Stereoselective Stabilization of Polymeric Vitamin E Conjugate

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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 Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01409 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 26, 2017

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Stereoselective Stabilization of Polymeric Vitamin E Conjugate Micelles Min Gao, 1,† Jian Deng, 1,† Huiying Chu, 2,† Yu Tang, 3 Zheng Wang, 1,4 Yanjun Zhao*,1,4 Guohui Li*,2 1

School of Pharmaceutical Science & Technology, Tianjin Key Laboratory for Modern Drug Delivery

& High Efficiency, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, China 2

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023, China

3

School of medicine and pharmacy, Ocean University of China, 5 Yunshan Road, Qingdao, 266003, China

4

State Key Laboratory of Medicinal Chemical Biology (Nankai University), Tianjin 300071, China

ACS Paragon Plus Environment

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ABSTRACT: Vitamin E (α-tocopherol) (TPGS) micelle is a robust nanocarrier in delivering hydrophobic active pharmaceutical ingredients, but 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.

KEYWORDS: Vitamin E, micelle, stability, stereoselective, self-assembly.


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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 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), i.e. 132 µM, which could induce severe premature drug release and altered biodistribution upon dose administration.6 Hence, increasing the stability of TPGS micelles are of great value for 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 the cargo activity might be compromised during the tedious and harsh chemical crosslinking 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 ultra-stable VE micelles. Therefore, the aim of this work was to investigate the role of


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stereochemistry of VE and its interplay with backbone conformation in engineering stable polymer-VE conjugate micelles (Scheme 1). Amphiphilic methoxy poly(ethylene)-poly(glutamic acid) (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 ultra-high 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).

Scheme 1. Interplay of poly(glutamic acid) backbone chirality and side chain (vitamin E) chirality on the stability of polymer-vitamin E conjugate micelles. D: dextrorotatory; L: levorotatory; D,L: racemic, D + L: physical mixture of D and L (1:1, molar ratio). 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 DCM and the mixture was


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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. Afterwards, DCM was condensed to ca. 10 mL under reduced pressure; the crude product was purified through precipitation in ice-cooled diethyl ether and dialyzing against water (molecular weight cut-off/MWCO: 1,000 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-CH2-CH2-), 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-, -O-C(CH2)2CH3 and -CH2-CH2-CH2-CH(CH3)-), 0.83 (m, 12H, -CH2-CH(CH3)-CH2- and -CH2-CH(CH3)2) (Figure S11, SI). The degree of polymerization was 25 and the VE grafting ratio was 32%, i.e. each conjugate contained ca. 8 VE. All the tocopherol conjugate polymers were synthesized in 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 dimethyl formamide (DMF) and the solution was dialyzed against deionized water (MWCO: 1,000 Da) for 24 h to get the polymeric conjugate micelles. The physically mixed mPEGPGlu(D+L)-VE(D) or mPEG-PGlu(D+L)-VE(D,L) polymeric micelles were obtained through combing equal molar mPEG-PGlu(D)-VE(D) and mPEG-PGlu(L)-VE(D) or mPEG-PGlu(D)-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, i.e. 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 sampled were filtered through a 0.22 µm PVDF filter prior to the measurement; the micellar aqueous solution was placed in a quartz cell with


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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 nm and 250 nm at 37oC. 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 nm to 420 nm. The excitation wavelength was 333 nm and the spectral slit bandwidth was 5 nm. All samples were measured at 25oC in a quartz cuvette with 1.0 cm path length. The intensity ratio of probe band at 384 nm and 373 nm was plotted against the logarithm of the conjugate micelles’ concentration and the CMC was obtained by getting the flexion 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 25oC 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


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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 mPEGPGlu(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 2 min; 50 mg polymer conjugate was dissolved in 3 mL 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. Afterwards, 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 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 30oC. 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 DMEM medium followed by 24 h’s incubation. Then, the medium was removed and followed by the addition of 1 mL medium containing probe-loaded micelles at a Nile red dose of 2 μg. The cells were cultured at 37oC and 5%


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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 h, 4h 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 PBS in triplicate. The cell nuclei were

stained with 500 μL DAPI solution (5 μg/mL) for 10 min and washed with 1 mL PBS three times ready for CLSM image-taking. The excitation and emission wavelength for DAPI was 405 nm and 450-540 nm, respectively. The excitation and emission wavelength for Nile red was 561 nm 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 hours, the excess micelles were cleaned by PBS washing in triplicate, which was labelled as “time point 0”. The cells were further incubated for 4 hours. At time point (0 h, 2 h, 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.


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2.8 Molecular dynamics simulation. Three micelles differing in stability were picked up for molecular dynamics (MD) simulations investigation, i.e. 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 out 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, i.e. 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, i.e. 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 10Å. 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 non-bonded 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.


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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 post-hoc 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, Figure S1-S6, Supporting Information/SI). Therefore, VE was chemically bonded to the backbone via the latter post-polymerization 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, Figure S7-S11, SI). The incomplete functionalization was thought 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 mPEGPGlu(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 secondary structure via the inhibition of charge repulsion, evidenced by the appearance of characteristic CD peak at 208 nm and 222 nm.28,37 However, the effect


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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, mPEGPGlu(L) and mPEG-PGlu(D) both adopted the α-helix conformation in contrast to the random coil at pH 7.0. Four types of VE-modified 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).

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 of micelle composition was shown in Table S1.


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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 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, i.e. mPEG-PGlu(D) and mPEG-PGlu(L), usually produced micelles with lower CMC, whereas the racemic mPEG-PGlu(D,L) generated micelles with higher CMC. The CMC of micelles with physically mixed backbone, e.g. 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.

Figure 2. The critical micelle concentration (CMC) of eight types of polymer conjugate micelles (n = 3). mPEG (10,000 Da), PGlu and VE indicates methoxy poly(ethylene glycol), poly(glutamic acid) and


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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. 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 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).


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Figure 3. The hydrodynamic diameters of eight types of mPEG-PGlu-VE conjugate micelles with different chirality (n = 3). 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 mPEGPGlu(D)-VE(D), mPEG-PGlu(D)-VE(D,L), and mPEG-PGlu(D,L)-VE(D,L) (Figure 4). Two NaCl concentrations were employed, i.e. 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 mPEGPGlu(D)-VE(D) micelles showed remarkable stability against NaCl concentration upsurge. mPEGPGlu(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 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 (Figure 1-2). 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).


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Figure 4. The 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) > mPEGPGlu(D)-VE(D,L) > mPEG-PGlu(D,L)-VE(D,L). 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 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,L-polymer 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


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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.

Figure 5. The 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 h, 4 h, 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 nm and 600-700 nm, respectively. Scale bar = 30 µm.


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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 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% (mPEGPGlu(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 trend was consistent with previous in vitro stability assay (Figure 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.


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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) The curve of FRET ratio against time for 3 types of selected micelles; the slope was compared statistically (n = 3). * p < 0.05. 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, i.e. 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 of 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 other with one chain exhibiting


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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.

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)


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PGlu(D,L)-VE(D,L). PGlu and VE indicates poly(glutamic acid) and vitamin E (α-tocopherol), respectively. 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. CONCLUSION In summary, we generated vitamin E-modified ultra-stable 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


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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. ASSOCIATED CONTENT Supporting Information. The synthesis and characterization of polymer conjugates and relevant intermediate products and other supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected] or [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. † M. Gao, J. Deng and H. Chu contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT 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 (91430110) and the innovation fund of Tianjin University (1706). ABBREVIATIONS


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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, dimethyl formamide; 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. REFERENCES (1) Brigelius-Flohe, R. E. G. I.; Traber, M. G. Vitamin E: Function and metabolism. FASEB J. 1999, 13, 1145-1155. (2) Muddineti, O. S.; Ghosh, B.; Biswas, S. Current trends in the use of vitamin E-based micellar nanocarriers for anticancer drug delivery. Expert Opin. Drug Deliv. 2017, 14, 715-726. (3) Hao, T.; Chen, D.; Liu, K.; Qi, Y.; Tian, Y.; Sun, P.; Liu, Y.; Li, Z. Micelles of d-alpha-tocopheryl polyethylene glycol 2000 succinate (TPGS 2K) for doxorubicin delivery with reversal of multidrug resistance. ACS Appl. Mater. Interfaces 2015, 7, 18064-18075. (4) Duhem, N.; Danhier, F.; Preat, V. Vitamin E-based nanomedicines for anti-cancer drug delivery. J. Controlled Release 2014, 182, 33-44. (5) Xin, K.; Li, M.; Lu, D.; Meng, X.; Deng, J.; Kong, D.; Ding, D.; Wang, Z.; Zhao, Y. Bioinspired coordination micelles integrating high stability, triggered cargo release, and magnetic resonance imaging. ACS Appl. Mater. Interfaces 2017, 9, 80-91.


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(6) Owen, S. C.; Chan, D. P. Y.; Shoichet, M. S. Polymeric micelle stability. Nano Today 2012, 7, 5365. (7) Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Micellar nanocontainers distribute to defined cytoplasmic organelles. Science 2003, 300, 615-618. (8) Talelli, M.; Barz, M.; Rijcken, C. J.; Kiessling, F.; Hennink, W. E.; Lammers, T. Core-crosslinked polymeric micelles: Principles, preparation, biomedical applications and clinical translation. Nano Today 2015, 10, 93-117. (9) Wieczorek, S.; Schwaar, T.; Senge, M. O.; Boerner, H. G. Specific drug formulation additives: revealing the impact of architecture and block length ratio. Biomacromolecules 2015, 16, 3308-3312. (10) Glavas, L.; Olsen, P.; Odelius, K.; Albertsson, A. C. Achieving micelle control through core crystallinity. Biomacromolecules 2013, 14, 4150-4156. (11) Wang, Z.; Chen, C.; Zhang, Q.; Gao, M.; Zhang, J.; Kong, D.; Zhao, Y. Tuning the architecture of polymeric conjugate to mediate intracellular delivery of pleiotropic curcumin. Eur. J. Pharm. Biopharm. 2015, 90, 53-62. (12) Priftis, D.; Leon, L.; Song, Z.; Perry, S. L.; Margossian, K. O.; Tropnikova, A.; Cheng, J.; Tirrell, M. Self-assembly of alpha-helical polypeptides driven by complex coacervation. Angew. Chem. Int. Ed. 2015, 54, 11128-11132. (13) Ding, J.; Li, C.; Zhang, Y.; Xu, W.; Wang, J.; Chen, X. Chirality-mediated polypeptide micelles for regulated drug delivery. Acta. Biomater. 2015, 11, 346-355. (14) He, T.; Li, D.; Yang, Y.; Ding, J.; Jin, F.; Zhuang, X.; Chen, X. Mesomeric configuration makes polyleucine micelle an optimal nanocarrier. Biomater. Sci. 2016, 4, 814-818.


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(15) Shen, K.; Li, D.; Guan, J.; Ding, J.; Wang, Z.; Gu, J.; Liu, T.; Chen, X. Targeted sustained delivery of antineoplastic agent with multicomponent polylactide stereocomplex micelle. Nanomedicine. 13, 1279-1288. (16) Mochida, Y.; Cabral, H.; Miura, Y.; Albertini, F.; Fukushima, S.; Osada, K.; Nishiyama, N.; Kataoka, K. Bundled assembly of helical nanostructures in polymeric micelles loaded with platinum drugs enhancing therapeutic efficiency against pancreatic tumor. ACS Nano 2014, 8, 6724-6738. (17) Perry, S. L.; Leon, L.; Hoffmann, K. Q.; Kade, M. J.; Priftis, D.; Black, K. A.; Wong, D.; Klein, R. A.; Pierce, C. F.; Margossian, K. O.; Whitmer, J. K.; Qin, J.; de Pablo, J. J.; Tirrell, M. Chiralityselected phase behaviour in ionic polypeptide complexes. Nat. Commun. 2015, 6, 6052. (18) Soleymani, A. H.; Vakili, M. R.; Shafaati, A.; Lavasanifar, A. Block copolymer stereoregularity and its impact on polymeric micellar nanodrug delivery. Mol. Pharmaceutics 2017, 14, 2487-2502. (19) Lalatsa, A.; Schatzlein, A. G.; Mazza, M.; Le, T. B.; Uchegbu, I. F. Amphiphilic poly(L-amino acids) - new materials for drug delivery. J. Controlled Release 2012, 161, 523-536. (20) Lavasanifar, A.; Samuel, J.; Kwon, G. S. Poly(ethylene oxide)-block-poly(l-amino acid) micelles for drug delivery. Adv. Drug Delivery Rev. 2002, 54, 169-190. (21) Lyu, P. C.; Sherman, J. C.; Chen, A.; Kallenbach, N. R. Alpha-helix stabilization by natural and unnatural amino acids with alkyl side chains. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 5317-5320. (22) Tang, H.; Zhang, D. General route toward side-chain-functionalized alpha-helical polypeptides. Biomacromolecules 2010, 11, 1585-1592.


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(23) Xiong, M.; Lee, M. W.; Mansbach, R. A.; Song, Z.; Bao, Y.; Peek, R. M.; Yao, C.; Chen, L.; Ferguson, A. L.; Wong, G. C.; Cheng, J. Helical antimicrobial polypeptides with radial amphiphilicity. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 13155-13160. (24) Zhao, Y.; Moddaresi, M.; Jones, S. A.; Brown, M. B. A Dynamic Topical hydrofluoroalkane foam to induce nanoparticle modification and drug release in situ. Eur. J. Pharm. Biopharm. 2009, 72, 521-528. (25) Gao, M.; Liu, S.; Fan, A.; Wang, Z.; Zhao, Y. Nitric oxide-releasing graft polymer micelles with distinct pendant amphiphiles. RSC Adv. 2015, 5, 67041-67048. (26) Li, X.; Gao, M.; Xin, K.; Zhang, L.; Ding, D.; Kong, D.; Wang, Z.; Shi, Y.; Kiessling, F.; Lammers, T.; Cheng, J.; Zhao, Y. Singlet oxygen-responsive micelles for enhanced photodynamic therapy. J. Controlled Release 2017, 260, 12-21. (27) Hans, M.; Shimoni, K.; Danino, D.; Siegel, S. J.; Lowman, A. Synthesis and characterization of MPEG-PLA prodrug micelles. Biomacromolecules 2005, 6, 2708-2717. (28) Lu, H.; Wang, J.; Bai, Y.; Lang, J. W.; Liu, S.; Lin, Y.; Cheng, J. Ionic polypeptides with unusual helical stability. Nat. Commun. 2011, 2, 206. (29) Murakami, M.; Cabral, H.; Matsumoto, Y.; Wu, S.; Kano, M. R.; Yamori, T.; Nishiyama, N.; Kataoka, K. Improving drug potency and efficacy by nanocarrier-mediated subcellular targeting. Sci. Transl. Med. 2011, 3, 64ra2. (30) Auton, M.; Holthauzen, L. M.; Bolen, D. W. Anatomy of energetic changes accompanying ureainduced protein denaturation. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 15317-15322.


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(31) Gu, Q.; Xing, J. Z.; Huang, M.; He, C.; Chen, J. SN-38 loaded polymeric micelles to enhance cancer therapy. Nanotechnology 2012, 23, 205101. (32) Case, D.A.; Betz, R.M.; Botello-Smith, W.; Cerutti, D.S.; Cheatham III, T.E.; Darden, T.A.; Duke, R.E.; Giese, T.J.; Gohlke, H.; Goetz, A.W. et al., AMBER 2014, University of California, San Francisco. (33) Maier, J.A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K.E.; Simmerling, C. ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theor. Comput. 2015, 11, 3696-3713. (34) Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald: An N×log(N) method for Ewald sums in large systems. J. Chem. Phys. 1993, 98, 10089-10092. (35) Kabsch, W.; Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 1983, 22, 2577-2637. (36) Deming, T. J. Synthesis of side-chain modified polypeptides. Chem.Rev. 2016, 116, 786-808. (37) Olander, D. S.; Holtzer, A. The stability of the polyglutamic acid alpha helix. J. Am. Chem. Soc. 1968, 90, 4549-4560. (38) Spek, E. J.; Gong, Y.; Kallenbach, N. R. Intermolecular interactions in .alpha. helical oligo- and poly(l-glutamic acid) at acidic pH. J. Am. Chem. Soc. 1995, 117, 10773-10774. (39) Yang, R.; Zhang, S.; Kong, D.; Gao, X.; Zhao, Y.; Wang, Z. Biodegradable polymer-curcumin conjugate micelles enhance the loading and delivery of low-potency curcumin. Pharm. Res. 2012, 29, 3512-3525.


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(40) Torchilin, V. P. Micellar nanocarriers: Pharmaceutical perspectives. Pharm. Res. 2007, 24, 1-16. (41) Morton, S. W.; Zhao, X.; Quadir, M. A.; Hammond, P. T. FRET-enabled biological characterization of polymeric micelles. Biomaterials 2014, 35, 3489-3496. (42) Huang, P.; Song, H.; Zhang, Y.; Liu, J.; Cheng, Z.; Liang, X. J.; Wang, W.; Kong, D.; Liu, J. FRET-enabled monitoring of the thermosensitive nanoscale assembly of polymeric micelles into macroscale hydrogel and sequential cognate micelles release. Biomaterials 2017, 145, 81-91. (43) Rivera, E.; Straub, J.; Thirumalai, D. Sequence and crowding effects in the aggregation of a 10residue fragment derived from islet amyloid polypeptide. