Synthesis of P4VP-b-PBLG Diblock Copolymers and Their Self-Assembly Behavior

Well-defined P4VP-b-PBLG diblock polymer composed of poly (4-vinylpyridine) (P4VP) and poly (γ-benzyl-L-glutamate) (PBLG) was synthesized by click reaction with alkyneand azide-functionalized homopolymers. Besides, P4VP blocks were synthesized by copper-mediated atom transfer radical polymerization (ATRP) with a chlorine-containing alkyne bifunctional initiator, and the azido-terminated PBLG homopolymers were synthesized by ring-opening polymerization (ROP) of γ-benzyl-L-glutamate with an amine-containing azide initiator. In addition, the synthesized P4VP-b-PBLG with different block ratios has been characterized by proton nuclear magnetic resonance (H NMR), Gel permeation chromatograph (GPC) and fourier transform infrated spectroscopy (FT-IR). Then, the self-assembly behaviors of P4VP-b-PBLG have been studied by changing parameters like dripping speed and block ratio. The morphologies of self-assembly of spherical, disk-like and ellipsoid-like shape particles have been observed and analyzed by scanning electron microscopy (SEM). These results have provided guidelines for the design of macromolecular self-assembly.


Introduction
Polypeptide copolymer was a kind of block copolymers based on polypeptide-hybrid which consist of polypeptide blocks and other polymer blocks [1] [2].The so called polypeptide with desirable biocompatibility usually owned specific characteristics of polypeptide.The main chain formed by amide bonds has the characteristic of secondary structure similar to proteins [3].Hence, polypeptide copolymer differed from traditional copolymer due to the unique sec- was dripped into the bottle under ice water bath.Thereafter, the reaction system was kept under negative pressure for 1.5 h at 60˚C, followed by adjusting pH to neutral level.Finally, the resulting product of γ-benzyl-L-glutamate was purified twice by recrystallization, collected and dried under vacuum.BLG-NCA was synthesized from γ-Benzyl-L-glutamate [11].In detail, γ-Benzyl-L-glutamate (34.5 mol, 7.67 g), dry-processed ethyl acetate (200 ml) and tri-phosgene (14.5 mol, 4.30 g) were transferred into a dry schlenk flask in vacuum, followed by reflux reaction for 3 h under 80˚C until the system was clear.
Then, the mixture was washed twice respectively by NaHCO 3 solution (2%), saturated NaCl solution and ultrapure water.The organic layer was transferred into a dry conical flask and MgSO 4 was added to remove water.Afterwards, the mixture was filtered and poured into a large amount of petroleum ether.The white products were collected by drying precipitation in a vacuum [12] [13] [14].PBLG-azidehomopolymers were prepared by ring-opening polymerization (ROP) of BLG-NCA.In a round-bottom flask, the dried BLG-NCA (1.09 mol, 0.270 g) and 1-azide 3-aminopropane (0.0328 mol, 1.21 μL) were dissolved in anhydrous DMF (3 mL).The solution was stirred under nitrogen at room temperature in dark for 3 days.Finally, the solution was precipitated into an excess amount of methanol and dried to collect the white products [14].

Characterization
The molecular weight and polydispersity of the polymers were recorded on a gel permeation chromatograph (GPC) which equipped with a refractive index detector (Perkin-Elmer Series 200) using DMF (1 g/L LiBr) as the eluent at 45˚C with a flow rate of 1 mL•min −1 . 1 H NMR spectra were measured by a 500 Bruker NMR instrument and TMS as the reference standard for chemical shifts.Fourier transform infrated spectroscopy (FT-IR) spectra were recorded on Nicolet iS50 and samples mixed with KBr were pressed into pellets.The elaborate morphologies of self-assembles were obtained from SEM (S-4800, HITACHI) operated at an accelerating voltage of 15.0 kV.Before the observation by electron microscope, the samples were coated by a thin layer of sputtered Au on silicon slice.

Results and Discussion
As shown in Figure 2, the GPC curves of P4VP 50 -b-PBLG 20 and P4VP 75 -b-PBLG 20 were unimodal and symmetrical, and the elution time were about 18.5 min and 17.5 min respectively.These two samples had narrow molecular weight distributions (M n /M w ), which were 1.21 and 1.28.When the degree of polymerization increased, the M n also increased from 10,100 to 13,300.It clearly indicated that the higher molecular weight was, the shorter elution time was.
The 1 H NMR spectra of P4VP-alkyne was illustrated in Figure 3  Therefore, the characterization results of GPC, 1 H NMR and FT-IR proved that P4VP-b-PBLG diblock copolymers had been synthesized successfully.

Self-Assembly Behavior Research
The main operations of self-assembly were divided into following steps.The And then, the self-assembly aggregates after dialysis were dripped on silicon then the self-assembly morphologies can be observed by scanning electron microscope.After many single factor changing tests, we found that stable self-assembly morphologies can only be prepared when the volume ratio of mother solvent and selective solvent was of 1:1.Besides, concentration of mother solvent, dialysis time and storage time have little influences on morphology changing.However, the morphologies were affected largely by the parameters of polymerization degree and dropping speed.
The dropping speed has a significant influence on morphology of P4VP 50 -b-PBLG 20 .The different morphologies resulted from different dripping speeds were illustrated in Figure 5.It was very obvious that the morphologies prefer to be ellipsoid particles at 0.75 ul/s (Figure 5(A)) and be a disk-like shape at 0.5 ul/s (Figure 5(B)), and the dimension of disk-like aggregates was larger than ellipsoid shape particles.It means that the packing method of self-assembly can be affected sensitively by dynamic factors [12].
We have also replaced P4VP 50 -b-PBLG 20 with P4VP 75 -b-PBLG 20 and studied the morphology changing result from dripping speed with different polymerization degree.As shown in Figure 6, the particles were disk-like at 0.75 ul/s (Figure 6(A)) and spherical at 0.5 ul/s (Figure 6(B)), however, the dilemmas were closed to each other.The self-assembly would like to be isotropic when the diffusion rate slower than the assembly rate and be anisotropic once the diffusion  Besides, comparing the differences of morphologies with different polymerization degrees under the same self-assembly parameters, we found that the chain length has a certain contribution to the sizes of self-assemblies although they owned the same hydrophobic PBLG chains.It also means that the PBLG segment acted as the role of core and P4VP as the shell of self-assembles in molecular packing [13].

Conclusion
In this article, ring-opening polymerization of N-carboxyanhydride (NCA-ROP), copper-mediated atom transfer radical polymerization (ATRP) and click reaction were used to synthesize diblock copolymers P4VP-b-PBLG, which were the first examples of diblock copolymers poly (4-vinylpyridine)-b-polypeptide combining pH-stimulating P4VP with biocompatible and biodegradable PBLG.Gel permeation chromatograph (GPC), proton nuclear magnetic resonance ( 1 HNMR) and fourier transform infrared spectroscopy (FT-IR) were used to characterize the structures.Then, self-assembly behaviors of PBLG-b-P4VP were studied by using scanning electron microscope (SEM).Some self-assembly aggregates were obtained from P4VP-b-PBLG, such as disk and sphere etc.We found that the dripping speed affected the morphologies sensitively which means that dynamic factor played a very important role in self-assembly of P4VP-b-PBLG.Because the dripping rate was the most direct parameter to change dynamic factors in self-assembly, which affected the speeds of stretching and curling of hydrophilic and hydrophobic segments.Besides, P4VP were not easy to form strong hydrogen bonds with water molecular which resulted in that the self-assembly process of P4VP-b-PBLG was "slower".On the other hand, the morphologies self-assembled by P4VP-b-PBLG with different hydrophilic polymerization degrees have obvious difference because P4VP segment wrapped outside the aggregates.