One-pot synthesis of PEG-poly(amino acid) block copolymers

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Characterization, Synthesis, and Modifications

One-pot synthesis of PEG-poly(amino acid) block copolymers assembling polymeric micelles with PEG-detachable functionality Takuya Miyazaki, Kazunori Igarashi, Yu Matsumoto, and Horacio Cabral ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01549 • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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ACS Biomaterials Science & Engineering

One-pot synthesis of PEG-poly(amino acid) block copolymers assembling polymeric micelles with PEG-detachable functionality Takuya Miyazaki1, Kazunori Igarashi2, Yu Matsumoto2, Horacio Cabral1* 1. Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 73-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 2. Department of Otorhinolaryngology and Head and Neck Surgery, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

*Corresponding author: Horacio Cabral, PhD [email protected]

Abstract Block copolymers comprised of poly(ethylene glycol) (PEG) and poly(amino acids) (PAA) segments have shown exceptional features for developing biocompatible nanostructures.

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While the conventional methods for synthesizing PEG-PAA provide excellent control of the degree of polymerization and polydispersity, these protocols involve several steps, which increase the time and costs, and reduce the number of possible block copolymer designs. In this study, we developed a one-pot synthetic method for PEG-PAA block copolymers by doing sequential ring-opening polymerizations (ROP) of ethylene oxide (EO) and the Ncarboxyanhydrides (NCAs) of amino acids promoted by the organic base 1,1,3,3tetramethylguanidine (TMG) as the catalyst. The procedure was effectively used to synthesize PEG-poly(benzyl-L-glutamate) (PEG-PBLG) and PEG-poly(L-Lysine) (PEG-PLL) with narrow molecular weight distribution, comparable to that of block copolymers synthesized by the conventional method initiating the ROP of NCA by amine-terminated PEG. The resulting block copolymers present an ester bond between the PEG and the PAA segments, which can be gradually hydrolyzed in physiological conditions, being advantageous for improving the biocompatibility. Besides, we confirmed that the one-pot PEG-PBLG self-assembled into micelles in aqueous conditions, which showed comparable blood circulation and biodistribution to the micelles prepared from conventional PEG-PBLG. These results indicate the high potential of our one-pot synthetic approach for preparing hydrolysable PEG-PAAs for constructing supramolecular assemblies.

Keywords Block copolymers, Organo-bases, One-pot synthesis, Polymeric micelles, Ring-opening polymerization


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1. Introduction Block copolymers are playing a central role in the progress of both basic and applied fields of chemistry, physics, materials, biology and medicine [1,2]. For biological and medical applications, block copolymers comprising poly(ethylene glycol) (PEG) and poly(amino acids) (PAA) segments have shown outstanding properties as building units of biocompatible supramolecular structures, such as polymeric micelles and vesicles, incorporating a myriad of bioactive agents [3-7]. For example, PEG-poly(L-glutamic acid) block copolymer is used for preparing platinum drug-loaded polymeric micelles, which are being evaluated in the clinic against solid tumors [8], and PEG-poly(L-lysine) is successfully employed for assembling polymeric micelles incorporating nucleic acids, such as plasmid DNA (pDNA) or small interfering RNA (siRNA), via polyion complex formation [9,10]. The usual procedure for synthesizing the PEG-PAA block copolymers involves several steps, including the ring-opening polymerization (ROP) of ethylene oxide (EO) to prepare the PEG segment, the amination of the ω-end of the PEG, and the ROP of the N-carboxyanhydride (NCA) of the particular amino acid by using the PEG as the macroinitiator [11,12]. Because of this laborious process, a handful of commercially available PEG blocks has been exploited for skipping the initial stages of the PEGPAA synthesis, though such approach increases the cost and limits the possibilities of the block copolymer design. On the other hand, the development of one-pot synthetic strategies for PEGPAA block copolymers will heighten the efficiency of the reaction, while minimizing time, costs and environmental burden. To synthesize PEG-PAAs in one-pot, here we propose a method starting from the controlled polymerization of EO followed by the initiation of the ROP of the NCA via the alcohol group of the fresh PEG. We hypothesized that we could conduct these sequential


