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Conformation-Directed Micelle-to-Vesicle Transition of Cholesterol-Decorated Polypeptide Triggered by Oxidation Hang Liu, Rui Wang, Jing Wei, Cheng Cheng, Yi Zheng, Yue Pan, Xueling He, Mingming Ding, Hong Tan, and Qiang Fu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b01873 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 3, 2018

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Journal of the American Chemical Society

Conformation-Directed Micelle-to-Vesicle Transition of CholesterolDecorated Polypeptide Triggered by Oxidation Hang Liu‡,a, Rui Wang‡,a, Jing Weia, Cheng Chenga, Yi Zhenga, Yue Panb, Xueling Hec, Mingming Ding*, a, Hong Tan a, Qiang Fua a

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China. b

Key Laboratory of Organic Synthesis of Jiangsu Province, College of Chemistry, Chemical Engineering and Materials Science & Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou, China. c

Laboratory Animal Center of Sichuan University, Chengdu 610041, China

KEYWORDS secondary conformation, hierarchical self-assembly, polypeptides, cholesterol, reactive oxygen species

ABSTRACT: Hierarchical self-assembly of synthetic polypeptides has attracted increasing interests due to its proteinmimetic structure and great potential in nanotechnology and biomedical applications. However, controlling the morphology and function of polymeric nanostructures via secondary structures remains largely unexplored. Here, we report an unusual micelle-to-vesicle transformation of cholesterol-decorated poly(L-cysteine) copolymer assemblies in response to reactive oxygen species (ROS). We found that the interesting morphological transition correlates with the alteration in conformations from β-sheet to α-helix, which grants an attractive “on-off” switch for triggered release and cellular interaction. We further demonstrated the usefulness of the conformation-regulated assembly strategy both in vitro and in vivo, taking cancer treatment as a model. The work offers a new insight on the folding and hierarchical assembly of polypeptides and a novel approach for the development of smart platforms in biosensing, disease treatment and diagnostic applications.

INTRODUCTION In nature, proteins are well known for their ability to fold into various structures, such as regular vesicle-like structures formed by virus capsids.1 The precise folding and specific conformations of subunits can direct the assembly and disassembly of proteins and regulate their biological activities.2 On the other hand, synthetic polymers can also self-assemble into a wide variety of bioinspired nanoscale morphologies.3 The high chemical tunability of polymers in principle allows for fine tailoring of the assembled structures.4 Hence, to mimic the ordered structures of sophisticated proteins using synthetic polymers is of great significance to understand the functions of proteins, and provide attributes in the development of functional biomaterials for biotechnology and medicine applications.1, 2, 5, 6 To facilitate self-assembly in aqueous media, polymers are usually designed with controlled balance of hydrophobicity/hydrophilicity and the ability to aggregate into a range of well-organized architectures, including spheres, cylinders, and vesicles.5, 7, 8 Moreover, the incorporation of rod-like segments (e.g., crystalline block, peptides with ordered α-helix or β-sheet conformations) can introduce additional order and shape anisotropy, and

impact on the aggregation behavior of macromolecules.9, Of particular interest is the ability of polymeric assemblies to undergo a transition between different morphologies as a result of an alteration in molecular characteristics in response to various environmental factors (pH, temperature, redox, etc.), which is promising for advanced applications in mimicking adaptive biological systems, delivering therapeutics and diagnostics, and amplifying signals in biosensors, etc.11 To date, the aggregation behaviors of most responsive polymer systems have been triggered mainly by the cleavage of labile linkages or the change of amphiphilicity.12 However, directing the morphology and function of polymeric assemblies by the conformation of synthetic polypeptides has been rarely achieved. 10

Here, we report an unusual micelle-to-vesicle transformation of polypeptides triggered by oxidation. Previous reports have shown that the oxidation of thioether groups to more polar sulfoxide or sulfone groups can enhance the solvation of poly(L-cysteine) and poly(L-methionine) derivatives in water, leading to the formation of random coil structures combined with the dissolution or disassembly of polymeric assemblies.13-15 Such a transformation from an ordered state to a disordered or lower ordered one also

