radicals.The M n (GPC) values estimated from GPC were only apparent values because sodium poly(styrenesulfonate) was used as a standard for molecular weight calibration.To verify the true molecular weight of PMPC m -PDEA n , SLS measurements were performed (Figure 3).The M n (NMR) for PMPC m -PDEA n was calculated from 1 H NMR in D 2 O at 25 °C (Figure 4a).The M w (SLS) values of PMPC m -PDEA n , determined by SLS, were in fair agreement with M n (NMR), using M w /M n .Apparent M w , R g , and A 2 for the diblock copolymers at 25 °C, determined by SLS measurement, are listed in Table 1.The dn/dC p values at 633 nm for the polymers at 25 °C are listed in Table 1.The same PMPC 64 macro-chain transfer agent of M n (NMR) = 1.89 × 10 4 was used to prepare a series of PMPC 64 -PDEA n with different PDEA block lengths.The DP values for PMPC and PDEA blocks were calculated based on 1 H NMR. Table 1 lists the molecular parameters of the polymers.To study heat-induced association of the diblock copolymers, 1 H NMR spectra for the block copolymers were measured at different temperatures in D 2 O containing 0.1 M NaCl. Figure 4 compares typical 1 H NMR spectra for PMPC 64 -PDEA 66 measured at 25 and 60 °C.At 25 °C the diblock copolymer chains are fully solvated and molecularly dissolved in water-i.e., a "unimer" state-and all signals expected for each block were observed.The resonance bands in the 0.8-1.4ppm region were attributed to the sum of the α-methyl protons in the main chain and the methyl protons in the pendant N,N-diethylamino group.The methylene protons in the main chain of the PMPC and PDEA blocks were observed at 1.6 and 2.0 ppm, respectively.The resonance peak at 2.6 ppm was attributed to the methine proton in the main chain of the PDEA block.The resonance bands at 3.1-3.5 ppm were assigned to the methyl protons of the pendant trimethyl ammonium group of the PMPC block and the methylene protons in the pendant N,N-diethylamino group of the PDEA block, respectively.The methylene protons in the pendant phosphorylcholine group of the PMPC block were observed at 3.6-4.5 ppm.The composition of the block copolymer was determined from the area intensity ratio of the resonance bands due to the methine proton in the main chain of the PDEA block at 2.6 ppm and the methylene protons of the pendant phosphorylcholine of the PMPC block at 3.6 ppm at 25 °C, because these peaks were relatively isolated from the other peaks.The intensity of resonance peaks corresponding to the PDEA block decreased at an elevated temperature of 60 °C, which implies poor solvation and reduced mobility of the PDEA block.Considering its chemical structure, the block copolymer should form a core-shell-type polymer micelle with dehydrated PDEA blocks forming a core and hydrophilic PMPC blocks forming a shell.Figure 5 shows the peak intensity ratio for the methine proton at 2.6 ppm in the main chain of the PDEA block and the methylene protons of the pendant phosphorylcholine group of the PMPC block at 3.6 ppm as a function of temperature.The peak intensity ratio is normalized with the ratio at 25 °C.When the temperature is increased from 24 °C, the normalized intensity ratios for PMPC 64 -PDEA 35 and PMPC 64 -PDEA 22 were almost constant, independent of the temperature.The normalized peak intensity ratios for PMPC 64 -PDEA 66 and PMPC 64 -PDEA 46 were practically constant below 37 and 47 °C, respectively.The intensity ratios for PMPC 64 -PDEA 66 and PMPC 64 -PDEA 46 decreased with increasing temperature, reaching minimum values of 0.4 and 0.6, respectively.These findings indicate that the motion of the methine protons in the PDEA blocks for PMPC 64 -PDEA 66 and PMPC 64 -PDEA 46 was restricted above a certain temperature.These results suggest that the onset of micellization occurs at lower temperatures for polymers with longer DEA block lengths.In 0.1 M NaCl aqueous solution, the diblock copolymer undergoes a transition from a molecularly dissolved unimer state at low temperatures to a micellar state above the critical association temperature (CAT).Figure 6 shows values of T% monitored at 600 nm for an aqueous solution of the diblock copolymers at C p = 5.0 g/L.The T% value for PMPC 64 -PDEA 66 is 100% below 38 °C and the solution shows no Tyndall scattering.T% decreases with increasing temperature, reaching 30% above 50 °C.Tyndall scattering at 50 °C, which is characteristic of micellar solutions, was visually confirmed.The solution, which was turbid above the CAT, became clear again when the solution was cooled below the CAT.The CAT values for the diblock copolymers were estimated from a break in the T% versus temperature plot.The CAT values for PMPC 64 -PDEA 66 , PMPC 64 -PDEA 46 , PMPC 64 -PDEA 35 , and PMPC 64 -PDEA 22 were estimated at 38, 43, 44, and 54 °C, respectively.The CAT values for PMPC m -PDEA n increased as the thermo-responsive PDEA block length decreased.These observations are consistent with the aforementioned 1 H NMR data (Figure 5).Heat-induced association behavior for the diblock copolymers was confirmed by DLS measurements.The hydrodynamic radius (R h ) for the diblock copolymer was measured in 0.1 M NaCl aqueous solution.Figure 7 shows DLS relaxation time distributions for PMPC 64 -PDEA 66 at C p = 5.0 g/L at 25, 42, and 60 °C.The distributions were unimodal at 25 and 60 °C with different relaxation times.A faster relaxation mode at 25 °C was attributed to a unimer state with R h = 3.2 nm, whereas the slower relaxation time at 60 °C was attributed to polymer aggregates with R h = 95 nm.At 42 °C, the relaxation time distribution was found to be bimodal.The fast and slow modes were attributed to unimers and polymer aggregates.The R h values for the diblock copolymers, determined by DLS measurement, are plotted as a function of temperature in Figure 8a.The R h values for the diblock copolymers below a certain temperature were in the order of ca. 5 nm, suggesting that all of the polymers existed in a unimer state.Upon an increase in temperature, the R h values for PMPC 64 -PDEA 66 , PMPC 64 -PDEA 46 , PMPC 64 -PDEA 35 , and PMPC 64 -PDEA 22 began to increase at 42, 44, 50, and 57 °C.These observations indicate the formation of polymer micelles above certain temperatures.As temperature was further increased, the R h values for PMPC 64 -PDEA 66 , PMPC 64 -PDEA 46 , and PMPC 64 -PDEA 35 started to decrease.These observations suggest that aggregates of the PDEA blocks became more compact due to further dehydration of the PDEA blocks as the temperature was increased beyond the CAT, or the aggregation number (N agg ) of the multipolymer aggregate decreased with increasing temperature.The scattering intensity is proportional to M w , because the intensity is linearly related to R θ /C p (i.e., R θ /C p  M w ).Therefore, the observation that the scattering intensities are nearly constant above CAT (Figure 8b) suggests that N agg is practically constant.The absolute scattering intensity is not important to obtain DLS data, however for SLS measurements the absolute scattering intensity is important to determine correct R θ .The accurate SLS data for the polymer micelle solutions at high temperature cannot be obtained, because there is multiple scattering from the turbid solution at 60 °C (Figure 6).Excitation spectra of pyrene in 0.1 M NaCl aqueous solution in the presence of the block copolymers at varying temperatures were obtained.The excitation spectrum in the case of PMPC 64 -PDEA 66 showed peaks associated with the (0-0) band of pyrene at 335 nm below the CAT, and the peak shifted to 338 nm at 60 °C.It is known that the (0-0) band in pyrene excitation spectra in water shifts to longer wavelengths when pyrene is solubilized in hydrophobic domains [15].Thus, we estimated the ratio of the intensity at 338 nm relative to that at 335 nm (I 338 /I 335 ), and this is plotted in Figure 9 as a function of temperature.It is known that the dithiobenzoate group at the polymer chain end prepared via RAFT is responsible for pyrene fluorescence quenching [16].We measured only the relative intensity ratio of the excitation emission spectra.Therefore, the quenching effect can be ignored to some degree.The I 338 /I 335 values for PMPC 64 -PDEA 35 and PMPC 64 -PDEA 22 were almost constant independent of the temperature.This observation indicates that the hydrophobic domain formed from the PDEA blocks could not incorporate the pyrene probes, presumably because of the low hydrophobicity of the core.As temperature was increased, the I 338 /I 335 value for PMPC 64 -PDEA 66 and PMPC 64 -PDEA 46 began to increase at 34 and 44 °C.When the temperature was increased from 25 to 60 °C and subsequently decreased to 25 °C, the thermo-responsive emission spectral changes were found to be completely reversible without hysteresis.