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polymerizations under relatively mild conditions with neutral organo-bases, which are known to activate both primary alcohols and heterocyclic monomers, including epoxides and NCAs [13,14]. Among neutral organo-bases, 1,1,3,3-tetramethylguanidine (TMG) with relatively high pKa has been recently used to activate primary alcohols by hydrogen bonding interactions to initiate N-buthylglycine-NCA [15]. However, the utility of TMG for promoting the polymerization of epoxy groups, as well as other types of NCAs, such as amino acids-NCAs, has not been explored so far. Thus, in this study, we developed a one-pot synthetic approach for PEG-PAAs by using TMG as the catalyst to start the ROP of EO, followed by the addition βbenzyl-L-glutamate-NCA (BLG-NCA) or ε-trifluoroaceticacid-L-lysine-NCA (Lys-TFA-NCA), and the subsequent ROP of the PEG-PAA block copolymers (Scheme 1). Compared to the conventional NCA polymerization initiated by amine-terminated PEG (PEG-NH2), where the PAA chain is attached to PEG segments via stable amide bonds, this reaction scheme results in the formation of PEG-PAAs bearing a hydrolysable ester bond between the PEG and PAA blocks, which could be further exploited for the gradual detachment of the PEG segment, allowing to reduce the prolonged accumulation in the living body of polymers and supramolecular structures. In this direction, the ability of the synthesized PEG-PAAs to hydrolyze in physiological conditions, as well as to self-assemble into supramolecular nanostructures, i.e. polymeric micelles, for in vivo application was further explored. Our results demonstrated the feasibility of our one-pot synthetic approach for preparing hydrolysable PEGPAAs capable of assembling into biocompatible supramolecular materials.

2. Materials and methods


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2.1. Materials EO was purchased from 3M Japan Co., Ltd. (Tokyo, Japan). 2-methoxy-ethanol (purity >99.0%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (purity >98.0%) and TMG (purity >99.0%) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), EO, 2-methoxy-ethanol and TMG were purified by distillation over CaH2. Lithium chloride (purity >99.0%) was bought from Nakalai Tesque Co., Inc. (Kyoto, Japan). Dehydrated N, Ndimethylformamide (DMF) and dehydrated tetrahydrofuran (THF) were purchased from Kanto Chemical, Co., Inc., (Tokyo, Japan). Disodium hydrogenphosphate (purity >99.0%), naphthalene (purity >98.0%) and sodium dihydrogenphosphate (purity >99.0%) were purchased from FUJIFILM Wako Pure Chemical Co. (Tokyo, Japan). Methanol (purity >99.5%), thiourea (purity >99.0%), potassium (purity >99.5%) and N-hydroxysuccinimide (purity >98.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Diethyl ether (purity >99.5%) was purchased from Showa ether (Tokyo, Japan). γ-benzyl-L-glutamate N-carboxyanhydride (BLGNCA) and ε-trifluoroacetyl-L-lysine N-carboxyanhydride (Lys-(TFA)-NCA) were purchased from Chuo Kaseihin Co. Inc. (Tokyo, Japan) and used as received. CF647 succinimidyl ester was purchased from Biotium (Hayward, CA, USA).

2.2. Measurements Gel permeation chromatography (GPC) measurements were performed by using a Tosoh HLC-8220. The GPC system was equipped with a TSKgel G3000HHR column (linear, 7.8 mm × 300 mm; pore size, 7.5 nm; bead size, 5 µm; exclusion limit, 6 × 104 g/mol) or a TSKgel G4000HHR column (linear, 7.8 mm × 300 mm; pore size, 20 nm; bead size, 5 µm; exclusion


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limit, 4 × 105 g/mol), a TSKgel guard column HHR-L and an internal refractive index (RI) detector. DMF containing was used as the eluent at a flow rate of 0.5 mL/min at 40 °C. A molecular weight calibration was done using a series of standard PEGs (Polymer Laboratories, Ltd., U.K.). The 1H-NMR spectra were measured by using a JEOL EX400 spectrometer (JEOL, Tokyo, Japan) at 400 MHz.

2.3. One-pot synthesis of α-methoxy-poly(ethylene glycol)-block-poly(γ-benzyl-L-glutamate) copolymer (PEG-OCO-PBLG) TMG (1.0 mmol, 130 µL) was added to 2-methoxyethanol (0.10 mmol, 7.9 µL) in 60 mL of THF. After 10 min stirring, liquid EO chilled below 0 °C (27 mmol, 1.4 mL) was added to the solution via a cooled syringe. The polymerization was monitored by 1H-NMR. The kinetics of polymerization of EO activated by TMG was compared to that of the ROP activated by potassium naphthalene (0.13 mmol, 270 µL), which was prepared as previously reported [13]. The mixture was allowed to react for 2 days at room temperature and dried under the reduce pressure to remove the unreacted monomers and THF. Then, TMG (1.0 mmol, 130 µL) and BLG-NCA (5.0 mmol, 130 mg) in 2 mL of DMF with 1 M thiourea were added to the polymer solution. The polymerization was monitored by 1H-NMR. After the reaction for 3 days at 35 °C, the reaction solution was added to the excess amount of diethyl ether (1.0 L) to precipitate the polymer. The recovered polymer was dried in vacuo to obtain PEG-PBLG. The yield of obtained polymer after purification was 86%, which is comparable to the yield of the PEG-PBLG synthesized by the conventional method. In the case of the synthesis with conventional method, PEG-NH2 (0.1 mmol, 1.2 g) with a 12,000 Mn in 18 mL of DMF was used as a macroinitiator to


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prepare the control polymer (PEG-NHCO-PBLG) having an amide bond between the PEG and PBLG blocks [17].