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occurs commonly among other responsive systems.16-18 Nonetheless, conformation-driven order-to-order morphological transition has not been realized so far. Keeping in mind that these reported systems did not possess Scheme 1. Representative synthetic scheme of PEG-PCysChol and PEG-PCys-Chol-O2.a Chol

Chol

O

S

S

HS H 3N Cl

O HN

HN

OH

a

OH

H2N

b O O

1 Chol

Chol

O HN

O

S N H

m

O

2

c

HN

d

O O

O

HN O

O

O

S

n

O

O

N H

m

O

4

PCys-Chol could self-assemble in water due to its amphiphilicity. The assembled nanostructure was first determined with 1H NMR. As shown in Figure S3, the characteristic peaks of PEG and polypeptide blocks were clearly detected in CDCl3, while those attributed to PCys-Chol segment were significantly weakened in D2O due to a restricted motion of the protons. The result suggests a core-shell micelle-like structure with a hydrophobic core formed by PCys-Chol segment and shielded by a PEG corona. The formation of micelle-like structure was also confirmed by the red-shift of (0,0) absorption band in the fluorescence excitation spectra of pyrene in polypeptide solutions (Figure S4).23 The fluorescence probe technique combined with surface tension measurement indicate a low critical aggregation concentration (CAC) of 10-7 M (Figure S5-S7). More direct structural evidence for the assembled polymeric micelles was obtained from TEM and cryo-TEM images,24 where dispersed individual particles around 60 nm were observed (Figure 1A and Figure S8).

n

O

3

a

Reagents and conditions: (a) Cholesteryl 3Bromopropylcarbamate (Chol-Br, Scheme S1), 2 N NaOH, tetra-n-butylammonium iodide, chloroform: ethanol 7:26, r.t., 48 h (60% yield); (b) triphosgene, THF, 50 °C, 4 h (70% yield); (c) PEG-NH2, THF, 35 °C, 72 h (70% yield); (d) 10% H2O2, 5% acetic acid, 37 °C, 16 h, dialysis. Chol represents cholesterol residue.

enough hydrophobicity to stabilize their secondary conformation after oxidation, we envisioned that highly hydrophobic side chains may retain both the secondary conformation and assembled structures of polypeptides. To verify this, we incorporated cholesterol, a lipophilic component of animal cells,19 into the side chains of poly(Lcysteine) copolymers with a β-sheet propensity.20, 21 Interestingly, the cholesterol-modified co-polypeptides display an unexpected morphological change from micelles to vesicular structures in response to reactive oxygen species (ROS). The mechanism of the transition was carefully investigated in this contribution. Additionally, the attractive transformation was observed to affect the interaction between polypeptide assemblies and cells and determine their fates in vivo, which holds great potential in biomedical applications. RESULTS AND DISCUSSION. To obtain the amphiphilic poly(L-cysteine) copolymers, we first synthesized a cholesterol modified L-cysteine (Cys-Chol, 1), and converted it to an L-cysteine-Ncarboxylic anhydride (Cys-Chol-NCA, 2) via a FuchsFarthing method (Scheme 1).22 Thereafter, the ringopening polymerization (ROP) of NCA was initiated by methoxypolyethylene glycol amine (PEG-NH2, MW 5000) to obtain an amphiphilic diblock copolymer (Scheme 1), PEG-PCys-Chol (3), with controllable molecular weights and quite narrow polydispersity (PDI 1.02-1.03, Figure S1). The chain lengths were calculated to be 14-27 residues by integration of the proton nuclear magnetic resonance spectra (1H NMR, Figure S2, Table S1). The prepared PEG-

Figure 1. Cryo-TEM images of PEG-PCys-Chol (A) and PEG-PCys-Chol-O2 (B) assemblies. The bars are 100 nm. (C) Hydrodynamic diameter associated functions at different incident angles. (D) Typical Berry plots of PEG-PCys-Chol and PEG-PCys-Chol-O2 assemblies measured at 25 °C using multi-angle SLS. (E, F) Fluorescence emission spectra (λex = 526 nm) and emission images (insets) of R6G in the presence of PEG-PCys-Chol and PEG-PCys-Chol-O2 assemblies, setting R6G in water as a control with the same R6G content as determined by UV-Vis spectra (Figure S12). The upper insets display the emission images of R6G in water while the lower ones present those of R6G in corresponding assemblies. The images were obtained using an IVIS Lumina Series III imager (PerkinElmer, MA, USA). In C-F, a and b represent PEGPCys-Chol and PEG-PCys-Chol-O2 assemblies, respectively.


