Stimuli-Responsive Zwitterionic Block Copolypeptides: Poly(N

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Biomacromolecules 2008, 9, 2670–2676

Stimuli-Responsive Zwitterionic Block Copolypeptides: Poly(N-isopropylacrylamide)-block-poly(lysine-co-glutamic acid) Jingguo Li, Tao Wang, Dalin Wu, Xiuqiang Zhang, Jiatao Yan, Song Du, Yifei Guo, Jintao Wang, and Afang Zhang* School of Materials Science and Engineering, Zhengzhou University, Daxue Beilu 75, Zhengzhou 450052, China Received April 13, 2008; Revised Manuscript Received July 23, 2008

Synthesis of novel zwitterionic block copolypeptides, poly(N-isopropylacrylamide)-block-poly(L-glutamic acidco-L-lysine) [PNiPAMn(PLGx-co-PLLysy)m, where n is the number-average degree of polymerization (DPn) of PNiPAM block, x and y are the mole fraction of glutamic acid and lysine residues, respectively, and m is the total DPn of the peptide block], and their stimuli-responsiveness to temperature and pH variation in aqueous solutions are described. Initiated with the amino-terminated poly(N-isopropylacrylamide) (PNiPAMn-NH2), ring-opening polymerization (ROP) of a mixture of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA), and Boc-L-lysine N-carboxyanhydride (BLLys-NCA) afforded the block copolypeptides PNiPAMn(PBLGx-co-PBLLysy)m, with a poly(N-isopropylacrylamide) block together with a random copolypeptide block, which was then deprotected with HBr/trifluoroacetic acid into the double hydrophilic block copolypeptides, PNiPAMn(PLGx-co-PLLysy)m. Their block ratios and lengths, as well as the amino acid residue ratios in the random copolypeptide block are varied (n ) 360, x ) 0.4-0.5, y ) 0.4-0.6, and m ) 220-252). The secondary structures of the copolypeptides in aqueous solution at different pH conditions were examined. Phase transitions in aqueous solutions induced by both pH and temperature variation were investigated by 1H NMR spectroscopy. The transitions induced by temperature were also explored by turbidity measurements using UV/vis spectroscopy for their lower critical aggregation temperature (LCAT) determination. Furthermore, these aggregation processes were followed by dynamic light scattering measurements.

Introduction Block copolypeptides (BCPs) have received much attention recently because of their tunable chemical structures and suprastructure formation.1 They have been found attractive for applications in different areas, such as tissue engineering, drug delivery,2 and biomimetic synthesis of ordered inorganic nanostructures.3 Seeing that polypeptides can fold into well-ordered secondary structures like R helices or β sheets, they are of special interest for promoting self-assembly and achieving high levels of suprastructural controllability when used as constituent blocks of block copolymers.4 These block copolypeptides can be synthesized either by ring-opening polymerization of amino acid N-carboxyanhydrides (NCAs) initiated by amino-terminated polymers5 or by stepwise amide coupling to form the sequenced peptide block(s).6 One intriguing class of block copolypeptides are those that show responsiveness to pH value.7 The conformation switching of polypeptide block between coil and helix by variation of solution pH controls their assembly8 and also potentially their functions in biomineralization.3c,9 Not only different morphologies, such as micelle,10 vesicle,7 and cylinder,6b were reported to be formed by the stimuli-induction, but also aggregate sizes were found to be dependent on solution pH.7e,g Nevertheless, most of the copolypeptides reported so far were constructed by a hydrophilic polypeptide block and a hydrophobic polymer block, such as glycopolymer, polyisoprene, polybutadiene, and poly(ε-caprolactone). Temperature has also been utilized as another useful external stimulus in the design of functional * To whom correspondence should be addressed. Fax: +86-37167766821. E-mail: [email protected]; [email protected].