2.4. One-pot synthesis of α-methoxy-poly(ethylene glycol)-block-poly(L-Lysine-TFA) copolymer (PEG-OCO-PLL(TFA)) TMG (1.0 mmol, 130 µL) was added to 2-methoxyethanol (0.10 mmol, 7.9 µL) in 60 mL of THF. After 10 min stirring, liquid EO chilled below 0 °C (27 mmol, 1.4 mL) was added to the solution via a cooled syringe. The polymerization was monitored by 1H-NMR. The mixture was allowed to react for 2 days at room temperature and dried under the reduce pressure to remove the unreacted monomers and THF. Then, TMG (1.0 mmol, 130 µL) and Lys-(TFA)-NCA (10 mmol, 2.7 g) in 41 mL of DMF with 1 M thiourea were added to the polymer solution. The polymerization was monitored by 1H-NMR. After the reaction for 3 days at 35 °C, the reaction solution was added to the excess amount of diethyl ether (1.0 L) to precipitate the polymer. The recovered polymer was dried in vacuo to obtain PEG-PLL(TFA). The yield of obtained polymer after purification was 81%, which is comparable to the yield of the PEG-PLL(TFA) synthesized by the conventional method. The degree of polymerization and molecular weight distribution of the PEG-PLL(TFA) were determined by 1H-NMR and GPC, respectively. In the case of synthesis with conventional method, PEG-NH2 (0.1 mmol, 1.2 g) with a 12,000 Mn in 18 mL of DMF was used as a macroinitiator to prepare the control polymer (PEG-NHCO-PLL(TFA)) having an amide bond between the PEG and PLL(TFA) blocks [18].

2.5. Detachment of the PEG segment from the block copolymer at physiological conditions


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PEG-OCO-PBLG (0.43 µmol, 10 mg) in 5 mL of DMF was added to 5 mL of 10 mM phosphate buffer (pH 7.4) and the solution was allowed to stir at 37 °C. After different time points, the solution was added to excess amount of water (100 mL) to remove the PEG-OCOPBLG and the detached Homo-PBLG. The supernatant was collected, and lyophilized to obtain the detached PEG. Then, the amount of PEG was determined by GPC measurement through the RI detector by using a standard curve prepared with different concentrations of PEG. As control, we used the PEG-PBLG prepared by using PEG-NH2 as the initiator (PEG-NHCO-PBLG), which has a stable amide bond between the PEG and PBLG blocks.

2.6. Preparation of PEG-PBLG micelles and stability in phosphate buffer PEG-OCO-PBLG or PEG-NHCO-PBLG (0.86 µmol, 20 mg) were dissolved in 20 mL of methanol, and the solution was evaporated to prepare a thin film on the bottom of a glass flask. After the addition of 20 mL of 10 mM phosphate buffer (pH 7.4), the solution was sonicated for 30 min. The hydrodynamic diameter and polydispersity index (PDI) of micelles were measured by dynamic light scattering (DLS) using a Zetasizer Nanoseries instrument (Malvern Instruments Ltd., UK). The measurement was performed at a detection angle of 173° and a temperature of 25 °C. The stability of the micelles was determined by DLS after incubating the micelles at 4 °C for 1 week.

2.7. Blood circulation profile and biodistribution of PEG-PBLG micelles


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The micelles were fluorescent-labeled for tracing their position in living tissues by using intravital confocal laser scanning microscopy (IV-CLSM). For fluorescently labeling of PEGPBLG prepared by one-pot or conventional methods, CF647 succinimidyl ester (1.0 µmol), 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.19 mg, 1.0 µmol) and Nhydroxysuccinimide (0.12 mg, 1.0 µmol) were added to PEG-PBLG (20 mg, 1.0 µmol) and the solution was allowed to react for 24 h. Then, the reaction solution was added to the excess amount of diethyl ether (300 mL) and was dried in vacuo to obtain PEG-PBLG-CF647. After preparing micelles with PEG-OCO-PBLG-CF647 or PEG-NHCO-PBLG-CF647, the solution (200 µL) was administrated in mice by intravenous injection, and the fluorescence intensities in the vein and the skin tissue were measured by IV-CLSM (Nikon, Japan) [19]. After 24 h circulation, the mice were sacrificed and the lungs, kidney, spleen, liver, muscle and heart were collects. The microdistribution of the micelles and the fluorescence intensity in these tissues was assessed ex vivo by IV-CLSM.

