Article pubs.acs.org/Macromolecules
Nanomorphological Diversity of Self-Assembled Cyclopeptisomes Investigated via Thermodynamic and Kinetic Controls Soo hyun Kwon, Woo-jin Jeong, Jun Shik Choi, Sanghun Han, and Yong-beom Lim* Department of Materials Science & Engineering, Yonsei University, Seoul 03722, Korea S Supporting Information *
ABSTRACT: The physicochemical and biological characteristics of vesicles are dependent on the type of self-assembly building blocks and methods of preparation. In this report, we designed a vesicle-forming linear and cyclic peptide building blocks and investigated the effect of molecular topology and thermodynamic and kinetic controls on the stability and morphological features of the self-assembled vesicles. Comparison of topological effect on self-assembly revealed that the strong association of the aromatic hydrophobic segments is observed only in the cyclic peptide, which is most likely the results of constrained structure along with the restriction in the molecular degree of freedom. Consequently, the formation of stable vesicles could be observed only with the cyclic peptide. Further investigation with cyclic peptide building blocks revealed that depending on the control methods, vesicles with a variety of structural features, such as polygonal, wrinkled, round, roundpatched, and round-fused vesicles, could be fabricated. Our results demonstrate that existing vesicle structures constitute only a fraction of the possible structural diversity and that macrocyclic peptides can provide a wealth of opportunities in vesicle engineering.
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INTRODUCTION
Self-assembly usually refers to a process that is poised to reach the thermodynamic equilibrium state, a global minimum in the potential energy surface. However, a kinetically trapped metastable state, i.e., a local minimum in the potential energy surface that is considered to be irreversible on the experimental time scale, can sometimes be utilized to expand the morphological and structural space of nanoassemblies. Although many studies have investigated the topic of thermodynamic and kinetic controls in assemblies of block copolymers and other building blocks,13 such controls in peptide assemblies remain elusive. Here, we report the studies on the topology effect and thermodynamic and kinetic controls in peptide vesicles based on self-assembling macrocyclic peptides (cyclopeptisomes). We observed strong aromatic interactions only in the cyclic form of the peptide, which contrasted with the results from the linear counterpart. As a consequence, the formation of stable vesicles could be possible only with the cyclic peptide. Depending on the control methods based on kinetic and thermodynamic principles, vesicles with various structures were obtained, suggesting a vast possible structural space of cyclopeptisomes.
Self-assembled peptide nanostructures (SPNs) are of interest in the control of nanostructure morphology,1 the surface functionalization of nanostructures,2 the exploration of novel building blocks,3 the structural control of peptide chain,4 and the utilization of SPNs in various applications. As with other fields of nanotechnology, the control of nanostructural morphology and substructure is a necessary starting point in SPN research. Especially, cyclic peptides offer the possibility to fabricate unique and stable molecular assemblies that are not obtainable by conventional linear peptides.5 One of the most important topological benefits of cyclic peptides is their conformationally constrained structure, which restrict the molecular degree of freedom and minimize the entropic penalty associated with the molecular self-assembly or the targeting binding.6 Vesicles can be defined as fluid-filled sacs formed by the selfassembly of molecular building blocks into a bilayered structure. The building blocks are typically amphiphilic molecules. Geometric packing considerations indicate that vesicular structures can form when the volume fraction of the hydrophilic segment is slightly larger than the volume fraction of the hydrophobic segment.7 The amphiphilic molecules that can form vesicles include lipids,8 the low-molecular-weight amphiphiles,7 block copolymers,9 amphiphilic dendrimers or hyperbranched polymers,10 rod−coils,11 and self-assembling peptides.5b−d,12 © XXXX American Chemical Society
Received: July 25, 2016 Revised: September 10, 2016
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DOI: 10.1021/acs.macromol.6b01603 Macromolecules XXXX, XXX, XXX−XXX
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Figure 1. Thermodynamic and kinetic controls in cyclopeptisome self-assembly.