copolypeptides. For example, thermoresponsive triblock proteins were reported by Chaikof to form monodispersed micelles, whose compactness and sizes were triggered by helix-to-sheet folding transition.8b Recently, several examples of double hydrophilic block copolypeptides (DHBCs) were reported. For instance, zwitterionic diblock copolypeptides from poly(L-lysine) and poly(L-glutamic acid) were reported by Lecommandoux to reversibly self-assemble into vesicles as a function of solution pH values.11 Very recently, we reported the synthesis of DHBCs constructed by thermoresponsive poly(N-isopropylacrylamide) (PNiPAM) block and pH-responsive poly(L-glutamic acid) block by the combination of reversible addition fragmentation chain transfer (RAFT) polymerization and ring-opening polymerization of NCA.12 Their thermo- and pH-induced aggregation behaviors in aqueous solutions were investigated and various assembly morphologies were observed. Both Liu13a and Chen’s13b groups also reported the synthesis and self-assembly of the similar copolypeptides, but with slightly short block lengths. Here we report on the synthesis of a novel class of DHBCs constructed by a PNiPAM block together with a random copolypeptide14 block from L-glutamic acid and L-lysine. Besides their thermoresponsiveness from PNiPAM block and pH-responsive properties from peptide block, especially interesting characteristics of these DHBCs are their tunable charge states at different pH conditions, which makes them unique compared to our previous system,12 and also would afford potential opportunities to mimic stimuli behaviors of zwitterionic biomacromolecules. Their conformation switching between helix and random coil was followed by circular dichroism (CD) spectroscopy, and their stimuli-responsiveness to solution pH and

10.1021/bm800394p CCC: $40.75  2008 American Chemical Society Published on Web 08/29/2008

Stimuli-Responsive Zwitterionic Block Copolypeptides

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Table 1. Conditions for and Results from ROP of BLG-NCA and BLLys-NCA Initiated with PNiPAM360-NH2 polymerization conditionsa

GPC resultsc

entries

[I]/[Z]/[K]b

time (h)

yield (%)

Mn (× 10-4)

PDI

DPPBLGd

DPPBLLyse

LCATf (°C)

PN360(PBLG0.54-co-PBLLys0.46)220 PN360(PBLG0.41-co-PBLLys0.59)252

1:190:197 1:200:240

120 120

88 87

4.77 4.01

1.70 1.42

120 104

100 148

34.2 35.0

a PNiPAM360-NH2 (PDI ) 1.24) was used as the macroinitiator. b [I]/[Z]/[K] represents [PNiPAMn-NH2]/[BLG-NCA]/[BLLys-NCA]. c DMF as eluent at 40 °C. d DPPBLG represents the number of γ-benzyl-L-glutamate residues of the copolypeptide block and was calculated based on the 1H NMR integration (see text). e DPPBLLys represents the number of Boc-L-lysine residues of the copolypeptide block and was calculated based on the 1H NMR integration (see text). f LCAT for the corresponding deprotected BCPs at pH 3.0 in aqueous solution.

temperature variation was investigated by means of 1H NMR and UV/vis spectroscopy, as well as dynamic light scattering (DLS).