3. Results and Discussion 3.1. One-pot synthesis of PEG-PAAs We began the one-pot synthesis by constructing the PEG segment initiated with a primary alcohol, 2-methoxyethanol, which does not have steric hindrance and electronwithdrawing substituents (Scheme 1). Both 2-methoxyethanol and TMG were dissolved in THF, and the EO monomer was added at 272 equivalents to 2-methoxyethanol for synthesizing PEGOH homopolymers with molecular weight (Mn) of 12 kDa. The reaction was allowed to proceed for 48 h. The resulting PEG-OH homopolymer was characterized by 1H-NMR after precipitation


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in ether, showing a peak at 3.5 ppm, which corresponds to the PEG (Figure 1a). Moreover, the Mn of the synthesized PEG was determined to be 11.5 kDa by GPC, which was close to the 12 kDa Mn target (Figure 1b). Besides, the PEG showed a narrow molecular weight distribution, indicating the uniform activation of 2-methoxyethanol by TMG. Thus, these results denoted the ability of TMG for effectively promoting the ROP of EO. After polymerizing the PEG, we proceeded to polymerize the PAA block by using the PEG-OH as the initiator. First, the unreacted EO monomers and the THF were removed from the reaction mixture by drying under the reduced pressure. Then, TMG was added again for catalyzing the ROP of the NCAs of the amino acids. For preparing PEG-OCO-PBLG, BLGNCA was added at 50 equivalents to PEG-OH, corresponding to block copolymers having 50 units of PBLG, and the reaction was allowed to proceed for 48 h. By 1H-NMR analysis, we determined the degree of polymerization by comparing the protons in the side chain ((CH2)6 and CH2-CH2-) and the backbone (-CH-) of PBLG with the protons in the PEG backbone (-CH2CH2-O-). Thus, the PBLG segment in the block copolymers was confirmed to be 40 units (Figure 1c). Moreover, the GPC results showed that the molecular weight of the PEG-OCOPBLG prepared by the one-pot approach was narrowly distributed, with a molecular weight distribution (Mw/Mn) of 1.03 (Figure 1d), indicating that the alcohol group in PEG was effectively activated by TMG to react with BLG-NCA. In addition, the distribution of PEGOCO-PBLG was comparable to that of PEG-NHCO-PBLG prepared by the conventional method. The total time for the one-pot synthesis of PEG-OCO-PBLG was only 5 days, which is significantly shorter than the typical multistep method usually lasting 1 month. To further check the utility of TMG in the synthesis of PEG-PAAs, we studied the polymerization of Lys-TFA-NCA in the presence of TMG for preparing PEG-OCO-PLL(TFA)


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(Scheme 1). Thus, after polymerizing the PEG by using 2-methoxyethanol and TMG, Lys-TFANCA was added at 100 equivalents to PEG-OH, aiming to produce PEG-PLL(TFA) block copolymers having 80 units of PLL(TFA), as the conversion of Lys-TFA-NCA for this degree of polymerization of PLL(TFA) is approximately 80%. The reaction was then allowed to proceed for 48 h. The degree of polymerization of PLL(TFA) segments in the polymers were determined to be 81 by 1H-NMR after comparing the protons in the side chain of PLL(TFA) (CH2-CH2CH2-) and PLL(TFA) backbone (-CH-) with the protons in the PEG backbone (-CH2-CH2-O-) (Figure 1e). A narrow molecular weight distribution for the PEG-OCO-PLL(TFA) (Mw/Mn = 1.03) was confirmed by GPC (Figure 1f), which was comparable to that of PEG-NHCOPLL(TFA) prepared by the conventional method. These observations indicate that the one-pot approach can be effectively extended to other NCAs, supporting the versatility of our one-pot synthesis approach for preparing different PEG-PAAs. It is important to note that, since the onepot block polymers present an ester bond between the PEG and PAA chains, the PAA deprotection protocol should be carefully designed. Thus, the usual acid- or alkali-based deprotection protocols may not be feasible for deprotecting PBLG and PLL(TFA) to obtain PEG-poly(L-glutamic acid) and PEG-poly(L-lysine), respectively, as previously reported [14, 15]. On the other hand, alternative deprotection methods, such as hydrogen reduction for PBLG or piperidine for PLL(TFA)), which are milder to ester bonds, could be employed to avoid the cleavage of the block copolymers [20]. In addition, the NCAs of the amino acids could be protected with other groups having orthogonal chemistry with ester bonds, such as Fmoc, thereby, preserving the ester linkage between the segments.