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(water/acetonitrile with 0.1% TFA). The molecular weight of the peptide was confirmed by MALDI-TOF mass spectrometry. The peptide concentration was determined spectrophotometrically in water/acetonitrile (1:1) using the molar extinction coefficient of tryptophan (5502 M−1 cm−1) at 280 nm. Vesicle Formation Protocols. The concentration of the peptide during vesicle formation was typically 20 μM. The structures of the self-assembled vesicles were controlled by the following protocols. Sonication. The peptide was sonicated using a Cup-horn sonicator (Qsonica, USA) for 20 min (∼180 W; 221 510 J). Extrusion. The peptide was extruded 10 times through the polycarbonate membranes (Whatman) with a pore diameter of 0.1 μm, using a mini-extruder (Avanti Polar Lipids, Inc.). Flash Injection. The peptide was freeze-dried and dissolved in HFIP. The peptide solution (400 μM, 20 μL) was rapidly injected into a large excess of distilled water (400 μL), followed by evaporation of HFIP. The final concentration of the peptide was 20 μM. Dialysis. The peptide was freeze-dried and hydrated with 30% HFIP (HFIP/water, vol %). The solution was transferred to a dialysis membrane (Spectra/Por 6 Standard RC Prewetted Dialysis Tubing, 1000 Da MWCO, Spectrum Laboratories, USA) and dialyzed against distilled water. Sonication of the Lyophilized Peptide from 30% HFIP. The peptide was lyophilized and dissolved in 30% HFIP (HFIP/water, vol %). The solution was freeze-dried, and the peptide was rehydrated with distilled water. Then, the solution containing the peptide was sonicated using a Cup-horn sonicator for 20 min. Circular Dichroism (CD) Spectroscopy. CD spectra were measured using a Chirascan circular dichroism spectrometer equipped with a Peltier temperature controller (Applied Photophysics, UK). The CD spectra of the samples were recorded from 260 to 190 nm using a 2 mm path length cuvette. The molar ellipticity was calculated per amino acid residue. Transmission Electron Microscopy (TEM). Two microliters of sample was placed onto a carbon-coated copper grid and dried completely. Next, 3 μL of a 1% (w/v) uranyl acetate solution was added, and after 1 min, and the excess solution was wicked off using filter paper. The specimen was observed using a JEOL-JEM 2010
EXPERIMENTAL SECTION
Peptide Synthesis and Macrocyclization. 2-Chlorotrityl resin (Novabiochem, Germany) was first preloaded with Fmoc-Ebes-OH (Anaspec, USA). The peptide was synthesized using standard Fmoc protocols in a Tribute peptide synthesizer (Protein Technologies, USA) on a 0.1 mmol scale. Standard amino acid protecting groups were used for the synthesis except Dde-Lys(Fmoc)-OH. For the headto-tail cyclization reaction, the N-terminal Fmoc group was deprotected following the completion of the final amino acid coupling. The protected peptide fragment (20 μmol) was liberated from the resin using a cleavage cocktail of acetic acid/2,2,2-trifluoroethanol (TFE)/methylene chloride (MC) (2:2:6). After an appropriate interval (∼1 to 2 h), the resin was removed by filtration and the filtrate was recovered (4 mL × 2). Finally, the resin was washed three times with the cleavage cocktail for complete recovery. Hexane was added to the filtrate to remove acetic acid as an azeotrope with hexane. The protected peptide fragment was obtained as a white powder following repeated evaporation cycles (dissolution of the peptide fragment in MC, hexane addition, and evaporation). The peptides were cyclized by head-to-tail cyclization between the N-terminal amine and C-terminal carboxylic acid groups. In a typical cyclization, 20 μmol (1 equiv) of the protected peptide fragment and 4 equiv of DIPEA were dissolved in DMF (20 mL) and transferred to a syringe. Then, 1 equiv of HATU was dissolved in DMF (20 mL) and transferred to another syringe. To achieve pseudohigh dilution conditions, the two solutions were added together to a stirred solution of 0.1 equiv of HATU and 1 equiv of HOBt in DMF (20 mL) at a rate of 0.06 mL/min using a syringe pump. Once the addition was complete, the reaction mixture was further stirred overnight. Following DMF evaporation, the residue was dissolved in MC, and tert-butyl methyl ether/hexane was added to triturate the cyclized protected peptide fragment (four times). The Dde group on lysine in the peptide was selectively removed using 2% (v/v) hydrazine/DMF four times (2 min each). The 2% hydrazine/DMF and free Dde were removed by evaporation. The peptide fragment was triturated with MC and tertbutyl methyl ether/hexane, treated with a cleavage cocktail (TFA/ TIS/water; 95:2.5:2.5) for 3 h, and then triturated with tert-butyl methyl ether. The peptides were purified by reverse-phase HPLC B