Experimental Section Materials.Amino-terminatedpoly(N-isopropylacrylamide)(PNiPAMnNH2) was synthesized according to our previous report.12 γ-BenzylL-glutamate N-carboxyanhydride (BLG-NCA) and Boc-L-lysine Ncarboxyanhydride (BLLys-NCA) of high purity were synthesized by phosgenation of γ-benzyl-L-glutamate and Boc-L-lysine with triphosgene from anhydrous ethyl acetate, respectively, and used immediately after recrystallization twice from ethyl acetate and hexane.12 Pure water was redistilled. Triethylamine (TEA) was dried over NaOH pellets. Other reagents and solvents were purchased and used as received unless otherwise stated. Instrumentation and Measurements. 1H and 13C NMR spectra were recorded on a Bruker 400 MHz spectrometer (1H, 400 MHz; 13C, 100 MHz). The temperature-dependent 1H NMR measurements were performed after the sample tube was kept at each preset temperature around 10 min for equilibrium. The solution pH values were adjusted with DCl or NaOD in D2O. Gel permeation chromatography (GPC) measurements were carried out at 40 °C by using a Waters 2414 GPC equipped with four Waters styragel columns (300 × 7.8 mm) and a differential refractive index detector. The system was operated with DMF (containing 1 g/L LiBr) as the eluent at a flow rate of 1 mL/min and calibrated with polymethyl methacrylate standards in the molar mass range of Mp ) 2.58 × 103 to 9.80 × 105 Da (Polymer Standards Service-USA Inc., U.S.A.). UV/vis turbidity measurements were carried out for lower critical aggregation temperature (LCAT) determination on a Varian Cary 1E (Australia) UV/vis spectrophotometer, equipped with a thermostatically regulated bath. A solution of the respective block copolymer (6-7 mg) in pure water (4 mL) was added into a cell (path length, 1 cm), which was placed in the spectrophotometer and heated at a rate of 0.2 °C · min-1. The solution pH values were adjusted with HCl or NaOH aqueous solutions. The measurement at each temperature was done after the solution was kept at the preset temperature around 5 min for equilibrium. The temperature of the phase transition was considered the one at which the transmittance at λ ) 500 nm had reached 50% of the value difference between the initial and final stages. DLS measurements were performed with a High Performance Zetasizer Nano instrument (Malvern, U.K.) using a light scattering apparatus equipped with a He-Ne (633 nm) laser and a thermoelectric Peltier temperature controller. The measurements were made at a scattering angle of θ ) 173° (“backscattering detection”). The autocorrelation functions were analyzed with the CONTIN method. Copolymer solutions (1.5 mg · mL-1) were filtered through a 0.45 µm PTFE filter prior to use. Temperature-dependent DLS experiments were equilibrated 10 min at each step. The data were reported according to volume distribution of the particles. Circular dichroism measurements were performed on a JASCO J-715 spectropolarimeter (continued scanning mode, scanning speed 20 nm · min-1, data pitch 1 nm, response 1 s, bandwidth 5.0 nm). A thermo-controlled Quartz cell with a path length of 1 mm was used with peptide solutions containing approximately 3-5 × 10-6 dmol · mL-1 per amino acid residue. CD data are given as mean molar ellipticity based on amino acid residues (θ in deg dmol-1

cm2). All samples were equilibrated for at least 12 h before measurement. The spectra are the result of five accumulations for the measurements in aqueous solutions. The blank spectrum of the solution was subtracted. PNn(PBLGx-co-PBLLysy)m, where n is the polymerization degree (DPn) of PNiPAM block, x and y are the mole fraction of glutamic acid and lysine residues, respectively, and m is the total DPn of the peptide block]: The macroinitiator PNiPAMn-NH2 (0.20 g, 0.0056 mmol) was dissolved in dry DMF (5.0 mL) inside a Schlenk tube. After the solution was degassed by three freeze-evacuate-thaw cycles, the required amounts of BLG-NCA and BLLys-NCA (according to the targeted molar mass) were added, and the polymerization was carried out at r.t. under N2 for 5 days. The copolymer was then precipitated in diethyl ether twice and dried in high vacuum at 50 °C for 24 h. Typical results are shown in Table 1. 1H NMR (CDCl3): δ (ppm) 1.12 (br, CH3), 1.37 (br, CH3), 1.53∼2.47 (br, CH3 + CH2), 3.04 (br, CH2), 3.95 (br, CH), 5.13 (br, CH2), 6.64 (br, NH), 7.27 (br, benzyl), 8.39 (br, NH). 13C NMR (CDCl3): δ (ppm) 22.35, 23.80, 26.11, 28.32, 40.71, 41.12, 65.99, 77.86, 128.02, 128.15, 128.68, 136.92, 156.17. PNn(PLGx-co-PLLysy)m: The copolymer PNn(PBLGx-co-PBLLysy)m (0.60 g) was dissolved in triflouroacetic acid TFA (7 mL). After stirring at rt for 5 h, HBr/acetic acid (33%, 5 mL) was then added. After stirring at rt for additional 3 h, the copolymer was precipitated out by adding excess amount of diethyl ether and then washed with ethyl ether at least four times. After evaporation of solvents in vacuum, the residue was dried in vacuum at rt and yielded the copolymer as gray powder (0.53 g, 88%). 1H NMR (D2O): δ (ppm) 1.01 (br, CH3), 1.13 (br, CH3), 1.30-1.80 (br, CH2 + CH3), 1.90-2.00 (br, CH2 + CH), 2.44 (br, CH), 2.93 (br, CH2), 3.67 (br, CH2), 3.76 (br, CH), 4.02 (br, CH), 4.20 (br, CH). 13C NMR (D2O): δ (ppm) 21.96, 22.51, 25.34, 26.67, 30.54, 39.56, 42.12, 68.17, 173.95, 175.70, 176.97.

Results and Discussion Synthesis and Characterization. The synthesis procedure of block copolypeptides is delineated in Scheme 1, and typical polymerization results are summarized in Table 1. Initiated with the amino-terminated poly(N-isopropylacrylamide) (PNiPAMnNH2), ring-opening polymerization of NCA mixture from γ-benzyl-L-glutamate and L-lysine afforded the corresponding block copolypeptides PNiPAMn(PBLGx-co-PBLLysy)m with a presumed random copolypeptide block. The residual homopolymers were eliminated by precipitation. The content of amino acid residues inside the polypeptide block can be tuned by changing the NCA ratio in the starting mixture. The composition of the block copolypeptides were estimated based on the integrals at δ (ppm) ) 1.12, 1.37, and 7.27 from the corresponding 1H NMR spectra for NiPAM, BLLys, and BLG units, respectively, and the molar masses of the copolypeptides were therefore calculated from the integral ratios together with the molar mass of the first block, PNiPAMn-NH2. Monomodal GPC elution curves in Figure 1 illustrated the absence of homopolymers and also the molar masses increase of the polymers upon growing with copolypeptide block. Boc group and benzyl group in PNiAPMn(PBLGx-coPBLLysy)m were quantitatively removed with TFA and HBr/

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Scheme 1. Synthesis of Diblock Copolypeptides PNiPAMn(PBLGx-co-PBLLysy)ma

a n is the polymerization degree (DPn) of PNiPAM block, x and y are the mole fraction of glutamic acid and lysine residues, respectively, and m is the total DPn of the peptide block. Reagents and conditions: (a) BLG-NCA, BLLys-NCA, DMF, room temperature, 5 d; (b) TFA, HBr/AcOH, room temperature, 8 h.

Figure 2. 1H NMR spectra of copolypeptide (Table 1, entry 2) in DMFd7 at 80 °C before (a) and after deprotection (b). The solvent signals are indicated with an asterisk (*). Figure 1. Typical GPC elution curves of PNiPAM360-NH2 and PNiPAM360(PBLG0.54-co-PBLLys0.46)220 (Table 1, entry 1).

glacial acetic acid by standard method15 (step b in Scheme 1), which afforded the zwitterionic double hydrophilic copolymers PNiAPMn(PLGx-co-PLLysy)m with positively-charged lysine residues at neutral or acidic conditions and negatively-charged glutamic acid residues at neutral or basic conditions. 1H NMR spectra in Figure 2 illustrate this clean deprotection process. The signals from the benzyl groups as well as Boc groups in PNiAPMn(PBLGx-co-PBLLysy)m at δ ) 5.13 and 7.27 ppm, as well as at δ ) 1.37 ppm, respectively, disappeared completely upon deprotection. Secondary Structures. It is well-known that poly(L-lysine) attains R-helical conformation at basic conditions but takes random coil conformation at acidic conditions.16a In contrast, poly(L-glutamic acid) adopts R-helical conformation in solution at acidic conditions and undergoes conformation transition into random coil at basic conditions.16b The secondary structure of PNiPAMn(PLGx-co-PLLysy)m in aqueous solution was therefore examined by circular dichroism (CD) at different pH values. The CD spectra are plotted in Figure 3. The behavior of PNiPAM360(PLG0.54-coPLLys0.46)220, for which glutamic acid residues are more numerous in the copolypeptide block, will be first discussed.