3.2. Kinetics of polymerization


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Because the kinetics of ROPs is related to the initiation and propagation state of polymers, the kinetics of the ROPs of EO was examined by using 2-methoxyethanol with TMG, or with potassium naphthalene, which is a traditional catalyst of the ROPs of EO [11,21], to clarify the effect of the catalyst on the kinetics of polymerization. At different time points, the conversion ratio of EO to the feeding amount was monitored by 1H-NMR. The ROPs of EO with TMG (kobs = 7.1 h-1) was slower than that of EO with potassium naphthalene (kobs = 9.5 h-1) until the conversion reached to the plateau, where the reaction was supposed to be terminated (Figure 2a). As potassium naphthalene is a stronger base than TMG regarding the formation of alkoxide during the initiation and the propagation state [22], TMG showed slower kinetics than potassium naphthalene probably because the alcohol initiators were mildy polarized by TMG to attack EO. These results indicate the potential of TMG as a mild catalyst to polymerize epoxides with reactive functional groups with no unfavorable side reactions. Next, the kinetics of the NCA polymerization was examined by using the PEG-OH macroinitiator with TMG or amine initiators to clarify the effect of the initiation state on the kinetics of polymerization. At different time points, the conversion ratio of BLG-NCA to the feeding amount was monitored by 1H-NMR. The kinetics of the ROPs of BLG-NCA with alcohol initiators and TMG to prepare PEG-OCO-PBLG was faster (kobs = 8.6 h-1) than that of BLG-NCA with amine initiators for preparing PEG-NHCO-PBLG (kobs = 6.2 h-1) (Figure 2b). Thus, the rate of reaction with the alcohol and TMG was faster than that of reaction with the amine, which may be related to the accelerated donation of the lone pair of electrons from alcohol to NCA by TMG via hydrogen bonding interactions, which was suggested by previous reports [15], or the promoted deprotonation of the primary amine in the ω-end of PBLG segments during the propagation state.


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To investigate the mechanism of the accelerated ROPs with TMG, we conducted the NCA polymerization using PEG-NH2 macroinitiator in the presence of TMG (Figure 2b; PEGNHCO-PBLG+TMG). At different time points, the conversion ratio of BLG-NCA to the feeding amount was monitored by 1H-NMR. The ROPs of BLG-NCA with TMG (kobs = 7.7 h-1) was faster than that of BLG-NCA with no TMG (kobs = 6.2 h-1) until the conversion reached a plateau, where the reaction was assumed to be terminated (Figure 2b), indicating that TMG affected the propagation state of PBLG chains with TMG, probably by deprotonating the primary amine in the ω-end of PBLG segments to accelerate the donation of the lone pair of electrons to the carbonyl carbon, which may also be polarized by TMG. These data support the acceleration of the reaction rate of TMG by increasing the reactivity of not only alcohol initiators, but also primary amine in the end of PBLG segments and carbonyl groups in NCA.

3.3. Hydrolytic detachment of PEG from PEG-PBLG Because the synthesized PEG-PAAs present an ester bond between the PEG and PAA blocks, which could be gradually hydrolyzed in physiological conditions, we studied the rate of the PEG detachment from the PEG-PBLG by incubating the polymer in 10 mM phosphate buffer at pH 7.4. The cleavage of the block copolymer was analyzed by using GPC at different time points. Thus, initially the chromatogram only showed the peak corresponding to PEG-OCOPBLG. With increasing incubation time, the formation of new peaks corresponding to PEG and PBLG chains became evident in the GPC chromatogram (Figure 3). These results indicate that the ester bond between the PEG and PBLG blocks was gradually hydrolyzed in physiological conditions into the forming homopolymers. On the other hand, the PEG-NHCO-PBLG, which


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has an amide bond between the PEG and PBLG segments, did not release the PEG even after 96 h incubation in 10 mM phosphate buffer at pH 7.4 (Figure 3). The possibility to remove the PEG block from the PAA segment could be a substantial benefit for reducing the long-term accumulation of the block copolymers in living tissues, by promoting the renal clearance of the relatively small PEG component [23]. In biological environments, the cleavage of ester bonds can be accelerated by enzymes, such as esterases, further improving the PEG detachment from our PEG-PAA block copolymers. It is also important to note that while a myriad of PEGylated materials have shown enhanced bioavailability and reduced immunogenicity in the clinic, the PEG component has also been indicated to trigger an unexpected immunogenic response known as the accelerated blood clearance (ABC) phenomenon, which is a prompt increased in the clearance of the PEGylated substances after repeated administrations [24]. Thus, cleavable PEGylation has been considered as a promising alternative for alleviating the induction of the ABC phenomenon. Previous studies have shown that liposomes modified with PEG-lipid derivatives with cleavable bonds, including ester bonds, could avoid the ABC phenomenon and efficiently prolong the circulation time of liposomes after repeated injections [25, 26]. These observations suggest that our PEG-PAAs with hydrolysable ester bonds could provide an effective way for reducing the incidence of the ABC phenomenon upon repeated injection. PEGylation also produces the so-called “PEG dilemma”, which is the reduced cellular uptake by the target cells and the limited ability to escape from endosomes due to the PEG shielding on the materials [27]. Thus, the development of PEGylated materials with cleavable PEG components is an effective strategy for overcoming the PEG dilemma [27]. The gradual PEG detachment from our PEG-PAAs by ester hydrolysis could be advantageous for developing strategies aimed to surmount this issue.


