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Article Cite This: Biomacromolecules 2017, 18, 4349−4356

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Stereoselective Stabilization of Polymeric Vitamin E Conjugate Micelles Min Gao,†,‡ Jian Deng,†,‡ Huiying Chu,‡,§ Yu Tang,∥ Zheng Wang,†,⊥ Yanjun Zhao,*,†,⊥ and Guohui Li*,§ †

School of Pharmaceutical Science and Technology, Tianjin Key Laboratory for Modern Drug Delivery and High Efficiency, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, China § Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023, China ∥ Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yunshan Road, Qingdao, 266003, China ⊥ State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China S Supporting Information *

ABSTRACT: Vitamin E (α-tocopherol; TPGS) micelle is a robust nanocarrier in delivering hydrophobic active pharmaceutical ingredients, but it is suffering from poor stability that is essential in terms of pharmaceutical and biomedical applications. Taking advantage of the chirality of vitamin E, this work reports the stereoselective stabilization of polymer-vitamin E conjugate micelles. Vitamin E was covalently linked to multivalent methoxy poly(ethylene glycol)-co-poly(glutamic acid), generating amphiphilic conjugates that could self-assemble into micelles. Eight types of micelles were produced via tailored combination of polymer backbone and side chain with different chirality. The particle size and critical micelle concentration analysis demonstrated a correlation between conjugate chirality and micelle stability. The most stable micelles were obtained when poly(glutamic acid) and vitamin E both are dextrorotatory, because of the high degree of α-helix revealed by both circular dichroism spectroscopy and molecular dynamics simulation. This phenomenon was further verified by the fluorescence resonance energy transfer (FRET) analysis in HepG2 cells. The current work not only provides a method to enhance the stability of vitamin E micelles, but also adds an additional facile tool in regulating the stability of polymer conjugate micelles without changing the conjugate composition.

1. INTRODUCTION Vitamin E (VE, α-tocopherol) is not only a daily nutrient, but also an important pharmaceutical agent.1 VE has been included in FDA inactive ingredients list for intravenous, oral and topical use. In addition, VE is often utilized as a drug delivery vehicle via the form of tocopherol polyethylene glycol succinate (TPGS) micelles.2 In the pharmaceutical area, TPGS micelles have been primarily employed to solubilize hydrophobic drugs as a biocompatible nanocarrier.3 However, the performance of TPGS micelles has been limited by its poor stability.4 The physical stability of micelles is one key parameter in determining micelles’ in vivo fate and therapeutic efficacy.5 As a major micelle stability indicator, the critical micelle concentration (CMC) of TPGS is fairly high at 0.02% (w/v), that is, 132 μM, which could induce severe premature drug release and altered biodistribution upon dose administration.6 Hence, increasing the stability of TPGS micelles is of great value for the therapeutics delivery purpose. Self-assembled polymer micelles are unique colloidal carriers that can be used for various medical areas.7 Covalent crosslinking has been an effective means for micelle stabilization, but © 2017 American Chemical Society

the cargo activity might be compromised during the tedious and harsh chemical cross-linking processes.8 In contrast, physical stabilization of micelles often involves the manipulation of amphiphilicity, architecture, and crystallinity that are related to the polymer composition, length of hydrophobic segment, and the block length ratio.9−11 Besides these, the chirality is another exploitable factor to manipulate the stability of polymer micelles.12−16 The effect of polymer backbone chirality on micelle self-assembly has been reported previously,16−18 but the influence of side chain spatial arrangement on micelle stabilization was not elucidated yet. Since VE displays different chiral structure, it was postulated that the generation of amphiphilic polymer−VE conjugate and tailored combination of the chirality of VE and polymer backbone could produce ultrastable VE micelles. Therefore, the aim of this work was to investigate the role of stereochemistry of VE and its interplay with backbone conformation in Received: September 29, 2017 Revised: November 16, 2017 Published: November 17, 2017 4349