Considering that the lysine unit has an average pKa of 10.5417 and the glutamic acid unit of 4.05,16a the charge states of the copolypeptides can be adjusted by solution pH. At pH 3.0, which is lower than the pKa of both glutamic acid and lysine, glutamic acid residues mostly neutralized and lysine residues must be positively-charged, resulting in a positivelycharged polypeptide block. CD spectrum at this pH condition showed helix-like features though the helix content is not so high (about 45%). Increasing the solution pH to 6.0, at which the lysine units should still be protonated but the glutamic acid units deprotonated, that is, both lysine and glutamic acid residues are charged, a characteristic CD spectrum for R-helix was still obtained, though the helix content reduced as indicated by the decrease of the signal intensities at 208 and 222 nm (about 37% of R-helix). Further increasing the solution pH resulted in precipitation of the copolypeptide due to the charged peptide-peptide interactions. A clear solution was again obtained when the solution pH reached 10. At pH 12, where both lysine and glutamic acid residues are expected to be deprotonated, and thus, the peptide block becomes negatively-charged, the CD spectrum showed only 16% of R-helix. The R-helix content decrease with the increase of solution pH was also observed for PN360(PLG0.41-coPLLys0.59)252, where lysine residues are more numerous in



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Figure4.Temperature-dependent 1HNMRspectraofPNiPAM360(PLG0.54co-PLLys0.46)220 (Table 1, entry 1, 2.0 wt %, pH ) 3.0) in D2O. The four arrows indicate the poly(NiPAM) signals whose intensities decrease with increasing temperature.

-1

Figure 3. CD spectra in aqueous solution (0.02 mg · mL ) at different pHconditions.(a)PNiPAM360(PLG0.54-co-PLLys0.46)220,(b)PN360(PLG0.41co-PLLys0.59)252. The helix content was estimated according to literature method.16a

the copolypeptide block (Figure 3b). The following conclusions can be made from the above results: (a) glutamic acid units have a dominant influence over lysine units on controlling the conformation of copolypeptide, irrespective of which amino acid residue is more numerous in the copolypeptide block, (b) solution pH for coil-helix transition is shifted from 5.2 for poly(L-glutamic acid) homopolymer to above 6.0, (c) the water-solubility of the copolypeptide is balanced by the positive and negative charges in the peptide block. The strong acidic and basic environments are expected to make the copolypeptide molecules positively- or negativelycharged enough to counter-balance peptide-peptide interactions and prevent the copolypeptides from strongly aggregating. Stimuli-Responsive Behaviors. The stimuli-responsiveness of PNiPAMn(PLGx-co-PLLysy)m to both temperature and solution pH was investigated, and their thermoresponsive behaviors were first examined by 1H NMR spectroscopy. Figure 4 shows representative temperature-dependent 1H NMR spectra of PNiPAM360(PLG0.54-co-PLLys0.46)220 in aqueous solution at pH 3.0 in the temperature range of 25 to 55 °C. The signal intensities from the copolypeptide block remained unaltered in the whole temperature range except for slight chemical shift changes, but the signal intensities from PNiPAM block (indicated by arrows) started to decrease markedly at around 33 °C, which is a clear indication that NiPAM chains start to collapse around this point. This temperature is close to that reported for the NiPAM homopolymer.18 These signal intensities continued decreasing upon further increase of solution temperature and disappeared nearly completely above 42 °C. At this stage, the solution was turbid by visual inspection, which suggests that the NiPAM chain collapse was followed by big or compact aggregate formation. The responsiveness of PNiPAMn(PLGx-co-PLLysy)m to solution pH was also checked with 1H NMR spectroscopy, and resulting spectra from PNiPAM360(PLG0.54-co-PLLys0.46)220