DOI: 10.1021/acs.macromol.6b01603 Macromolecules XXXX, XXX, XXX−XXX
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Figure 2. Characterization of self-assembly behavior by CAC measurement. Concentration-dependent fluorescence emission spectra (figures on the left) and the CAC calculation (figures on the right) of (a) LP in pure water, (b) MCP in pure water, (c) LP in PBS, and (d) MCP in PBS. PBS: 20 mM potassium phosphate, 150 mM KF, pH 7.4. instrument operating at 120 kV. The data were analyzed using DigitalMicrograph software. Leakage Experiment. Vesicles containing 5(6)-carboxyfluorescein (FAM) were prepared in the presence of a large excess of FAM, and the untrapped FAM was removed. The vesicles were treated with 1% (vol %) Triton X-100 with gentle shaking at room temperature. The fluorescence emission from the sample was recorded in a Victor X5 plate reader (PerkinElmer, USA) in a 384-well plate. The samples were excited at 495 nm, and the emission filter was set to 535 nm. To analyze time-course release data, the percentage of released FAM at different time points was calculated using the formula
tryptophan-rich hydrophobic segment (Figure 1 and Figure S1). In this new design of MCP, lysine, connected via its εamino group, is located in the middle of the tryptophan-rich segment, increasing the flexibility of the hydrophobic segment. The flexibility of the hydrophobic segment is critical for cyclopeptisome formation by reducing the rigidity of the polypeptide chain and thus enhancing the internal hydrophobic packing of the molecular assembly and chain entanglement.5d The hydrophilic peptide segment is derived from the Rev protein of human immunodeficiency virus type-1 (HIV-1), which binds RRE RNA of HIV-1 and mediates nucleocytoplasmic export of HIV-1 RNA. The Rev peptide contains numerous arginines, and thus the hydrophilic segment is positively charged. The tryptophan-rich segment is supposed to drive the selfassembly of LP and MCP building blocks because of the hydrophobic nature and π−π stacking capability of the amino acid. Because of the high sensitivity of the tryptophan’s indole chromophore to the change in its local environment, the intrinsic fluorescence of the amino acid was used to monitor the progress of molecular self-association.5b,14 We first dissolved the peptides in pure water, and the fluorescence emission from tryptophans was monitored with excitation at 280 nm. As shown in Figures 2a and 2b, there was a concentration-dependent increase in fluorescence intensity for both building blocks and the emission maxima observed were around 360 nm. Plotting the emission intensity values at 360 nm as a function of peptide concentrations revealed the sudden increase in fluorescence (Figure 2a,b; figures on the right). This discontinuous point likely represents the critical aggregation concentration (CAC).5b The CAC was calculated using the intersection between the regression straight lines of the linearly
% release = 100(It − I0)/(I∞ − I0) where It is the fluorescence intensity at a specific time point, I0 is the fluorescence intensity before surfactant treatment, and I∞ is the fluorescence intensity at complete vesicle disruption. Tissue Culture and Intracellular Delivery. For microscopic observation of the intracellular delivery of the vesicle and entrapped cargos, HeLa cells (1 × 104) were seeded in an 8-well Lab-Tek II chambered cover-glass system (Nunc, USA) in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% pen/strep and cultured overnight at 37 °C with 5% CO2. The cells were washed with Opti-MEM (ThermoFisher, USA) and treated with the vesicles (20 μM peptide) entrapped with 5(6)-carboxyfluorescein (FAM) for 16 h. Then, the sample solution was removed and replaced with Opti-MEM. The cells were visualized under a confocal microscope (LSM 800, Carl Zeiss, Germany).