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3.4. Self-assembly of polymeric micelles from PEG-PBLG The utility of our block copolymers to self-assemble into supramolecular nanostructures was evaluated by using PEG-PBLG, which is amphiphilic and can form core-shell polymeric micelles in aqueous solution [28]. The preparation of the PEG-PBLG micelles was done by the thin-film hydration method by first dissolving the block copolymer in methanol, followed by the evaporation of the methanol to form a thin layer and the subsequent addition of 10 mM phosphate buffer at pH 7.4. The resulting micelle solution was then sonicated to narrow the size distribution. The size and the polydispersity index (PDI) were measured by dynamic light scattering (DLS). The micelles obtained from PEG-OCO-PBLG showed sharp size distribution, a diameter of 83 nm (polydispersity index: 0.1) (Figure 4a). The features of these micelles were comparable to those of the micelles prepared from the PEG-NHCO-PBLG synthesized from previous method (Figure 4b), suggesting that the ester bond in the one-pot PEG-OCO-PBLG did not affect the self-assembly. Since the hydrolysis of the ester bond will gradually detach the PEG from the PEG-OCO-PBLG, the PEG shell of the micelles may be compromised, resulting in aggregation. Thus, we studied the stability of the micelles after 1-week incubation in 10 mM phosphate buffer at pH 7.4. The results showed that both the micelles prepared from the PEGOCO-PBLG and the micelles made from the conventional PEG-NHCO-PBLG showed comparable size distribution (Figure 4c). Moreover, the size distribution of the micelles was comparable to that at the starting point, indicating that the micelles assembled from the PEGOCO-PBLG synthesized by one-pot reaction can preserve their size even after 1-week. These results indicate that the micelles can maintain their PEG shielding capacity in buffer conditions.


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The unique advantages of PEG-PAAs for constructing polymeric micelles as drug carriers with controlled physicochemical and biological properties have been demonstrated in both preclinical and clinical studies [3, 8]. Of interest, polymeric micelles based on PEG-PBLG block copolymers were able to load hydrophobic antitumor agents, i.e. the proteasome inhibitor MG132, in their core and promote their antitumor efficacy against several cervical cancer models, while reducing the side effects of the loaded drugs [28]. The diameter of the MG132loaded micelles is comparable to that of the micelles reported in current study, suggesting the possibility of preparing biologically relevant formulations based on our one-pot synthetic strategy.

3.5. Blood circulation and tissue microdistribution The blood circulation and tissue microdistribution of the micelles were evaluated by using IV-CLSM to confirm that the one-pot synthesis scheme and the hydrolysable PEG segment did not affect their behavior in the bloodstream. Thus, fluorescent-labeled micelles were prepared by using both PEG-PBLGs having Cy5 conjugated at the amino group at the ω-end of the block copolymer. The fluorescent-labeled micelles showed comparable size distribution to their non-labeled counterparts. Then, the micelles were intravenously injected to mice, and their circulation in blood was followed by real-time IV-CLSM in the skin tissue of the mice. According to the time-lapse fluorescence intensity in the blood vessels, both the micelles prepared from the one-pot PEG-OCO-PBLG and the micelles prepared by the conventional PEG-NHCO-PBLG were able to stably circulate in blood in a comparable manner (Figure 5a and 5b), avoiding aggregation and interaction with cellular components. Moreover, both


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micelles remained in the blood compartment, showing minimal extravasation into the skin due to their relatively large size, as well as because of their sufficient stability, which prevented their disassembly into the forming block copolymers (Figure 5a and 5b). These observations are in good agreement with our previously reported blood circulation profiles and stability of drugloaded polymeric micelles [3, 29]. The microdistribution of the fluorescent-labeled micelles in different organs was studied by ex vivo fluorescence microscopy at 24 h after injection. Thus, the liver, kidney, spleen, lungs, muscle and heart were collected, stained with Hoechst for marking the nuclei of cells, and set under the microscope. The results showed that both micelles distributed to the organs in a comparable manner 24 h after injection (Figure 5c). Thus, these results support the potential of the one-pot-synthesized block copolymers for constructing effective nanocarriers.

4. Conclusion We have developed a method to polymerize PEG-PAA block copolymers in a one-pot manner by taking advantage of the catalyst TMG, which activated the ROP of both EO and NCAs. The obtained PEG and PAA segments showed narrow molecular weight distribution and controlled degree of polymerization. Moreover, the ester bond between PEG and PAA chains can be hydrolyzed in buffer mimicking physiological conditions, while it did not affect the selfassembly of micelles in aqueous solution nor the in vivo behavior after intravenous injection in mice. These capabilities indicate the potential of our PEG-PAAs for the development of functional nanostructures with increased biocompatibility for a wide range of biomedical applications. From an industrial perspective, our synthetic approach is a convenient way for


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reducing the time and costs to synthesize PEG-PAA block copolymers with good manufacturing practice grade, which is key for developing biomedical materials.

Acknowledgements This research was supported by Grants-in-Aid for Scientific Research B (JP16H03179; H.C.) from the Japan Society for the Promotion of Science (JSPS) and the Project for Cancer Research And Therapeutic Evolution (P-CREATE) (JP17cm0106202) from Japan Agency for Medical Research and Development (AMED).

Conflict of Interest Disclosure The authors declare no conflict of interest.