DOI: 10.1021/acs.biomac.7b01409 Biomacromolecules 2017, 18, 4349−4356

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VE(D,L) and mPEG−PGlu(L)-VE(D,L), respectively. Eight types of micelles were produced (Table S1, SI). 2.3. Secondary Structure Analysis. Circular dichroism (CD) analysis was used to determine the secondary structure of the polymeric conjugates. Eight types of mPEG−PGlu-VE conjugate micelles (Table S1, SI) and four types of mPEG−PGlu polypeptides, that is, mPEG−PGlu(L), mPEG−PGlu(D), mPEG−PGlu(D,L), and mPEG−PGlu(D + L) were tested. Each sample was dissolved in phosphate buffer solution at pH 7.0 or pH 2.0 at a concentration of 0.01 mM. The samples were filtered through a 0.22 μm PVDF filter prior to the measurement; the micellar aqueous solution was placed in a quartz cell with a path length of 1 cm. The spectra were recorded using a Bio-Logic MOS-450 CD spectrometer with the scan rate at 100 nm min−1. All the CD data were collected between 190 and 250 nm at 37 °C. The spectra of conjugates with racemic or mixed backbone at pH 7.0 were shown in Figure S13 (SI). 2.4. Critical Micelle Concentration and Particle Size Determination. The determination of CMC was carried out using a fluorescence method with pyrene as the probe.27 Micellar aqueous solution (0.4−400 μg mL−1) was supplemented with the pyrene probe at a constant concentration (0.5 μM). The emission spectra were recorded from 350 to 420 nm. The excitation wavelength was 333 nm and the spectral slit bandwidth was 5 nm. All samples were measured at 25 °C in a quartz cuvette with 1.0 cm path length. The intensity ratio of probe band at 384 and 373 nm was plotted against the logarithm of the conjugate micelles’ concentration and the CMC was obtained by getting the inflexion point of the sigmoidal curve. Data presented were the average of three individual measurements (mean ± standard deviation; Figure S12, SI). The hydrodynamic diameters of all conjugate micelles were measured by dynamic light scattering. The measurements were taken at 25 °C by a Zetasizer Nano ZS (Malvern Instruments). All samples were prepared at a concentration of 1 mg mL−1 in deionized water. The measurements were performed in triplicate. The core size and morphology of both types of micelles were determined by a JEM-100 CXII transmission electron microscope (TEM). In brief, two drops of aqueous micelle solution (1 mg mL−1) were transferred to carbon-coated copper grids. The excess solution was blotted away with filter paper and the samples were left to dry in the air. Imaging was taken via a Gatan model 782-CJ01 CCD camera (Figure S14, SI). 2.5. Salt and Urea Treatment. Based on the stability screening, three conjugate micelles differing stability were selected for salt (NaCl) or urea challenge experiment, including mPEG−PGlu(D)-VE(D), mPEG−PGlu(D)-VE(D,L), and mPEG−PGlu(D,L)-VE(D,L). The conjugate micelles were treated at two salt concentrations (150 mM and 2 M). The former represents the salt concentration in the extracellular environment and the latter indicates a harsh condition under which the stability of different types of micelles could be readily discriminated.28,29 Likewise, the micelles were also treated by 1 M urea that could interact preferentially with the polypeptide backbone, disrupt the hydrogen bond, and destroy the micelles.30 The hydrodynamic diameters of micelles treated by different destabilizer were monitored 1 day post salt/urea supplementation. 2.6. Nile Red-Loaded Micelles and Probe Release Analysis. To test the stability of polymer-VE conjugate micelles differing in chirality, a fluorescent probe (Nile red) was selected and physically encapsulated in three types micelles: mPEG−PGlu(D)-VE(D), mPEG−PGlu(D)-VE(D,L), and mPEG−PGlu(D,L)-VE(D,L). The encapsulation employed a published film hydration method.31 Briefly, 5 mg Nile red was dissolved in 2 mL ethanol and sonicated for 2 min; 50 mg polymer conjugate was dissolved in 3 mL of ethanol. These ethanol solutions were well-mixed via vortexing. Then, ethanol was evaporated via a vacuum-rotary evaporator to obtain a Nile red-loaded polymer film. The thin film was then hydrated with 6 mL deionized water, followed by vortexing for 2 min and sonication for 5 min. Afterward, the hydrated system was centrifuged for 15 min to remove the excess Nile red. Finally, the Nile red-loaded micelle solution was obtained and freeze-dried for probe content quantification. The loading content of Nile red was determined by high performance liquid chromatography (HPLC) system coupled with a UV detector at

engineering stable polymer−VE conjugate micelles (Scheme 1). Amphiphilic methoxy poly(ethylene)-co-poly(glutamic acid) Scheme 1. Interplay of Poly(glutamic acid) Backbone Chirality and Side Chain (Vitamin E) Chirality on the Stability of Polymer−Vitamin E Conjugate Micellesa

a

D: dextrorotatory; L: levorotatory; D,L: racemic, and D + L: physical mixture of D and L (1:1, molar ratio).