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RESULTS AND DISCUSSION Cyclopeptisomes, fabricated by the self-assembly of macrocyclic peptides, have recently been developed and exhibit high structural and thermal stability as well as molecular recognition capability.5b,d The linear peptide (LP) or macrocyclic peptide (MCP) building blocks used in this study consists of a hydrophilic and charged segment, a linker segment, and a C
DOI: 10.1021/acs.macromol.6b01603 Macromolecules XXXX, XXX, XXX−XXX
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Figure 3. Structural elucidation of peptide assemblies by CD spectroscopy. (a) Thermal ramp CD data for LP. Thermal ramp CD data for MCP: (b) forward scan; (c) backward scan. (d) Plot of the molecular ellipticity of MCP at 229 nm as a function of temperature. [Peptide] = 20 μM.
dependent regions. The calculated CACs for LP and MCP are 1.7 and 1.4 μM, respectively. Thus, there was no significant difference between the linear and cyclic building blocks in aggregation propensity. Next, the CAC measurement was performed in phosphate buffered saline (PBS), a physiological buffer. The results showed that CACs for LP and MCP were 0.5 and 0.7 μM, respectively. Because hydrophobic interactions get stronger as the ionic strength increases,15 CAC values became smaller in PBS solution. Again, the differences in CACs between the peptides was not significant. The decrease in the CAC values at the high ionic strength solution gives an indirect evidence that hydrophobic interactions play important roles in the self-assembly of these peptide building blocks. To further elucidate the structural characteristics of these assembles, we performed circular dichroism (CD) spectroscopy. It should be noted that the study was conducted at concentrations above the CACs of the both peptides. The CD spectrum of LP showed a strong negative minimum at approximately 200 nm, indicating that the peptide did not have a clearly defined secondary structure (Figure 3a). The negligible changes in the CD spectra of LP following the temperature ramp suggest that the peptide is either resistant to
heat denaturation or highly unstructured even at low temperatures. The CD spectrum of MCP, in contrast with that of LP, showed an unusual positive maximum at 229 nm (Figure 3b). This exciton-coupled band indicates interaction between the aromatic chromophores, especially tryptophans.16 Hence, the interaction between tryptophan residues in MCP is significantly stronger than those in LP. The band at 229 nm gradually disappeared at the temperature increased, indicating thermal destabilization of the MCP assembly (Figure 3b). Notably, the MCP assembly showed a reversible thermal transition behavior (Figure 3c,d). We next investigated the morphologies of LP assemblies. LP was dissolved in pure water and vigorously sonicated using a high-intensity Cup horn sonicator. Because water is a selective solvent for the hydrophilic segment, the tryptophan-rich hydrophobic segment is likely to form the core of the molecular assembly. The transmission electron microscopy (TEM) analysis at a concentration just above the CAC of LP showed the formation of spherical objects with diameters approximately 40−45 nm (Figure 4a). Considering the molecular length of LP at fully extended state, the observed nano-objects are likely to be vesicles.5b Further investigation at D
DOI: 10.1021/acs.macromol.6b01603 Macromolecules XXXX, XXX, XXX−XXX
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weaker due to charge screening (Figure 5b and Figure S4). The vesicles formed under this condition were highly irregular and far from perfect spheres (wrinkled vesicles). The formation of these wrinkled vesicles implies incomplete rearrangement among the tryptophan residues due to the enhanced crystallinity of the hydrophobic segment at high ionic strength.15a,17 Therefore, the wrinkled vesicles represent a further extreme of kinetically trapped states compared to polygonal vesicles. Next, we prepared vesicles by extrusion. The observed morphologies were a mixture of irregular and round vesicles (Figure 5c,d and Figure S5). The disruptive energy supplied for molecular rearrangement might not be homogeneously distributed during extrusion, resulting in the mixed vesicle populations. The vesicles with mixed morphological states likely represent the kinetically trapped state (Figure 5c), whereas the morphologically homogeneous vesicles in Figure 5d should show the nanostructure in the thermodynamic equilibrium state. Therefore, depending on the method of preparation and the nature of the solvent, vesicles of varying morphology and roughness can be prepared. Intrigued by the possibility of structural controls, we further explored the structural space of the cyclopeptisomes and attempted to devise the methods for the preparation of more homogeneous vesicles. We employed modified cosolvent methods using hexafluoroisopropanol (HFIP) as a common solvent. HFIP is a good solvent for both hydrophilic and hydrophobic segments. The cosolvent method (or a solvent switch method) has been commonly applied to fine-tune the thermodynamic and kinetic parameters of block copolymer assemblies.13a,18 HFIP is a strong inducer of α-helices.19 Thus, MCP is expected to have an α-helical conformation regardless of the thermodynamically stable conformation of the peptide. The consequence is the complete disruption of the conformation of MCP, thus promoting its reorganization into a thermodynamically stable conformation during self-assembled structure formation. To quantify the conformational susceptibility of MCP in HFIP, the molecule was dissolved in a water:HFIP mixture, and the secondary structure of the peptide was investigated using circular dichroism (CD) spectroscopy (Figure 6). In pure water, the conformation of MCP was nearly random coil. MCP exhibited a substantial shift in secondary structure when the HFIP content reached to 20%. The dual negative minima at
Figure 4. TEM investigation of the self-assembly behavior of LP in pure water. (a) [LP] = 2 μM. (b) [LP] = 5 μM.