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[11] Harris, J. M.; Struch, E. C.; Case, M. G.; Paley, M. S. Synthesis and Characterization of Poly(ethylene glycol) Derivatives. J. Polym. Sci. 1984, 22, 341-352. [12] Deng, C.; Wu, J.; Cheng, R.; Meng, F.; Klok, H.A.; Zhong, Z. Functional Polypeptide and Hybrid materials: Precision Synthesis via α -amino acid N-carboxyanhydride Polymerization and Emerging Biomedical Applications. Prog. Polym. Sci. 2014, 39, 330-364.. [13] Xia, Y.; Zhao, J. Macromolecular Architectures based on Organocatalytic Ring-Opening (co)Polymerization of Epoxides. Polymer 2018, 143, 343-361. [14] Kricheldorf, H. R. α-Aminoacid-N-Carboxyanhydrides and Related Heterocycles; SpringerVerlag: Germany, 1987. [15] Chan, B. A.; Xuan, S.; Horton, M.; Zhang, D. 1,1,3,3-Tetramethylguanidine-Promoted Ring-Opening Polymerization of N-buthyl N-carboxyanhydride Using Alcohol Initiators. Macromolecules 2016, 49, 2002-2012. [16] Scott, N. D.; Walker, J. F.; Hansley, V. L. Sodium Naphthalene. A New Method for the Preparation of Addition Compounds of Alkali Metals and Polycyclic Aromatic Hydrocarbons. J. Am. Chem. Soc. 1936, 58, 2442-2444. [17] Nishiyama, N.; Okazaki, S.; Cabral, H.; Miyamoto, M.; Kato, Y.; Sugiyama, Y.; Nishio, K.; Matsumura, Y.; Kataoka, K. Novel Cisplatin-Incorporated Polymeric Micelles Can Eradicate Solid Tumors in Mice. Cancer Res. 2003, 63, 8977-8983. [18] Kataoka, K.; Togawa, H.; Harada, A.; Yasugi, K.; Matsumoto, T.; Katayose, S. Spotaneous Formation of Polyion Complex Micelles with Narrow Distribution from Antisense


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[25] Xu, H.; Wang, K. Q.; Deng, Y. H.; Chen, D. W. Effects of Cleavable PEG-Cholesterol Derivatives on the Accelerated Blood Clearance of PEGylated Liposomes. Biomaterials 2010, 31, 4757-4763. [26] Chen, D.; Liu, W.; Shen, Y.; Mu, H.; Zhang, Y.; Liang, R.; Wang, A.; Sun K.; Fu, F. Effects of a Novel pH-responsive Liposome with Cleavable Esterase-Catalyzed and pH-Responsive Double Smart mPEG Lipid Derivative on ABC Phenomenon. Int. J. Nanomedicine 2011, 6, 2053-2061. [27] Hatakeyama, H.; Akita, H.; Harashima, H. The Polyethyleneglycol Dilemma: Advantage and Disadvantage of Pegylation of Liposomes for Systemic Genes and Nucleic Acids Delivery to Tumors. Biol. Pharm. Bull. 2013, 6, 892-899. [28] Matsumoto, Y.; Miyamoto, Y.; Cabral, H.; Matsumoto, Y.; Nagasaka, K.; Nakagawa, S.; Yano, T.; Maeda, D.; Oda, K.; Kawana, K; Nishiyama, N.; Kataoka, K.; Fujii, T. Enhanced Efficacy against Cervical Carcinomas through Polymeric Micelles Physically Incorporating the Proteasome Inhibitor MG132. Cancer Sci. 2016, 107, 773-781. [29] Quader, S.; Cabral, H.; Mochida, Y.; Ishii, T.; Liu, X.; Toh, K.; Kinoh, H.; Miura, Y.; Nishiyama, N.; Kataoka, K. Selective Intracellular Delivery of Proteasome Inhibitors through pH-Sensitive Polymeric Micelles directed to Efficient Antitumor Therapy. J. Control. Release 2014, 188, 67-77.


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Figures

Scheme 1. One-pot synthesis scheme of PEG-PAAs (PEG-OCO-PBLG and PEG-OCOPLL(TFA)).


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Figure 1. Characterization of block copolymers synthesized by one-pot approach. 1H-NMR spectra of PEG (a), PEG-OCO-PBLG (c) and PEG-OCO-PLL(TFA) (e), and GPC chromatogram of PEG (b), PEG-OCO-PBLG (d; full line) and PEG-NHCO-PBLG (d; dotted line), and PEG-OCO-PLL(TFA) (f; full line) and PEG-NHCO-PBLG (f; dotted line).


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Figure 2. (a) Kinetics of EO polymerization by using TMG (black circles) or potassium naphthalene (open circles). (b) Kinetics of NCA polymerization of BLG-NCA by the one-pot approach (black circles), the conventional approach initiated by PEG-NH2 (open circles; full line) and the conventional approach initiated by PEG-NH2 plus TMG (open circles; dotted line).