are compiled in Figure 5. At pH 3.0 and 6.0, copolypeptides are soluble in D2O due to the protonation of lysine residues. Therefore, the proton signals from lysine residues [δ (ppm) ) 1.2-1.8 and 3.0] are visualized. In contrast, the proton signals from the glutamic acid residues [δ (ppm) ) 2.5-2.8] almost disappeared due to the strong hydrophobicity of the protonated glutamic acid units. Thus, the copolypeptide solubility at acidic conditions is dominated by the protonated lysine residues. Interestingly, the copolypeptides are also soluble in water at strong basic conditions. As evidenced by the disappearance of the δ (ppm) ) 3.0 and change in intensity of the δ (ppm) ) 1.2-1.8 proton signals of lysine at pH 12.0, the lysine residues were deprotonated (neutralized) under this condition and became less-soluble in water. The copolypeptide solubility at this stage is dominated by the deprotonated (thus, negatively-charged) glutamic acid residues. Between pH 6.0 and 10.0 both glutamic acid and lysine residues were partially protonated or deprotonated. The copolypeptides became insoluble in water as evidenced by their turbid solutions, which should be caused by the large and compact aggregate formation. Therefore, the random copolypeptides from lysine and glutamic acid behave quite differently in aqueous solutions than the zwitterionic poly(L-lysine)-block-poly(L-glutamic acid).11 In the latter case, block copolypeptides formed schizophrenic vesicles in water at acidic (pH < 4) and basic (pH > 9) conditions, but the aggregates disassembled and caused precipitation around neutral conditions. Thermoresponsive transition of PNiPAMn(PLGx-co-PLLysy)m was also investigated with turbidity measurements, aiming at determining their lower critical aggregation temperature (LCAT). The LCATs of the two polymers are listed in Table 1. The transmittance curves for PNiPAM360(PLG0.54co-PLLys0.46)220 are plotted in Figure 6. Three different solution pH conditions (pH 3.0, 4.5, and 12.0) were selected to ensure that the copolypeptide was soluble at room temperature. At lower temperatures, the solutions are clear, but turn turbid when solution temperatures increase above the LCAT. This temperature for PNiPAM360(PLG0.54-coPLLys0.46)220 is 34.2 °C, which is in good agreement with the results from the temperature-dependent 1H NMR measurements, where PNiPAM chains started to significantly collapse around 33 °C. This agreement shows that NiPAM

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Figure 5. 1H NMR spectra of PNiPAM360(PLG0.54-co-PLLys0.46)220 (Table 1, entry 1, 2.0 wt %) in D2O at different solution pH values (25 °C).

Figure 6. Plots of transmittance vs temperature for 1.0 wt % aqueous solutions of PNiPAM360(PLG0.54-co-PLLys0.46)220. Heating and cooling rate ) 0.2 °C · min-1.

chain collapse is instantaneously followed by the aggregation of the BCP, which contrasts with the behavior of diblock copolypeptides constructed from NiPAM and glutamic acid blocks at basic conditions.12 In the latter case, a significant delay of BCPs’ aggregation after NiPAM chain collapse was observed. The transmittance curves are superimposed for the heating processes at different pH, indicating that the thermoresponsive transition of the copolypeptide shows no significant pH dependence during the heating process. Below the LCAT, the transmittance at each pH condition was nearly 100%, while the transmittance above the LCAT was nearly zero at each pH condition. For the cooling process, however, hysteresis increased markedly with solution pH, and transmittance below the LCAT was much less than 100% for the case of pH 12.0. All these suggest a more complicated rehydration process at basic conditions. The following conclusions can be made: (a) The BCP is fully soluble in water at the selected pH when solution temperature is lower than its LCAT. (b) Large or compact aggregates are formed at elevated temperatures. (c) Hysteresis between heating and cooling process increases with solution pH. The responsive behaviors of copolypeptides to temperature and pH variation were further investigated with DLS, aiming at determining aggregate size. The results for temperature

Figure 7. Plots of hydrodynamic diameters (Dh) of BCP aggregates in aqueous solution from DLS measurements as a function of (A) temperature [PNiPAM360(PLG0.54-co-PLLys0.46)220, 0.1 wt %, pH 3 and 12] and (B) pH variations [0.1 wt %, 25 °C, line a, PNiPAM360(PLG0.54co-PLLys0.46)220; line b, PNiPAM360(PLG0.41-co-PLLys0.59)252].