Table 1. Characteristics of poly(2-(methacryloyloxy
The CAT values for the block copolymers were measured using various methods, including 1 H NMR, T%, R h , scattering intensity, and pyrene fluorescence.These CAT values did not always coincide, depending on the measurement method.For example, the CATs for PMPC 64 -PDEA 66 estimated from 1 H NMR, R h , and fluorescence methods were 37, 42, and 36 °C, respectively, presumably due to the difference in the sensitivity of the measurement methods.The capture and release of small hydrophobic guest drugs by PMPC m -PDEA n in 0.1 M NaCl aqueous solution may be easily controlled by changing the temperature.We studied the thermo-responsive release behavior of PMPC 64 -PDEA 66 , applying a dialysis method, with ADR as a guest drug [17].Above the CAT, PMPC 64 -PDEA 66 formed polymer micelles with a hydrophobic core, sufficient to keep ADR captured in the interior.Below the CAT, however, the polymer micelles dissociated, and ADR was released from the core into the aqueous bulk phase.Figure 10 represents the time course of the cumulative permeation of ADR through the dialysis membrane at 25 and 50 °C.For comparison, data without PMPC 64 -PDEA 66 at 25 and 50 °C are presented along with control data.At 25 °C, the rate of permeation of ADR through the membrane was almost same in the presence and absence of PMPC 64 -PDEA 66 ; however, the permeation rate of ADR without PMPC 64 -PDEA 66 at 50 °C was much faster than that at 25 °C.At 50 °C, the permeation rate of ADR from the polymer micelle was slow compared to the control experiment without the polymer.From these observations, it was concluded that the PMPC 64 -PDEA 66 polymer micelle can retain captured ADR in its core at 50 °C.It is noteworthy that the release rate from the PMPC 64 -PDEA 66 polymer micelle at 50 °C was slightly higher than that at 25 °C.This observation suggests that the polymer micelle cannot completely incorporate ADR into its core at 50 °C, because the diffusion coefficient of ADR at 50 °C is much higher than at 25 °C.To create a temperature-responsive micelle containing such a molecule without leaking the hydrophobic medication at temperatures higher than the CAT, it is necessary to improve the molecular design of the diblock copolymer-for example, by extending the hydrophobic block length.