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Journal of the American Chemical Society The thioether groups in the side chains of PEG-PCysChol provide attractive sites for further functionalization, e.g., oxidation, alkylation and other click-type modifications.15, 25 Here we treated the polypeptide assemblies with 10% H2O2 in the presence of 5% acetic acid for 16 h to attain oxidated polymers (PEG-PCys-Chol-O2, 4). The oxidative condition has been demonstrated to give quantitative conversions of thioether to sulfone derivatives.13, 14 We did not detect any polymer degradation occurring with oxidation (Figure S9). The structure of oxidized polymer was characterized using 1H NMR (Figure S10). It was found that the methylene protons near the sulfur atom of PEG-PCys-Chol-O2 were remarkably faded in CDCl3, owing to the increased polarity of –S=O groups after oxidation. In the Fourier transform infrared (FTIR) spectra, the peak at ~1280 cm-1 associated with –C-S-C- groups disappears after H2O2 treatment, implying that all thioether groups have been oxidized (Figure S11). The new absorption bands at ~1100 and ~1350 cm-1 were assigned to the symmetric and antisymmetric S=O stretch vibrations, respectively, and consistent with the formation of sulphone moieties (Figure S11). Moreover, an additional absorption at ~1040 cm-1 evidences the coexistence of sulphoxide groups in PEG-PCys-Chol-O2.17 The transformation of –C-S-C- to higher polar –S=O groups has been demonstrated to increase the solubility of polymers and result in the disassembly or dissolution of the polymer aggregates.13-15, 17 Surprisingly, we found that the oxidation of PEG-PCys-Chol induced a micelle-to-vesicle transition. As elucidated by dynamic light scattering (DLS) measurements, the solutions before and after oxidation showed hydrodynamic radius RH of 69 and 72 nm (Figure 1C and Figure S12), which appeared larger than those determined by TEM and cryo-TEM observations, because of the collapse of assemblies in TEM and invisibility of hydrophilic corona in cryo-TEM.26, 27 With additional static light scattering (SLS) measurements, the radius of gyration (RG) of PEG-PCys-Chol and PEG-PCys-Chol-O2 were determined to be 55 and 70 nm (Figure 1D), respectively. Accordingly, the ratio RG/RH which is sensitive to the particle morphology8, 28 was found to be 0.80 and 0.97, respectively, for polymers before and after H2O2 treatment. The result suggests a morphological transformation from micelle to vesicle triggered by ROS. TEM imaging confirmed the vesicular morphology of PEG-PCys-Chol-O2 (Figure S8). A good membrane visualization was achieved by cryo-TEM imaging of the vesicles (Figure 1B), where the wall thickness was estimated to be 4.1 nm, in good accordance with doubled theoretical length of polypeptide segments (14 residues, with a pitch of 0.54 nm per 3.6 amino acid residues for α-helix).29 Hence, the result implies a bilayer membrane structure for the PEG-PCys-Chol-O2 vesicles. To further investigate the uncommon morphological change using host-guest features of different assemblies, we incorporated both hydrophilic rhodamine 6G (R6G), doxorubicin hydrochlorate (DOX·HCl) and hydrophobic fluorescein isothiocyanate isomer I (FITC) within the polymer dispersions. As expected, PEG-PCys-Chol solutions could not accommodate hydrophilic dyes, with a fluores-