the slightly higher concentration revealed that LP aggregated into irregular objects (Figure 4b). The weak association between tryptophans as revealed by the CD investigation is likely responsible for this heterogeneous aggregation of LP at the higher concentration. Next, MCP was dissolved in distilled water and vigorously sonicated similarly to LP. As shown in Figure 5a and Figure S3,
Figure 5. Self-assembly of MCP building block into vesicles. Negativestaining TEM images for MCP assemblies (a) in pure water with sonication, (b) in phosphate-buffered saline (PBS; 20 mM potassium phosphate, 150 mM KF, pH 7.4) with sonication and (c, d) in pure water after extrusion through a membrane with a pore size of 100 nm. [MCP] = 20 μM.
the TEM analysis confirmed the formation of vesicles. The sizes of vesicles were bigger than those of LP vesicles. Importantly, MCP did not show irregular aggregate formation even at high concentration. The constrained cyclic structure of MCP should be the reason behind the strong aromatic interactions and the consequent stable molecular assembly formation. Notably, the thickness of the vesicle walls was not highly uniform, and the shape of the vesicles was polygonal rather than spherical (polygonal vesicles). This result indicates that MCP has a suitable molecular structure and composition for vesicle formation in water; however, the observed inhomogeneity in morphology suggests that the vesicles are in kinetically trapped metastable states. We then performed self-assembly under physiologically relevant buffered saline conditions in which hydrophobic interactions would become stronger and the charge repulsion in the hydrophilic segment would become
Figure 6. Investigation of the susceptibility of MCP conformation in HFIP by CD spectroscopy. [MCP] = 20 μM. E
DOI: 10.1021/acs.macromol.6b01603 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules 204 and 221 nm indicated that MCP was mostly in an α-helical conformation. There was a slight increase in ellipticity at 221 nm at an HFIP content of 40%, whereas the further increases in HFIP content up to 80% resulted in a similar degree of helix stabilization. These results indicate that an HFIP content of approximately 20−40% is sufficient to disrupt the peptide’s conformation and propensity toward aggregation. We first dissolved MCP in pure HFIP and performed flash injection13b,20 into excess water. Highly round vesicles with heterogeneous size distributions were obtained by this rapid solvent switch method (Figure 7a and Figure S6). Interestingly,
Figure 8. Thermodynamic control of cyclopeptisome formation. Sonication of lyophilized MCP from 30% HFIP.
We regard this method as being most advantageous among various methods investigated in this study because it is simple and gives desired homogeneous vesicles. The vesicles used in following leakage assay and cell internalization studies have been prepared by this method. Having established the experimental conditions to fabricate homogeneous vesicles at thermodynamic equilibrium, we then entrapped fluorescent probes (FAM) within the vesicles and monitored the time-dependent release profile of FAM following surfactant (Triton X-100) treatment to disrupt vesicular membranes. The release profile from typical liposomes was very rapid; the release of the encapsulated probes was nearly complete in less than 20 s (Figure 9a, inset). By contrast, the release profile of the cyclopeptisomes was comparatively very slow (Figure 9b). The time window for the cyclopeptisome disruption was on the order of hours. The slow release kinetics indicates that the cyclopeptisomes were far more structurally robust than liposomes. Most arginine-rich peptides have cell penetration capability.21 Because the MCP cyclopeptisomes are decorated with the arginine-rich hydrophilic segment, the cyclopeptisomes might be able to mediate intracellular transport of entrapped molecules. To address this question, HeLa cells were treated with the MCP cyclopeptisomes entrapped with FAM molecules, and the intracellular distribution of fluorescence was monitored using confocal laser scanning microscopy (CLSM). As shown in Figure 9c, all the cells displayed bright green fluorescence of FAM, verifying the efficient intracellular delivery of cyclopeptisomes.