(mPEG−PGlu) copolymer was chosen as the model polymer in the current work due to its biodegradability, biocompatibility, and multivalent effect.19,20 Various investigations have been documented to produce stable α-helical polypeptide via controlling the hydrophobicity, charge repulsion, and intramolecular hydrogen bonding.21−23 Chiral VE shows ultrahigh hydrophobicity (Log P = 10).24 The presence of a hydroxyl group in VE offers an active conjugation site for facile linking to the polymer backbone via ester bond. PGlu can be produced to exhibit four chirality including D, L, (D,L) and D + L (i.e. equal mixture of chiral polymers), whereas there are only two commercially available VE (D and D,L). Hence, eight types of polymer conjugates were generated (Table S1, Supporting Information, SI).

2. EXPERIMENTAL SECTION 2.1. Synthesis of mPEG−PGlu-VE. The synthesis of the mPEG− PGlu-VE employed a previously published method with slight modification.25 In short, mPEG−PGlu (0.1 g, 0.0076 mmol), EDC· HCl (1.09 g, 5.68 mmol), and DMAP (0.69 g, 5.68 mmol) were mixed in 10 mL of DCM, and the mixture was stirred at room temperature for 1 h. Then the mixture was transferred dropwise to a VE-containing (2.45 g, 5.68 mmol) round-bottom flask and continually stirred at ambient temperature for 24 h. Afterward, DCM was condensed to about 10 mL under reduced pressure; the crude product was purified through precipitation in ice-cooled diethyl ether and dialyzing against water (molecular weight cutoff/MWCO: 1000 Da) for 24 h before freeze-drying. Yield: 0.095 g (76.1%). 1H NMR (400 MHz, DMSO-d6) δ [ppm]: 4.14 (m, 1H, -CO-CH(CH2-)-NH-), 3.51 (m, 4H, -O-CH2CH2-), 3.24 (s, 3H, -CH3), 2.26 (m, 2H, Ar-CH2-CH2-), 2.01 (m, 9H, (CH3)3-Ar), 2.0−1.04 (m, 30H, -CH2-CH2-CO-, Ar-CH2-CH2-, -OC(CH2)2-CH3 and -CH2-CH2-CH2-CH(CH3)-), 0.83 (m, 12H, -CH2CH(CH3)-CH2- and -CH2-CH(CH3)2) (Figure S11, SI). The degree of polymerization was 25 and the VE grafting ratio was 32%, that is, each conjugate contained about 8 VE. All the tocopherol conjugate polymers were synthesized using the same protocol. 2.2. Preparation of Conjugate Micelles. The preparation of chiral or racemic mPEG−PGlu-VE polymeric micelle was carried out using a typical dialysis method.26 Briefly, 0.1 g mPEG−PGlu-VE was dissolved in 10 mL of dimethylformamide (DMF) and the solution was dialyzed against deionized water (MWCO: 1000 Da) for 24 h to get the polymeric conjugate micelles. The physically mixed mPEG− PGlu(D + L)-VE(D) or mPEG−PGlu(D + L)-VE(D,L) polymeric micelles were obtained through combining equal molar mPEG− PGlu(D)-VE(D) and mPEG−PGlu(L)-VE(D) or mPEG−PGlu(D)4350

DOI: 10.1021/acs.biomac.7b01409 Biomacromolecules 2017, 18, 4349−4356

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Biomacromolecules 553 nm. The sample was dissolved in methanol and diluted with appropriate ratio. A Gemini C18 reverse phase column (5 μm, 250 mm × 4.6 mm) (Phenomenex, Beijing, China) was used for the analysis at 30 °C. The mobile phase was methanol and the injection volume was 20 μL. All the measurements were carried out in triplicate. HepG2 cells were seeded on confocal laser scanning microscopy (CLSM) plates at a density of 1 × 105 cells/well containing 1 mL of DMEM medium followed by 24 h’s incubation. Then, the medium was removed and followed by the addition of 1 mL of medium containing probe-loaded micelles at a Nile red dose of 2 μg. The cells were cultured at 37 °C and 5% CO2 for 2 h. The micelle-containing medium was replaced with 1 mL fresh medium to remove the micelles that located outside the cells; this time point was defined as “0 h”. The cells were cultured for another 8 h. At 3 different time points (0, 4, and 8 h), the cells were rinsed with PBS in triplicate and fixed with 1 mL paraformaldehyde (4%) for 20 min with light protection. Subsequently, the paraformaldehyde was removed following the cell rinse by 1 mL of PBS in triplicate. The cell nuclei were stained with 500 μL of DAPI solution (5 μg/mL) for 10 min and washed with 1 mL of PBS three times ready for CLSM image-taking. The excitation and emission wavelength for DAPI was 405 and 450−540 nm, respectively. The excitation and emission wavelength for Nile red was 561 and 600−700 nm, correspondingly. 2.7. FRET Analysis. Cy3 and Cy5 were mixed with 3 different polymer-VE conjugates at a feeding ratio of 1:1:10 (w/w/w): mPEG− PGlu(D)-VE(D), mPEG−PGlu(D)-VE(D,L), and mPEG−PGlu(D,L)-VE(D,L). The probe encapsulation and purification process were similar to that of Nile red loading in section 2.6 (n = 3). The probe loading was quantified by a Cary 60 UV−vis absorption spectrophotometer. The micelles were incubated with HepG2 cells under normal culturing conditions at a fixed Cy5 dose of 5 μg/mL. After 2 h, the excess micelles were cleaned by PBS washing in triplicate, which was labeled as “time point 0”. The cells were further incubated for 4 h. At time point (0, 2, and 4 h), the cells were fixed using 1 mL paraformaldehyde (4%) for 20 min followed by imaging at two wavelengths: 568 nm (Cy3 excitation) and 633 nm (Cy5 excitation); the emission wavelength was the same at 650−750 nm. The FRET percentage was taken as the ratio between the fluorescence intensity excited at 568 nm and that excited at 633 nm. For different micelles, the FRET ratio was plotted against incubation time to obtain and compare the slope. DAPI probe was used to stain nucleus throughout the FRET study. 2.8. Molecular Dynamics Simulation. Three micelles differing in stability were picked up for molecular dynamics (MD) simulations investigation, that is, mPEG−PGlu(D)-VE(D) > mPEG−PGlu(D)VE(D,L) > mPEG−PGlu(D,L)-VE(D,L). The parameters of Glu-VE residue was built following the AMBER tutorials. Considering our computation capability, a 12-residue peptides of poly(glutamic acid) (PGlu) were selected to carry out the MD simulations. Two chiral versions of PGlu backbone were used, that is, a homochiral PGlu(D) and a racemic PGlu(D,L). For the latter, the sequence was defined as LLDDDLLLDDLD by shuffling a sequence with six L and six D chiral centers. The side chains were kept fully charged for the carboxyl sites without VE modification. For each backbone, four VE were conjugated at the position of 2, 5, 8, and 11 in the constructed sequence, that is, LLDDDLLLDDLD. Either homochiral D version VE(D) or racemic VE(D,L) was employed for one single polymer conjugate. Regarding the racemic VE, the chirality of VE was set at LDLD corresponding to the 2, 5, 8, and 11 positions. These single chains were initialized into a random coil conformation. To assess the interactions between different polymer conjugates, two coiled chains were placed interlaced to each other with 1 nm distance apart. All systems were solvated in a periodic box of TIP3P water model, and the minimum distance from the edges of the solvent box to the closest atom the complex was set at 10 Å. An appropriate number of neutralized ions were added to the entire systems. AMBER14 was used for MD simulations at the periodic boundary condition,32 and AMBER14SB force field was employed for all simulations.33 Energy minimization was carried out using the steepest descent minimization of 1000 steps followed by 9000 step conjugate gradient minimization. The step size was

maintained at 2 fs under the condition of a direct-space and nonbonded cutoff of 10 Å. The SHAKE algorithm was adopted to constrain all bonds involving the hydrogen. The particle mesh Ewald method was applied to treat the long-range electrostatic interactions,34 for which the time course of molecular dynamics was 500 ns (single chain) and 200 ns (double chain) at a constant pressure of 1 bar and a fixed temperature at 300 K. The secondary structure was determined using the DSSP (Define Secondary Structure of Proteins) criteria for all simulations.35 2.9. Statistical Analysis. The data were expressed as the mean ± standard deviation (SD). A statistically significant difference was determined at a minimal level of significance of 0.05 via one-way ANOVA (analysis of variance) coupled with Turkey’s posthoc test.

3. RESULTS AND DISCUSSION 3.1. Conjugate Synthesis and Characterization. mPEG−PGlu was synthesized via the ring-opening polymerization of glutamic acid N-carboxyanhydride (NCA) monomers. Both functional monomer route and post-polymerization modification route were employed to prepare VE-modified mPEG−PGlu.23 Regarding the former approach, VE was successfully linked to the NCA monomer, but the ultimate polymerization step failed possibly due to the steric effect from the bulky side chain (Scheme S1, Figures S1−S6, Supporting Information, SI). Therefore, VE was chemically bonded to the backbone via the latter postpolymerization modification method. The average degree of polymerization was 25 and each conjugate contained ca. 8 side chain VE molecules, which was kept constant for all samples (Scheme S2, Figures S7−S11, SI). The incomplete functionalization was thought to be due to the steric crowding and inefficiency in coupling, which was a typical phenomenon in post-polymerization modification.36 3.2. Secondary Structure Analysis. The circular dichroism (CD) analysis showed that both mPEG−PGlu(L) and mPEG−PGlu(D) backbone displayed a random coil structure at neutral pH because of the electrostatic repulsion between the excess carboxyl groups in the backbone (Figure 1). The presence of VE(D) converted the conjugate to α-helix

Figure 1. Secondary structure of six conjugate micelles at pH 7.0 and 2.0. The circular dichroism spectra of D-polymer (a, b) and L-polymer (c, d) micelles were presented. Except chirality, all conjugated exhibited the same composition, molecular weight, and degree of polymerization and conjugation. mPEG, PGlu, and VE indicates methoxy poly(ethylene glycol), poly(glutamic acid), and vitamin E (αtocopherol), respectively. Detailed information on micelle composition was shown in Table S1. 4351

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for example, mPEG−PGlu(D + L)-VE(D) was a reflection of the CMC of the two constituent polymer. These data demonstrated that simple manipulation of the chirality of backbone and side chain (e.g. VE) could tune the micelle stability without changing the copolymer composition. 3.4. Particle Size Analysis. The hydrodynamic sizes of polymer−VE conjugate micelles showed a noticeable correlation with their CMC values (Figure 3). A lower CMC indicated

secondary structure via the inhibition of charge repulsion, evidenced by the appearance of characteristic CD peak at 208 and 222 nm.28,37 However, the effect of racemic VE(D,L) on inducing α-helix structure was less superior to its counterpart, VE(D), which signified that the spatial arrangement of side chain could make an impact on the secondary structure of polymer conjugates. The medium pH also influenced the polymer conformation. At pH 2.0, mPEG−PGlu(L) and mPEG−PGlu(D) both adopted the α-helix conformation in contrast to the random coil at pH 7.0. Four types of VEmodified conjugates with a chiral backbone all demonstrated an enhanced helicity upon pH reduction from 7.0 to 2.0. This was contributed by the suppression of charge repulsion due to the shift of unconjugated carboxyl group (pKa: ca. 4.0) from ionized −COO− to neutral −COOH.38 Irrespective of medium pH, right-handed α-helix formed in L-polymer and left-handed α-helix developed in D-polymer, which agreed well with the previous finding.16 At pH 7.0, there was no detectable helicity for conjugates with racemic or mixed backbone (Figure S13, SI). 3.3. Critical Micelle Concentration Determination. The physical stability of conjugate assembled micelles was assessed using the critical micelle concentration (CMC) as the index (Figure 2, Figure S12, SI). A lower CMC implies better micelle

Figure 3. Hydrodynamic diameters of eight types of mPEG−PGlu-VE conjugate micelles with different chirality (n = 3).

a stronger driving force of micelle formation with the hydrophobic segment packing together within the particle interior and hydrophilic PEG facing the aqueous media.40 Thus, more stable micelles would have a relatively dense core at the premise of the same hydrophobic moiety, leading to a smaller hydrodynamic diameter. For example, the most stable mPEG− PGlu(D)-VE(D) displayed the smallest micelle diameter at 122.7 ± 13.2 nm; the least stable mPEG−PGlu(D,L)-VE(D,L) exhibited the largest micelle size at 168.7 ± 9.3 nm. The transmission electron microscope (TEM) images revealed that the micelle core sizes were smaller than the corresponding hydrodynamic sizes (Figure S14, SI). 3.5. Salt and Urea Challenge. It was expected that micelle integrity would be compromised with increasing salt concentration, which could be used for indirect micelle stability assessment.28 The micelle stability against salt (NaCl) and urea was investigated using three samples including mPEG− PGlu(D)-VE(D), mPEG−PGlu(D)-VE(D,L), and mPEG− PGlu(D,L)-VE(D,L) (Figure 4). Two NaCl concentrations were employed, that is, 150 mM and 2 M. The former mimics the concentration of NaCl in the extracellular environment.29 Although NaCl was known for its helix-destabilizing effect,28 mPEG−PGlu(D)-VE(D) micelles showed remarkable stability against NaCl concentration upsurge. mPEG−PGlu(D,L)-VE(D,L) micelles displayed the worst stability at the presence of salt; even at the condition of 150 mM NaCl, two peaks were apparent, indicating micelle aggregation. mPEG−PGlu(D)VE(D,L) micelles seemed able to moderately tolerate the low

Figure 2. Critical micelle concentration (CMC) of eight types of polymer conjugate micelles (n = 3). mPEG (10000 Da), PGlu, and VE indicates methoxy poly(ethylene glycol), poly(glutamic acid), and vitamin E (α-tocopherol), respectively. The degree of glutamic acid polymerization is 25; for each backbone, there are 8 vitamin E molecules in the side chain for all conjugates. D + L represents the physically mixed backbone with equal molar ratio.

stability. The CMC determination employed a standard fluorescence method with pyrene as the probe.39 Interestingly, tailoring the chirality of VE and backbone indeed affects the stability of conjugate micelles. The most and least stable conjugate micelle was mPEG−PGlu(D)-VE(D) and mPEG− PGlu(D,L)-VE(D,L), respectively. The conjugates with chiral backbones, that is, mPEG−PGlu(D) and mPEG−PGlu(L), usually produced micelles with a lower CMC, whereas the racemic mPEG−PGlu(D,L) generated micelles with a higher CMC. The CMC of micelles with physically mixed backbone, 4352

DOI: 10.1021/acs.biomac.7b01409 Biomacromolecules 2017, 18, 4349−4356

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Figure 4. Effect of salt (NaCl) or urea challenge on the hydrodynamic size of three different conjugate micelles. The stability of tested micelles ranked as follows: mPEG−PGlu(D)-VE(D) > mPEG−PGlu(D)-VE(D,L) > mPEG−PGlu(D,L)-VE(D,L).

Figure 5. Release kinetics of a fluorescent probe, Nile red from three conjugate micelles with different stability in HepG2 cells. The same probe dose was applied for all samples. Two hours post micelle treatment, the medium was replaced with fresh one to remove those micelles remaining outside the cells and this time point was recorded as “0 h”; the cells were further incubated for 8 h. Images were taken at 0, 4, and 8 h by confocal laser scanning microscope. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus (Ex = 405 nm, Em = 450−540 nm). The excitation and emission wavelength of Nile red was 561 and 600−700 nm, respectively. Scale bar = 30 μm.

the probe release from all three micelles upon internalization (Figure 5). The number of very bright red dots (as an indicator of probe-loaded micelles) gradually reduced over time and the red color spread through the whole cell for all samples. However, the rate of such reduction is much higher for D,Lpolymer micelles without secondary structure in comparison to the other two micelles. There was only tiny difference regarding probe release kinetics between mPEG−PGlu(D)-VE(D) and mPEG−PGlu(D)-VE(D,L). The stable D-polymer micelles with the α-helical structure were presumed to experience a gradual disintegration via an erosion-like process, hence resulting in sustained probe release. In contrast, the unstable D,L-polymer micelles without the helical structure would show an accelerated disassembly and rapid cargo release. This phenomenon coincided with the observation in previous report.16 Performing the release study in cells is superior to that conducted in aqueous buffers because the stability of micelles in a biological system could be revealed. In addition, there is no need to incorporate the solubilization agents in cells to maintain the sink conditions. 3.7. FRET Analysis. FRET has been an advantageous biomedical tool to assess the stability of micelles.41,42 Theoretically, the FRET phenomenon is extremely sensitive to the distance between donor and acceptor. When the both chromophores were confined in a micellar carrier, the efficiency

concentration of salt (150 mM), but at the 2 M NaCl condition, the stability was destroyed with the appearance of large particles over 1 μm. These results occurred well with the CMC and CD data (Figures 1 and 2). Namely, micelles with higher degree of helicity exhibited better stability that was proven by the lower CMC and greater capability against salt challenge. Similarly, upon the supplementation of a denaturing agent, urea (1 M), the coiled mPEG−PGlu(D,L)-VE(D,L) micelle lost its integrity in contrast to the other two stable counterparts: mPEG−PGlu(D)-VE(D) and mPEG−PGlu(D)VE(D,L) (Figure 4). 3.6. Fluorescent Probe Release. To assess the interplay of backbone chirality and VE chirality on the micelle stability, we performed the cellular internalization study in the human hepatocellular liver carcinoma (HepG2) cells. A fluorescent molecule, Nile red, was encapsulated in three selected micelles differing in stability. The probe loading was determined at 1.0 ± 0.2%, 1.0 ± 0.2%, and 1.5 ± 0.2% (w/w) for mPEG− PGlu(D)-VE(D), mPEG−PGlu(D)-VE(D,L), and mPEG− PGlu(D,L)-VE(D,L), respectively. The relatively higher probe loading in D,L-polymer conjugate micelles was assumed due to the fairly loose packing of hydrophobic segment and the availability of more void space for cargo entrapment. This could be indirectly proven by the particle size analysis (Figure 3). The confocal laser scanning microscope (CLSM) images showed 4353

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Figure 6. (a) Kinetic FRET images of three types of Cy3/Cy5-loaded vitamin E (VE) micelles differing in stability: mPEG−PGlu(D)-VE(D) > mPEG−PGlu(D)-VE(D,L) > mPEG−PGlu(D,L)-VE(D,L) (scale bar: 20 μm); (b) Curve of FRET ratio against time for 3 types of selected micelles; the slope was compared statistically (n = 3). *p < 0.05.

Figure 7. Molecular dynamics (MD) simulations of three types of vitamin E-modified peptide conjugates. Visualization images (a−c) and residue maps (d−f) indicate the secondary structure of conjugates from representative MD simulations for single chain conjugate with a backbone polymerization degree of 12 and each backbone contains 4 vitamins. All conjugates are initially set to adopt a random coil conformation and allowed to equilibrate for 500 ns. (a) A typical simulation of homochiral PGlu(D)-VE(D) dictates the presence of helix structure; (b) The conjugate PGlu(D)-VE(D,L) containing racemic side chains display no helix conformation; (c) the conjugate PGlu(D,L)-VE(D,L) with both racemic backbone and side chains shows a mostly turn and bend structure. Maps of secondary structures as a function of time was also shown in (d) PGlu(D)-VE(D), (e) PGlu(D)-VE(D,L), and (f) PGlu(D,L)-VE(D,L). PGlu and VE indicates poly(glutamic acid) and vitamin E (α-tocopherol), respectively.

of energy transfer would be very high. In contrast, when the donor and acceptor are released from the micelles, the FRET phenomenon will be less evident. The rate and extent to which cargos are released from micelles are dependent on micelle stability, which enables FRET as an indirect method for assessing micelle stability. In the current work, Cy3 and Cy5 were selected as the FRET pair. The Cy3 loading in micelles was 2.0 ± 0.9% (mPEG−PGlu(D)-VE(D)), 2.3 ± 0.6% (mPEG−PGlu(D)-VE(D,L)), and 1.8 ± 0.1% (mPEG−PGlu(D,L)-VE(D,L)), respectively. The corresponding Cy5 loading was 1.4 ± 0.6% (mPEG−PGlu(D)-VE(D)), 1.3 ± 0.4% (mPEG−PGlu(D)-VE(D,L)), and 1.2 ± 0.3% (mPEG−PGlu(D,L)-VE(D,L)). It was clear that relatively stable micelles displayed slower probe release compared to the less stable micelles (Figure 6a). As an indirect index of micelle stability, the slope of kinetic FRET ratio curve was obtained (Figure 6b). The absolute slope values of mPEG−PGlu(D)-VE(D) and mPEG−PGlu(D)-VE(D,L) were significantly smaller than that of mPEG−PGlu(D,L)-VE(D,L) (p < 0.05). Such a trend was consistent with previous in vitro stability assay (Figures 2−5). Similar to the release study, carrying out FRET analysis in a cell model other than aqueous buffers would demonstrate the influence of chirality on the micelle stability in a more biologically relevant environment. 3.8. Molecular Dynamics. Molecular dynamics (MD) simulations was performed to investigate how the tailored combination of backbone and side chain with different chirality could affect the secondary structure of conjugates and, hence,

the stability of corresponding assembled micelles (Figure 7). The same conjugate systems were selected, that is, PGlu(D)VE(D), PGlu(D)-VE(D,L), and PGlu(D,L)-VE(D,L). To match the composition of these conjugates, simulations were run for a 12-residue PGlu coupled with a 4-residue VE over the time course of 500 ns. The reason for selecting 12-residue other than 25-residue was to control the simulation work load. As expected, PGlu(D)-VE(D) formed α-helix structure as well as the intermediary 3-helix conformation, which agreed well the CD spectrum of mPEG−PGlu(D)-VE(D) (Figure 1). Both PGlu(D)-VE(D,L) and PGlu(D,L)-VE(D,L) remained mostly in coil, bend, and turn conformation. Interestingly, MD simulations demonstrated that PGlu(D)-VE(D,L) did not display apparent helicity in contrast to the CD result. Such discrepancy might be due to the employment of shorter sequence of backbone in simulation.17,43 Another trend was that VE tended to pack together in one direction upon equilibrium, which could be a consequence of the strong hydrophobic interaction between VEs (Log P = 10).24 It was evident that VE distribution was a little scattered for PGlu(D,L)-VE(D,L) when compared to the other two counterparts, which indicated this conjugate would occupy more space within the micellar core. The particle size analysis justly concurred with this postulation (Figure 3, Figure S14, SI). To determine the assembly performance of conjugates, the interaction between two conjugates was investigated via simulation over 200 ns. Analogous to the behavior of single chain, two chains of PGlu(D)-VE(D) bound tightly to each 4354