cence intensity similar to that of the free probe in water (Figure 1E). By contrast, R6G and DOX·HCl were comfortably encapsulated within the vesicles formed by PEGPCys-Chol-O2, as evidenced by the decrease of fluorescence intensity and lifetime resulted from the selfquenching effect of dyes within the vesicular interior with a high local concentration (Figure 1F, Figure S13,S14).18 The result was further confirmed by confocal laser scanning microscopy (CLSM) observation, where DOX·HCl fluorescence was detected only in the oxidized assemblies, while FITC signal was visible in all the samples due to the fact that both hydrophobic micellar core and vesicular membranes are able to sequester lipophilic guests (Figure S16,S17). Despite the relatively low resolution of images, the indirect CLSM approach is promising and informative compared with conventional microscopic techniques due to less interference caused by drying artefacts. Additionally, we also included a hydrophobic superparamagnetic iron oxide nanoparticle (SPION) into the assemblies in view of its great potential in magnetically guided delivery and imaging.30 Of interest, the SPIONs notwithstanding their invasive character were found to aggregate together and form spherical clusters and toroid morphologies, respectively, in the presence of PEG-PCysChol and PEG-PCys-Chol-O2 assemblies (Figure S18), further verifying the stable micellar and vesicular structures. It is worth noting that such a characteristic can also be utilized to accommodate and manipulate other hydrophobic guests such as chemotherapeutic drugs and imaging agents for tailored applications.31

Figure 2. (A) CD spectra, (B) FTIR spectra and (C) SAXS curves of PEG-PCys-Chol assemblies before (a) and after (b) oxidation. (D) A schematic representation of the selfassembled amphiphilic polypeptides before and after oxidation.

The morphological transition reported here seems contradictory to traditional theory, since the enhancement of polarity and hydrophilicity commonly favors micelles or solutions over vesicular structures.17, 32, 33 Considering the high hydrophobicity of the polymers to stabilize their secondary structures, we postulated that the morphological change might be associated with their conformations, inspired by the fact that the secondary structures of poly-



peptides promote the folding of proteins into various three-dimensional architectures.20, 34 To confirm this hypothesis, we analyzed the polymer solutions with circular dichroism (CD). As shown in Figure 2A, PEG-PCys-Chol micelles display a CD pattern similar to that of a standard β-strand structure, with a positive band at 190 nm followed by a single minima at λ ≈ 209 nm. In contrast, PEGPCys-Chol-O2 adopts a helical structure, revealed by the presence of two negative peaks around 207 and 222 nm.35 Moreover, the FTIR spectrum of PEG-PCys-Chol presents an amide I band at ~1630 and ~1690 cm-1, suggesting the existence of an antiparalled β-sheet conformation.36 The shift of the peak from ~1630 cm-1 to ~1650 cm-1 verifies the conformation transition from β-sheet to α-helix after oxidation (Figure 2B and Figure S11). The α-helical hydrophobic segments of PEG-PCys-Chol-O2 favors the side-byside packing with each other, which may decrease the interfacial curvature between the hydrophobic and hydrophilic domains and result in the formation of membrane structures.1, 5 To further explore the assembly behavior of polypeptides, the polymer solutions were analyzed using small-angle X-ray scattering (SAXS). The local minimum at low q (≈ 0.07 nm-1) shifts slightly to lower q after oxidation (Figure S18), revealing an increase of the overall particle diameter. The SAXS data at low q region could be reasonably fitted to sphere (64 nm in diameter) and vesicle models (120 nm in diameter, 4 nm in thickness), respectively, for PEG-PCys-Chol and PEG-PCysChol-O2 assemblies (Supporting Information), which agrees well with cryo-TEM and DLS/SLS measurements (Figure 1). At large q region, the Bragg peaks at q = 1.3 and 2.6 nm-1 in the spectrum of PEG-PCys-Chol correspond to a layered structure with a period of 49.5 Å, which may be assigned to β-sheet spacing distance.37 Moreover, the peak shifted to a lower q after oxidation (Figure 2C and Figure S20), and the increased spacing (52.4 Å) may reflect the size of α-helical PEG-PCys-Chol-O2 domains, which includes the size of two extended cholesteryl side chains (~23.3 Å per residue, Figure S21) and the diameter of a helix core (~5 Å).38 Based on the above analyses, Figure 3D depicts a schematic illustration for the organization of the amphiphilic polypeptides before and after oxidation.