Figure 7. Thermodynamic control of cyclopeptisome formation. (a) Flash injection of MCP in HFIP into a large excess of water. (b) Sonication of vesicles from the flash injection. (c) Dialysis of MCP in 30% HFIP. (d) Dialysis of MCP in 50% HFIP.
most of the vesicles were covered with a patch of small lamella, suggesting a morphological transition from lamellae during vesicle formation. Sonication of these round vesicles resulted in the formation of irregular aggregates consisting of smaller vesicles (Figure 7b and Figure S7). Thus, the round vesicles formed by flash injection should represent one of the deep local minima rather than the global minimum on the potential energy surface. For a gradual shift of solvent from HFIP to water, MCP was dissolved in 30% HFIP and dialyzed against pure water. This slow and continuous solvent shift resulted in the formation of fairly homogeneous and round vesicles (Figure 7c and Figure S8). We then used a higher concentration of HFIP (50%) to further disrupt MCP building block, and the sample was dialyzed as above. As shown in Figure 7d and Figure S9, highly round vesicles were formed in this condition. In addition, the fusion of vesicles into an elongated shape was observed, possibly due to enhanced mixing of the hydrophobic domains mediated by the common solvent. Next, we devised a simpler version of the cosolvent method. MCP was dissolved in 30% HFIP and lyophilized with the expectation that the disrupted peptide conformation would be maintained after the lyophilization process. The dried sample was rehydrated with water and then vigorously sonicated (ca. 2 × 105 J). As shown in Figure 8 and Figure S10, highly round and homogeneous peptide vesicles were obtained with this method. These vesicles should represent the global minimum because MCP was disrupted with both HFIP and sonication.
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CONCLUSIONS We synthesized linear and cyclic peptides that can assemble into vesicles and investigated their self-assembly behaviors via kinetic and thermodynamic controls. The peptide vesicles showed the high colloidal stability as judged by zeta-potential measurement (Figure S13), and no identifiable precipitate could be observed even after several months of storage at room temperature. Comparison of topological effect on self-assembly revealed that the strong association of the aromatic hydrophobic segments is observed only in the cyclic peptide, which is most likely the result of constrained structure and the resulting restriction in the molecular degree of freedom. As a consequence, the linear peptide, even though it had a tendency to form vesicles, nonspecifically aggregated as the concentration increased. The cyclic peptide, on the contrary, could form stable vesicles even at high concentration. Depending on the control methods, various types of vesicles with polygonal, wrinkled, round, round-patched, and round-fused shapes were obtained with the cyclic peptide. These results suggest that the structural space of peptide vesicles or cyclopeptisomes is very F
DOI: 10.1021/acs.macromol.6b01603 Macromolecules XXXX, XXX, XXX−XXX
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Figure 9. Leakage assay. (a) Liposome. (b) Cyclopeptisome. Vesicles entrapped with 5(6)-carboxyfluorescein (FAM) were treated with 1% Triton X-100, and the fluorescence emission was monitored at 535 nm. Insets: release profiles at earlier time points. Liposomes were prepared using l-αphosphatidylcholine from egg yolk. (c) Intracellular delivery of cyclopeptisomes and the entrapped FAM molecules. Left: bright-field image; right: fluorescence image.
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ACKNOWLEDGMENTS This work was supported by grants from the National Research Foundation (NRF) of Korea (2014R1A2A1A11050359, 2014M3A7B4051594), the Agency for Defense Development, and the Yonsei University Future-leading Research Initiative.
large. It is likely that not all structural possibilities of cyclopeptisomes were observed in the current study. The wider possible structural space of cyclopeptisomes should be explored using peptide structural controls and methods of preparation, and their various physicochemical and biological properties should be investigated. Because cyclopeptisomes are covered with bioactive peptides, they can be used in various applications, including intracellular delivery and molecular recognition. Moreover, the structural robustness of cyclopeptisomes should facilitate the development of nanocarriers with slow release kinetics.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b01603. Peptide chemical structures, MALDI-TOF MS spectra, DLS and zeta-potential data, additional TEM data (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (Y.L.). Notes
The authors declare no competing financial interest. G
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DOI: 10.1021/acs.macromol.6b01603 Macromolecules XXXX, XXX, XXX−XXX