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Biomacromolecules other with one chain exhibiting considerably enhanced helicity and the other remaining as disordered conformation (Figure S15, SI). The interaction between two PGlu(D)-VE(D,L) conjugates revealed that the bend conformation was dominant in one chain and the coil structure was prevailing in the other (Figure S16, SI). In terms of PGlu(D,L)-VE(D,L), one chain mainly adopted coil and bend conformation, whereas 3-helix together with coil and bend was evident in the other chain (Figure S17, SI). Irrespective of the conjugate type and chirality, both chains seemed able to adjust their own spatial arrangement to aid the interaction with others.44 Therefore, each polymer conjugate might adopt different structure upon self-assembling into micelles. These MD data further supports the direct and indirect assays of micelle stability. 3.9. Biocompatibility Assay. The effect of three selected micelles on the viability of 3T3 model cells was investigated (Figure S18, SI). Regardless of the type and stability of micelles, they all exhibited good biocompatibility up to a dose of 500 μg/ mL. Since the building blocks of the micelles (e.g. mPEG, glutamic acid, and vitamin E) are common pharmaceutical excipients, the absence of cytotoxicity is expected. As a biocompatible vehicle, the stability of polypeptide-VE conjugate micelles could be tailored simply by chirality manipulation to balance the premature drug release during systemic circulation and intracellular drug release and hence enhance the delivery efficiency.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yu Tang: 0000-0001-8224-4639 Yanjun Zhao: 0000-0001-5739-1960 Author Contributions ‡

M.G., J.D., and H.C. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the funding support from the open fund from State Key Laboratory of Medicinal Chemical Biology (Nankai University; 2017030), National Natural Science Foundation of China (21625302 and 91430110), and the innovation fund of Tianjin University (1706).

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ABBREVIATIONS TPGS, α-tocopherol polyethylene glycol succinate; FRET, fluorescence resonance energy transfer; mPEG, methoxy poly(ethylene glycol); PGlu, poly(glutamic acid); VE, vitamin E; CMC, critical micelle concentration; NCA, N-carboxyanhydride; CD, circular dichroism; HepG2, human hepatocellular liver carcinoma; CLSM, confocal laser scanning microscope; MD, molecular dynamics; DMF, dimethylformamide; DCM, dichloromethane; DMAP, 4-dimethylaminopyridine; MWCO, molecular weight cutoff; HPLC, high performance liquid chromatography; DMEM, dulbecco’s modification of eagle’s medium; ANOVA, analysis of variance

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CONCLUSION In summary, we generated vitamin E-modified ultrastable polymer conjugate micelles and demonstrated that the chirality patterns of both backbone and side chain could play a significant role in tailoring the micelle stability without altering the chemical composition of conjugates. When both the backbone and VE are dextrorotatory, the obtained conjugate displayed the highest helicity and hence produced the most stable micelles, which was proven by the lowest CMC, the superior capability to resist salt and urea challenge, and the sustained release of a model fluorescent probe in HepG2 cells. In contrast, the combination of both racemic backbone and racemic VE resulted in the micelle with the worst stability. These experimental observations were well supported by the MD simulations and FRET analysis in HepG2 cells. The homochiral mPEG−PGlu(D)-VE(D) was liable to adopt a stable helical conformation. Such ordered hierarchical structure could facilitate the self-assembly and micelle formation, which was not observed in other types conjugates without the perfect chirality matching. The approach to tune the micelle stability via facile manipulating the spatial conformation of side chain and its interplay with the backbone provides a novel concept in improving the performance of polymer conjugate micellar nanomedicine. In addition, such simple strategy would also find application in designing tailored polymer-drug conjugates or other types of materials that could be used in a broad variety of pharmaceutical and biomedical fields.

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The synthesis and characterization of polymer conjugates and relevant intermediate products and other supplementary figures (PDF).

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REFERENCES

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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.biomac.7b01409. 4355

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