1

1

Figure 3. H- H NOESY spectra of PEG-PCys-Chol (A, C) and PEG-PCys-Chol-O2 (B, D).

To better understand the mechanism of conformation transition, we carried out 1H-1H nuclear Overhauser enhancement spectroscopy (NOESY) experiment on the polypeptide solutions. Obviously, an intense NOE signal between CαH-CαH protons (δ = 4.5 ppm) was observed for PEG-PCys-Chol (Figure 3A), which is indicative of an antiparallel β-sheet conformation.39 The correlation was negligible in the spectrum of PEG-PCys-Chol-O2 (Figure 3B), revealing the conformation variation.40 This result agrees well with CD, FTIR and SAXS analyses. Moreover, there are strong correlations between CαH and cholesterol residues (δ = 1.0, 1.1, 1.5, 1.85, 2.3, 5.35 ppm) in the spectra of PEG-PCys-Chol (Figure 3C), and these crosspeak patterns were significantly diminished after oxidation (Figure 3D). A possible explanation is that the presence of high polar –S=O may force the hydrophobic cholesterol moieties away from the peptide chain. It has been shown that both the bulky steric hindrance and ionic groups can disturb the formation of α-helical structure.28, 41, 42 Therefore, increasing their distance from the polypeptide backbones would be beneficial to the stabilization of α-helix conformation.42 In addition, the change of heteroatom character and possibility of –S=O groups participating in hydrogen bonding to stabilize the helical structure should also be taken into consideration and need further examination.14, 43 Our study provides a novel approach to regulate polypeptide conformations, which is helpful to understand the folding behavior of proteins and explore the mechanism of conformational diseases. More work is needed to justify the universality of this strategy and further explore the influence of oxidation degree on conformation regulation. Considering the overproduction of ROS associated with a variety of diseases including cancer, cardiovascular, inflammatory, diabetes, and degenerative diseases,44 we reasoned that the interesting conformation switch and morphological transition triggered by ROS are potential useful in biosensing, biodiagnostics, and controlled delivery applications. To test this potential, a model chemotherapeutic agent DOX was encapsulated into the polymer assemblies with high loading content of 27.0% (Figure S22). The nanocarriers displayed sustained and slow release profile under normal physiological conditions, with less than 25% of drugs released after 120 h. In contrast, DOX could be released completely in the presence of 10% H2O2 (Figure S23), while the spectroscopic characters of DOX were unaffected in the release media for quantitative analysis (Figure S24). This result suggests a highly sensitive on-off switch for controlled release. As a control, DOX was also loaded into PEG-PCys-Chol-O2 where the thioether groups have been pre-oxidized to – S=O moieties. The formulation possessed a relatively lower loading content (16.7%) and did not show significant acceleration of drug release anymore under oxidation environment (Figure S25), confirming that the “switch” was actually caused by the change of polymer structures. Different from the previous strategies that triggering the


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Journal of the American Chemical Society release of payloads by the change of amphiphilicity or the cleavage of polymer chains,30, 45 our work offers a novel conformation-specific approach to controlled delivery. On the other hand, inspired by natural cell-penetrating peptides (CPPs), α-helical polypeptides have been developed and showed potent membrane activities related to their helical structure.46 To investigate the impact of ROStriggered conformation transition on the interaction of polypeptide and cells, HeLa tumor cells were treated with DOX loaded polymer nanovehicles before and after oxidation (i.e., DOX@PEG-PCys-Chol and DOX@PEG-PCysChol-O2, respectively). CLSM imaging and flow cytometry analysis reveal that the oxidized PEG-PCys-Chol-O2 entered tumor cells more efficiently than its un-oxidated counterpart, with much stronger intracellular DOX fluorescence located mainly in the nucleus after 4 h of incubation (Figure 4 and Figure S26). Such a cell internalization efficiency is also much higher than that reported for commercial liposomal doxorubicin (Doxil).47 In attempt to elucidate the mechanism of cell internalization, we conducted experiments at 4 °C and 37 °C with different endocytosis inhibitors. We found that the uptake of both nanovehicles at 4 °C was much less effective than that at 37 °C, indicating that the entries of the formulations were mainly energy-dependent processes (Figure S27). In addition, chlororpromazine and colchicine could also reduce the cell internalization of DOX@PEG-PCys-Chol, implying clathrin-mediated endocytosis and micropinocytosis (Figure S27). Surprisingly, all the inhibitors had an imperceptible effect on the oxidative samples. This result suggests a high efficient translocation of DOX@PEG-PCysChol-O2 across cell membranes, which may be attributed to the cholesterol-bearing rigid and α-helical structure that can interact with and destabilize the cell lipid bilayers for enhanced uptake and subsequent endosome escape.48 Keeping in view the lack of cationic property in our systems compared with most CPPs,49 the highly promoted cell internalization is of great interest and worth further exploration.

Figure 4. (A) CLSM images and (B) flow cytometry histograms of HeLa cells incubated with DOX@PEG-PCys-Chol (a) and DOX@PEG-PCys-Chol-O2 (b) for 4 h. Nuclei of cells were stained with DAPI. (C) Cell viability of HeLa cells incubated for 48 h with DOX@PEG-PCys-Chol (a) and DOX@PEG-PCys-Chol-O2 (b) at different DOX concentrations. Free DOX (c) was used as a positive control.

Taking into account the conformation and morphology transitions of polypeptide that turn on both the “release switch” and “internalization switch” in response to ROS, we further explored their potential applications in biomedical field, taking cancer treatment as a model. We found that both the empty assemblies were cytocompatible (Figure S28), while DOX@PEG-PCys-Chol-O2 exhibited much higher inhibition effect against tumor cells, with a median inhibitory concentration (IC50) value (0.66 µg mL-1) five times lower than that of DOX@PEG-PCys-Chol (Figure 4C), which is consistent with its higher internalization capacity (Figure 4A). However, after intravenously injected to nude mice bearing HeLa tumors, DOX@PEGPCys-Chol-O2 showed significantly lower tumor inhibitory effect than DOX@PEG-PCys-Chol, as verified by the tumor weight inhibition (TWI) and histological analysis (Supporting Information). This result is expectable, since the oxidized assemblies (DOX@PEG-PCys-Chol-O2) lack stimuli-responsivity and functional switch (Figure S25), which may result in nonspecific release and fast clearance in vivo, while PEG-PCys-Chol formulations could provide conformation-specific and ROS-activatable triggers for on-demand cell internalization and drug release in target site. Nonetheless, the structure and shape effect should also be considered and studied in future work. We will further explore the conformation-regulated assembly strategy for other bio-mimicking and biomedical applications. CONCLUSIONS In summary, we have successfully designed and synthesized a cholesterol modified poly(L-cysteine) copolymer.



The oxidation of the thioether groups in the side chains can change the packing characteristics of cholesterol groups and the peptide backbone, resulting in a β-sheet to α-helix transformation, combined with an interesting morphological transition from micelle-like structures to vesicles. The changes of secondary structure and morphology endow the polymer assemblies with excellent specificity for controlled payload release and improved cell interaction in response to ROS. As a result, these smart formulations display excellent biological performance (e.g., anticancer) both in vitro and in vivo. Our work provides a facile strategy for the construction of responsive systems for nanotechnology and biomedical applications.

ASSOCIATED CONTENT Supporting Information. Materials and methods, experimental details, and characterization data (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] or [email protected] Author Contributions ‡These authors contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The research was supported by the National Natural Science Foundation of China (21474064, 51203101); the National Science Fund for Distinguished Young Scholars of China (51425305); and the Project of State Key Laboratory of Polymer Materials Engineering (sklpme2015-3-02). We acknowledge Prof. Zhibo Li and Yuhan Wei at Qingdao University of Science and Technology for cryo-TEM measurement.

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