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Figure 3. PEG-detachment rate from PEG and PBLG segments determined in 10 mM phosphate buffer (pH 7.4) from PEG-OCO-PBLG (black circles; full line) and PEG-NHCO-PBLG (open circles; dotted line).


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Figure 4. (a) Size distribution of micelles prepared from PEG-OCO-PBLG (black circles; full line) and PEG-NHCO-PBLG (open circles; doted line). (b) Size distribution of micelles prepared from PEG-OCO-PBLG (black circles; full line) and PEG-NHCO-PBLG (open circles; doted line) 1-week incubation in 10 mM phosphate buffer (pH 7.4).


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Figure 5. Blood circulation and tissue microdistribution of PEG-PBLG micelles. Blood circulation profile of micelles prepared from fluorescent-labeled PEG-OCO-PBLG (a) and PEGNHCO-PBLG (b) determined by real-time IV-CLSM. From the microscopic image, regions of interest (ROI) were selected in the blood vessel (red trapezoids) and the skin (green trapezoids) for quantifying the time-lapse fluorescence intensity (a and b; left panel). The fluorescence intensity was analyzed as the relative fluorescence intensity relative to the maximum fluorescence intensity in the blood vessels (a and b; right panel). The microdistribution of the


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micelles in the tissues (liver, kidney, spleen, muscle and lung) was analyzed ex vivo by IVCLSM (c; upper panel). The nuclei of cells were stained by using Hoechst (blue). The fluorescence intensities in the tissues were analyzed as the relative fluorescence intensity relative to the maximum fluorescence intensity in the blood vessels (c; lower panel). Scale bars = 100 μm.


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For Table of Contents Use Only One-pot synthesis of PEG-poly(amino acid) block copolymers assembling polymeric micelles with PEG-detachable functionality Takuya Miyazaki, Kazunori Igarashi, Yu Matsumoto, Horacio Cabral


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Scheme 1. One-pot synthesis scheme of PEG-PAAs (PEG-OCO-PBLG and PEG-OCO-PLL(TFA)). 80x36mm (300 x 300 DPI)



Figure 1. Characterization of block copolymers synthesized by one-pot approach. 1H-NMR spectra of PEG (a), PEG-OCO-PBLG (c) and PEG-OCO-PLL(TFA) (e), and GPC chromatogram of PEG (b), PEG-OCO-PBLG (d; full line) and PEG-NHCO-PBLG (d; dotted line), and PEG-OCO-PLL(TFA) (f; full line) and PEG-NHCO-PBLG (f; dotted line). 80x119mm (300 x 300 DPI)


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Figure 2. (a) Kinetics of EO polymerization by using TMG (black circles) or potassium naphthalene (open circles). (b) Kinetics of NCA polymerization of BLG-NCA by the one-pot approach (black circles), the conventional approach initiated by PEG-NH2 (open circles; full line) and the conventional approach initiated by PEG-NH2 plus TMG (open circles; dotted line). 80x150mm (300 x 300 DPI)



Figure 3. PEG-detachment rate from PEG and PBLG segments determined in 10 mM phosphate buffer (pH 7.4) from PEG-OCO-PBLG (black circles; full line) and PEG-NHCO-PBLG (open circles; dotted line). 80x80mm (300 x 300 DPI)


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Figure 4. (a) Size distribution of micelles prepared from PEG-OCO-PBLG (black circles; full line) and PEGNHCO-PBLG (open circles; doted line). (b) Size distribution of micelles prepared from PEG-OCO-PBLG (black circles; full line) and PEG-NHCO-PBLG (open circles; doted line) 1-week incubation in 10 mM phosphate buffer (pH 7.4). 80x146mm (300 x 300 DPI)



Figure 5. Blood circulation and tissue microdistribution of PEG-PBLG micelles. Blood circulation profile of micelles prepared from fluorescent-labeled PEG-OCO-PBLG (a) and PEG-NHCO-PBLG (b) determined by realtime IV-CLSM. From the microscopic image, regions of interest (ROI) were selected in the blood vessel (red trapezoids) and the skin (green trapezoids) for quantifying the time-lapse fluorescence intensity (a and b; left panel). The fluorescence intensity was analyzed as the relative fluorescence intensity relative to the maximum fluorescence intensity in the blood vessels (a and b; right panel). The microdistribution of the micelles in the tissues (liver, kidney, spleen, muscle and lung) was analyzed ex vivo by IV-CLSM (c; upper panel). The nuclei of cells were stained by using Hoechst (blue). The fluorescence intensities in the tissues were analyzed as the relative fluorescence intensity relative to the maximum fluorescence intensity in the blood vessels (c; lower panel). Scale bars = 100 μm. 80x149mm (300 x 300 DPI)


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88x25mm (300 x 300 DPI)


One-pot synthesis of PEG-poly(amino acid) block copolymers

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