and pH-dependent aggregation are plotted in Figure 7a and b, respectively. Temperature-dependent aggregation of PNiPAM360(PLG0.54-co-PLLys0.46)220 will be discussed first. The polymer aqueous solution was fully clear at 25 °C by visual inspection at solution pH 3.0 or 12, and the hydrodynamic diameters (Dh) were around 5 and 10 nm, respectively. The former could be the size for a single chain of the given molar mass, while the latter could correspond to small aggregates. With temperature increasing, the aggregate sizes showed a sharp transition around the LCAT of the copolymer and reached 400 and 200 nm for the solutions at pH 3.0 and 12, respectively (Figure 7a). These sizes were maintained in the temperature range of 36-55 °C. The size distributions of the aggregates are monomodal and very narrow, with


polydispersity indexes in the range of 0.04-0.08. For comparison, the cooling process was also followed by DLS, and the aggregate sizes were slightly larger than those found during the heating process when solution temperatures were below the LCAT, which indicates a slow deaggregation process. The pH-induced aggregation of BCPs was also followed by DLS, and the results are plotted in Figure 7b. In the following, the behavior of PNiPAM360(PLG0.54-coPLLys0.46)220, for which glutamic acid residues are more numerous in the copolypeptide block, will be discussed. Below pH ) 6.0, the solution was clear and the corresponding Dh was around 5 nm (unimer). An abrupt transition appeared above pH 6.0, with solution becoming turbid by visual inspection and aggregates with Dh of 300 nm being observed. The size distributions of the aggregates are multimodal and the corresponding polydispersity indexes are in the range of 0.24-0.68. The aggregate size reached its maximum (around 800 nm) at pH 7.5 and then started to decrease with the increase of solution pH. The solution became clear again when the pH reached 10.0, and the Dh reduced to 6 nm, as for the size of the unimer. For PNiPAM360(PLG0.41-coPLLys0.59)252, where lysine residues are more numerous in the copolypeptide block, the aggregation process resembled very much that of PNiPAM360(PLG0.54-co-PLLys0.46)220, the major difference being that the aggregate sizes are much larger than those obtained from the latter BCP. This unique aggregation and deaggregation process induced by solution pH can be understood to be caused by competitive protonation and deprotonation between glutamic acid and lysine residues in the random copolypeptide block. At low pH conditions, both lysine and glutamic acid residues are protonated, with the former being water-soluble and the latter being waterinsoluble, but water solubility of the copolypeptide is dominated by protonated lysine units at this stage. With the increase of solution pH above 6.0, both lysine and glutamic acid residues are oppositely charged, considering their pKa to be 10.54 and 4.05, respectively, which induces aggregation due to a strong charged peptide-peptide interaction. This situation is maintained until very basic conditions where the complete deprotonation of the glutamic acid residues confers the solubility to the copolypeptide, causing complete deaggregation.

Conclusion The efficient synthesis of novel zwitterionic block copolypeptides constructed with a random copolypeptide block and a NiPAM block has been presented. The random copolypeptide block contains both negatively-charged glutamic acid and positively-charged lysine residues, whose ratio can be tuned by changing polymerization conditions. The conformations of these copolypeptides are switchable between helix and random coil by changing solution pH values. Their unique responsive behaviors to pH are controlled by the protonation and deprotonation competition between lysine and glutamic acid residues. Their responsiveness to temperature in aqueous solutions, as evidenced with 1H NMR spectroscopy, turbidity, and DLS measurements, reveals that (1) the dehydration of NiPAM block is similar to that of PNiPAM homopolymer, (2) hysteresis between heating and cooling process increases with solution pH, and (3) condition for the largest aggregate formation can be mediated by the ratio of glutamic acid and lysine residues. These zwitterionic structures together with their tunable dually responsive characteristics make this kind of novel block copolypeptides


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promising candidates in self-assembly, nanotemplating, as well as biomineralization. Acknowledgment. We cordially thank the reviewers for their helpful comments and suggestions. This work has been financially supported by the National Natural Science Foundation of China (Grant Nos. 20374047 and 20574062).

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