Conclusions
Thermo-responsive diblock copolymers were prepared via RAFT controlled/living radical polymerization.Diblock copolymers, composed of hydrophilic biocompatible PMPC blocks of the same DP (= 64) and thermo-responsive PDEA blocks with different DPs (= 22, 35, 46, and 66), were synthesized.These diblock copolymers formed polymer micelles composed of a hydrophobic PDEA core and hydrophilic PMPC shells in water above the CAT.The CAT values decreased as the DP of the PDEA block increased.PMPC 64 -PDEA 66 and PMPC 64 -PDEA 46 can incorporate hydrophobic guest molecules such as pyrene into the hydrophobic PDEA core above the CAT.Guest molecules such as ADR can also be incorporated into the hydrophobic PDEA core above the CAT, which allows controlled release from the core to the bulk aqueous phase below the CAT.However, leakage of ADR from the hydrophobic core above the CTA cannot be entirely prevented.Therefore, to avoid the leakage of guest molecules from the core above the CAT, it is necessary to improve the system by increasing the chain length of PDEA block.

Figure 2 .
Figure 2. Time-conversion (○) and the first-order kinetic plots (△) for polymerization of DEA in the presence of PMPC 64 (M n (NMR) = 1.89 × 10 4 , M w /M n = 1.19) in methanol at 70 °C.[M] 0 and [M] are the concentrations of the monomer at polymerization time = 0 and the corresponding time, respectively.

Figure 3 .
Figure 3.Typical example of a Zimm plot for PMPC 64 -PDEA 46 in 0.1 M NaCl at 25 °C.The polymer concentration was varied from 5 to 10 g/L.The Rayleigh ratio (R θ ) is determined by subtracting the solvent scattering from the total scattering for the solutions.

Figure 4 .
Figure 4. Comparison of 1 H NMR spectra for PMPC 64 -PDEA 66 at 25 °C (a) and 60 °C (b) in D 2 O containing 0.1 M NaCl at C p = 5.0 g/L.Asterisks represent peaks due to solvent.