pubs.acs.org/Langmuir © 2009 American Chemical Society
Self-Assembly of Hyperbranched Multiarmed PEG-PEI-PLys(Z) Copolymer into Micelles, Rings, and Vesicles Zhaopei Guo,† Yanhui Li,‡ Huayu Tian,† Xiuli Zhuang,† Xuesi Chen,*,† and Xiabin Jing† †
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China, and ‡Changchun University of Science and Technology, Changchun 130022, China Received March 17, 2009. Revised Manuscript Received April 30, 2009
Self-assembling of synthesized novel biodegradable hyperbranched amphiphilic poly(ethylene glycol)-polyethyleniminepoly(ε-benzyloxycarbonyl-L-lysine) (PEG-PEI-PLys(Z)) in aqueous media is studied. In aqueous media, PLys(Z) is the hydrophobic segment, with PEG and PEI as the hydrophilic segments. It will self-assemble into spherical shape when the selected solvent water is dropped into the common solvent tetrahydrofuran (THF). And when PEG-PEI-PLYS in common solvent is dropped into mixed solvent water and THF, rings will come into being. The spherical and rings are observed by environmental scanning electron microscopy (ESEM) and transmission electron microscopy (TEM). It shows that the size of the sphere is about 100 nm, and the diameter of ring distributes from 400 nm to 10 μm and bigger with the time roll around. It also forms a large vesicle with a thick edge by another method, which is good for drug delivery.
1. Introduction Self-assembly of amphiphilic block copolymers in dilute solution has attracted more and more attention during the past 10 years because of the medical applications of the resulting products as drug carriers and in gene delivery. Up to now, a large number of morphological structures, such as spheres, vesicles, fibers, and tubules have emerged.1-6 The morphological structure will change with different selectivity of solvents for the blocks in a triblock copolymer.7 If a mixed solvent is used, the morphological structures will be diverse because of the different selectivities of the solvents toward different segments. It was reported that when a diblock copolymer, such as polystyrene-b-poly(ethylene oxide) (PS-b-PEO) or polystyrene-b-poly(acrylic acid) (PS-b-PAA), dissolved in a water-DMF mixture, tubules, rings, vesicles or complex interconnected structures would appear.2,8,9 Lately, more details of triblock copolymers that have been used in biological and technology have been studied. Poly(styreneb-2-vinyl pyridine-b-ethylene oxide) (PS-P2 VP-PEO) selfassembled into corona micelles in water, which was pH dependent. When pH5.10 Cylinders and spheres appeared from PS-P2 VP-PEO when the solvents were DMF-benzene and DMF, respectively.11 *Corresponding author. Tel & Fax: +86-431-85262112. E-mail: xschen@ ciac.jl.cn;
[email protected]. (1) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728. (2) Yu, K.; Zhang, L.; Eisenberg, A. Langmuir 1996, 12, 5980. (3) Desbaumes, L.; Eisenberg, A. Langmuir 1999, 15, 36. (4) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5133. (5) Lazzaro, D.; Michtchenko, T.; Carvano, J. M.; Binzel, R. P.; Bus, S. J.; Burbine, T. H.; Mothe-Diniz, T.; Florczak, M.; Angeli, C. A.; Harris, A. W. Science 2000, 288, 2033. (6) Fredric, K. D. G.; Menger, M. Angew. Chem., Int. Ed. 1995, 34, 2091. (7) Hadjichristidis, N.; Iatrou, H.; Pitsikalis, M.; Pispas, S.; Avgeropoulos, A. Prog. Polym. Sci. 2005, 30, 725. (8) Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6359. (9) Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168. (10) Gohy, J. F.; Willet, N.; Varshney, S.; Zhang, J. X.; Jerome, R. Angew. Chem., Int. Ed. 2001, 40, 3214. (11) Lei, L.; Gohy, J. F.; Willet, N.; Zhang, J. X.; Varshney, S.; Jerome, R. Macromolecules 2004, 37, 1089.
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Some attention has been given to the self-assembly of hyperbranched copolymers in recent years.12-14 Large micrometersized vesicles in aqueous solutions have been obtained by higher generations of poly(propylene imine) dendrimers functionalized with aliphatic chains.15 Deyue Yan et al. reported the direct formation of macroscopic tubes of hyperbranched poly(3-ethyl3-oxetanemethanol) core (HBPO)-star-poly(ethylene glycol) arms (PEG) in acetone, which was a selective solvent.16 In this paper, we report the self-assembly behavior of a novel biodegradable hyperbranched amphiphilic polymer, poly(ethylene-glycol) - polyethylenimine - poly(ε-benzyloxycarbonylL-lysine) (HA-PEG-PEI-PLys(Z)), in aqueous solution. Micelles, rings, and vesicles were observed with different preparation processes in selective solvents. The formation of large rings from the small ones was also tracked.
2. Experimental Section Materials. Methoxypolyethylene glycol (mPEG) with a molecular weight of 5000 Da (mPEG 5000) was purchased from Aldrich. It was purified by precipitation into hexane from tetrahydrofuran (THF), and then the vacuum-dried precipitate was further dried by azeotropic distillation with toluene. Hyperbranched polyethylenimine (Hy-PEI) with a molecular weight of 25 000 Da (PEI 25 000) was purchased from Aldrich and was dried in vacuum at 60 °C for 24 h before use. ε-Benzyloxycarbonyl-L-lysine (LLys(Z)) was purchased from GL Biochem, Ltd. (Shanghai, China). Hexamethylene diisocyanate (HMDI) was purchased from Fluka. Toluene, hexane, and THF were dried and distilled with sodium before use. Synthesis of r-Amino Acid N-Carboxyanhydrides (NCAs). LLys(Z)-NCA was synthesized according to the reported
method.17 LLys(Z) and 1.2 equiv of triphosgene reacted in THF at (12) Zhou, Y. F.; Yan, D. Y.; Dong, W. Y.; Tian, Y. J. Phys. B. 2007, 111, 1262. (13) Hong, H. Y.; Mai, Y. Y.; Zhou, Y. F.; Yan, D. Y.; Cui, J. Macromol. Rapid Commun. 2007, 28, 591. (14) Hong, H. Y.; Mai, Y. Y.; Zhou, Y. F.; Yan, D. Y.; Chen, Y. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 668. (15) Tsuda, K.; Dol, G. C.; Gensch, T.; Hofkens, J.; Latterini, L.; Weener, J. W.; Meijer, E. W.; De Schryver, F. C. J. Am. Chem. Soc. 2000, 122, 3445. (16) Yan, D.; Zhou, Y.; Hou, J. Science 2004, 303, 65. (17) Daly, W. H.; Poche, D. Tetrahedron Lett. 1988, 29, 5859.
Published on Web 06/12/2009
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Figure 1. Synthesis of PEG-PEI-Plys(Z). 60 °C for 2 h. Then, the mixture was precipitated in excess of petroleum ether and recrystallized three times with ethyl acetate and petroleum ether. Synthesis of PEG-PEI-PLys(Z). The first step was to synthesize the hyperbranched PEG-PEI block copolymer according to a similar method.18 The following step was the ring-opening polymerization of LLys(Z)-NCA with a macromolecular initiator PEG-PEI. To be brief, Hy-PEI 25 000 was dissolved in CHCl3, and mPEG 5000 was allowed to react with excess HMDI. Then, an mPEG solution was added dropwise into the PEI solution to obtain PEG-PEI. In a dry glass flask, a certain amount of a mixture of PEG-PEI and LLys(Z)-NCA with a weight ratio of 2:8 was dissolved in dried chloroform and stirred for 72 h at 25 °C. Then the mixture was condensed and dialyzed against CHCl3 for three days to remove low molecule reactants. After that, it was precipitated with an excess of diethyl ether and dried under vacuum. The synthesis process and the structure of PEG-PEI-PLys(Z) are shown in Figures 1 and 2, respectively. Measurement of the Copolymers. The structures of PEGPEI and PEG-PEI-PLys(Z) were characterized by 1H NMR spectroscopy in D2O and CDCl3, respectively, at room temperature by using an AV-400 NMR spectrometer from Bruker. The molecular weights of PEG-PEI and PEG-PEI-PLys(Z) were calculated through element analysis by element N. The 1H NMR results of PEG-PEI and PEG-PEI-PLys(Z) are shown in Figure 3. By comparing the two figures, we can find the peak at 5.05 ppm of methylene protons in P(-CH2-C6H5) and the peak of benzene rings in PLys(Z) at 7.2 ppm in Figure 3B, indicating the successful introduction of PLys(Z) onto PEG-PEI. The N element contents in PEI (NPEI%), PEG-PEI (NPEG-PEI%), Plys(Z) (NPlys(Z)%), and PEG-PEI-PLys(Z) (NPEG-PEI-PLys(Z)%) are summarized in Table 1; the PEI content (PEI%) in PEG-PEI can be calculated according to eq 1, and the Plys(Z) content (PZ%) in PEG-PEI-PLys(Z) can be calculated according to eq 2. In eqs 1 and 2, NPEI% and NPEG-PEI-PLys(Z)% were obtained from their structure information. The composition information of the copolymer is listed in Table 1. (18) Petersen, H.; Fechner, P. M.; Fischer, D.; Kissel, T. Macromolecules 2002, 35, 6867.
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Figure 2. Schematic representation of PEG-PEI-Plys(Z).
Self-Assembly into Micelles. The micelles were prepared by following literature procedure, that is, the copolymer PEGPEI-PLys(Z) (25 mg) was first dissolved in 10 mL of THF, which is a common solvent for the three blocks. Then 40 mL of doubly distilled water was added dropwise with gentle agitation in the solution for about 1 h. THF was removed by using a rotary evaporator at 30 °C for about 4 h. Afterward, the micellar solution was diluted to 50 mL to a concentration of 0.5 mg/mL. Self-Assembly into Rings. The copolymer PEG-PEI-PLys (Z) (12.5 mg) was first dissolved in 10 mL of THF. Then the copolymer solution was dropped into the THF/H2O mixture (5 mLTHF+15 mL H2O) for 40 min or so with gentle agitation, and the agitation was continued for 48 h. Then THF was removed as the mentioned above. Finally, the solution was diluted by water to 25 mL to a concentration of 0.5 mg/mL. Self-Assembly into Doughnut-Like Vesicles. The copolymer PEG-PEI-PLys(Z) (5.0 mg) was first dissolved in 2 mL of THF. DOI: 10.1021/la900932j
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Figure 3. The 1H NMR spectra of PEG-PEI (A) and PEG-PEI-PLys(Z) (B). Table 1. Composition of the Copolymer weight content of each segment (%) copolymer PEG-PEI PEG-PEI-PLys(Z)
PEG Mw (K) PEI Mw (K) N content in copolymer (%) 5 5
25 25
17.69 12.74
Then the solution was directly poured into THF/H2O mixture (5 mLTHF+8 mL H2O) in a vial, and it was left still and airproof for 2 weeks. THF was removed by evaporation into air for 4 weeks. Finally, the solution was diluted by water to 15 mL. Fluorescence Measurement. Pyrene probe is used to prove the micelles’ formation. Steady-state fluorescence spectra could be obtained by a Perkin-Elmer LS50B luminescence spectrometer. The copolymer solution was added into the volumetric flasks containing pyrene, and the concentration of the copolymer was from 10-3 to 0.5 g/L. The pyrene concentration of final solution was 6 10-7 mol/L. The emission wavelength of fluorescence 9692 DOI: 10.1021/la900932j
PEG
PEI
Plys(Z)
Mw of the copolymer (K)
45.60 13.40
54.40 15.99
0 70.61
46 156
excitation spectra was 388 nm. The spectra was recorded at a scan rate of 240 nm/min.19 Characterization of Morphology and Size. The micelles’ morphology was examined by environmental scanning electron microscopy (ESEM), which was performed on an XL 30 ESEM FEG scanning electron microscope (Micrion FEI PHILIPS). A drop of the diluted micelle solution was deposited on a silicon flake and dried at room temperature. A thin layer of Au was coated on the sample surface before measurement. The structure of the (19) Sun, J.; Deng, C.; Chen, X. S.; Yu, H. J.; Tian, H. Y.; Sun, J. R.; Jing, X. B. Biomacromolecules 2007, 8, 1013.
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Figure 5. TEM image of the spherical micelles.
Figure 4. Excitation spectra of pyrene as a function of the PEGPEI-PLys(Z) concentration in water (A), and plot of I339/I333 versus logarithm of the PEG-PEI-PLys(Z) concentration (B). The CMC was 0.004 mg/mL. Figure 6. Size distribution of the spherical micelles measured by micelles was also studied by transmission electron microscopy (TEM JEM-2010 electron microscope, JEOL, Japan). A drop of the dilute aqueous (about 12 μL) solution was placed onto a copper grid. The sample was kept and measured at room temperature. PEI% NPEI % ¼ NPEG-PEI %
ð1Þ
ðPEG-PEIÞ% NPEG-PEI % þ ð1 -ðPEG-PEIÞ%Þ NPLysðZÞ %
¼ NPEG-PEI-PLysðZÞ % ð2Þ
3. Results and Discussion Different morphological structures of the assembled aggregation were obtained by different preparation processes. The spherical micelles were obtained in the following process. First, copolymer HA-PEG-PEI-PLys(Z) was dissolved in a common solvent, then a selective solvent was added, followed by the removal of the common solvent, and the spherical micelles came into being. Critical micelle concentrations (CMCs) were measured to confirm the micelle formation of PEG-PEI-PLys(Z) using pyrene as a hydrophobic probe. Figure 4A shows the excitation spectrum of pyrene in PEG-PEI-PLys(Z) aqueous solution of various concentrations. A red shift from 333 nm to 339 nm was observed with the increasing PEG-PEI-PLys(Z) concentration, indicating the formation of micelles. Figure 4B showed the intensity ratios (I339/I333) of pyrene excitation spectra versus the logarithm of the copolymer concentration. The CMC of 0.004 mg/mL for PEG-PEI-PLys(Z) was determined from the intersection of two straight lines: the baseline and the rapidly rising I339/I333 line in Figure 4B. Langmuir 2009, 25(17), 9690–9696
DLS.
The spherical micelles were observed by TEM (Figure 5). The size of the micelles observed was about 100 nm. The size distribution of the micelles was measured by dynamic light scattering (DLS). It also shows a uniform size of about 100 nm (Figure 6), which is consistent with the size shown by TEM. Another assembling method was examined to form rings. After the removal of THF, the assembling solution was left for a long time. Then the TEM measurements were carried out at different intervals, which were 5 days, 30 days, and 60 days. The TEM images in different periods are listed in Figure 7. An interesting phenomenon can be seen from these pictures. The assembled rings became bigger with increasing time. At 5 days, the mean diameters of the rings were about 100-600 nm. After 30 days, the rings’ diameters increased to 1.0-1.5 μm. And after 60 days, the diameters increased to 2.5-3.0 μm. From the size-changing trend, it is easy to infer that the bigger rings came from the smaller ones by merging. The merging process is shown in Figure 7A-D. It could be seen that two rings approached each other and merged into one bigger ring. Although the rings in Figure 7A-D were not the same ones, they were representative of the merging process. Another proof for the merging process was that thickness of the wall was nearly fixed (about 50 nm) when the rings became bigger and bigger. Although there have been some reports on the vesicles’ merging process, this is the first report for the rings’ merging process to our best knowledge. By comparing the methods for the spherical micelles and the ring preparation, several factors were critical for the ring formation. The copolymer PEG-PEI-PLys(Z) in THF should be DOI: 10.1021/la900932j
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Figure 7. The process of rings changed from smaller to bigger. A and B were observed at the fifth day after THF was removed; C and D were observed one and two months later, respectively; E shows the edge of the ring two months later; B shows the course of two smaller rings combining to a bigger one.
dropped slowly into the mixed solvent (THF/H2O) with gentle agitation. After the dropping wasfinished, the agitation was continued for 48 h. Then THF was removed rapidly by rotary evaporation. In addition, enough placement time for the assembling solution was essential for the rings becoming bigger. A further study was carried out for the assembly by a modified process, and large collapsed doughnut-like vesicles were obtained at this time (Figure 8). The sizes of the doughnut-like vesicles ranged from one to several micrometers with a thick wall. A merging process of doughnut-like vesicles was also observed (Figure 8E,F). By comparing the preparation processes of spheres, rings, and doughnut-like vesicles, it can be seen that mixed solvent was the necessary condition for rings’ and doughnut-like vesicles’ formation. For the sphere-like micelle preparation, we use the traditional preparation process. We dropped selected solvent water into the copolymer’s THF solution. Then, THF was removed rapidly. It is not strange that the copolymer can form spherical micelles in such process. Many spherical micelles have been reported by other groups using a similar process.20,21 The ratio between common solvent and selected solvent is important for the formation of different morphologies. According to Adi Eisenberg’s report, when the content of selected solvent, such as water, in the mixed solvent is very low, the amphiphilic copolymer will self-assemble into micelles. When the selected solvent content is high, the amphiphilic copolymer will self-assemble into vesicles. When the selected solvent content is in the middle area, it will be mixed assemblies.22 (20) Du, H. B.; Zhu, J. T.; Jiang, W. J. Phys. Chem. B 2007, 111, 1938. (21) Zhu, J. T.; Hiang, W. Mater. Chem. Phys. 2007, 101, 56. (22) Shen, H. W.; Eisenberg, A. J. Phys. Chem. B 1999, 103, 9473.
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In the first phase of preparation of rings, we dropped THF dissolved with copolymer into the mixed solvent (THF/H2O). In the mixed solvent, the water content was 75 wt %, and, after the THF was dropped into the mixed solvent completely, the water content was 50 wt %, so the amphiphilic copolymers mainly selfassembled into vesicles in the mixed solvent. It is the same with the first phase’s preparation of doughnut-like vesicles. In the first phase of preparation of rings and doughnut-like vesicles, a large number of small vesicles are formed. But the next step is different. For the second phase of preparation of rings, we only keep copolymer in the mixed solvent for 48 h, followed by rapid removing of THF; this led to vesicle collapse, and a large number of rings appeared. The third phase of the preparation of rings is the small rings merge into bigger ones. A large number of small rings move and collide, and some of the rings possibly join together, then merge into bigger rings to reduce their free energy. For the second phase of the preparation of doughnut-like vesicles, we keep the copolymer in the mixed solvent for about 2 weeks. During this period, the walls of the vesicles become thicker and thicker, and, at the same time, two or several smaller vesicles may merge and become bigger. But the third phase of the preparation of doughnut-like vesicles is a little complicated. Because of the THF volatilizing slowly for about four weeks, the composing of the solvent changed constantly, which can also lead to vesicles becoming bigger,23 in addition to the merging course. When THF volatilizes completely, the morphology of the vesicles are fixed. So, in the end, we obtain different sizes of doughnut-like vesicles, which shows a wide distribution. Figure 8E, (23) Luo, L. B.; Eisenberg, A. Langmuir 2001, 17, 6804.
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Figure 8. Large vesicles were observed by ESEM (A,B,C,D,E); Panels E and F (TEM) show two vesicles joining together.
F shows two vesicles merged together, and B shows a smaller vesicle embedding into the wall of a larger one. According to the analysis above, we give the model of merging and growing of rings (Figure 9) and vesicles (Figure 10) from smaller ones. In the mixed solvent (THF/H2O), when water content is up to a certain value, the copolymer is prone to selfassemble into vesicles. The key factor for large vesicles’ formation was long placement time for copolymer in mixed solvent (THF/H2O) followed by very slow removal of THF. For the assembled rings, small rings grew into bigger ones in remaining aqueous solution, but for the assembled vesicles, they became large ones in the mixed solvent (THF/H2O). In addition, there were also some possibilities for two or more vesicles to merge into one complex vesicle (Figure 8 B) Although many structural issues and forming mechanism remain to be studied, such a merging process represents a significant advance in mimicking cells, and it also has potential Langmuir 2009, 25(17), 9690–9696
Figure 9. Self-assembly of PEG-PEI-PLys(Z) into rings: Step 1 is the assembly into small vesicles in mixed solvent H2O/THF; the step from 1 to 2 shows the formation of small rings with the THF vanishing; the steps from 2 to 5 show the small rings merging into bigger ones in H2O. DOI: 10.1021/la900932j
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4. Conclusion HA-PEG-PEI-PLys(Z) in aqueous media can self-assemble into different morphologies using different micellar preparation methods. Spheres, rings and gigantic vesicles were observed by TEM and ESEM. The mixed solvent is the precondition to form rings and vesicles. The bigger rings come from the incorporation of smaller rings. The size of large vesicles ranged from one to several micrometers. This finding has potential application in drug and gene delivery. Figure 10. Self-assembly of PEG-PEI-PLys(Z) into doughnutlike vesicles. The steps from 1 to 5 show the small vesicles merging into bigger ones in the mixed solvent H2O/THF; steps 5-7 are the process of slow THF evaporation and the vesicles starting to collapse in the center (6) and the completion of collapse in the center (7).
applications in polymer vesicles. Further studies concerning properties and precise control of vesicle assembly and the dynamics parameters are in progress.
9696 DOI: 10.1021/la900932j
Acknowledgment. The authors are thankful to the National Natural Science Foundation of China (20604028, 50873102, Key Project 50733003), the National Natural Science Foundation of China - A3 Foresight Program (20621140369) and the Jilin Science and Technology Bureau, Science and Technology Development Project (20060701) for financial support to this work. The International Cooperation Fund of Science and Technology (Key Project 20071314) from the Ministry of Science and Technology of China is also acknowledged.
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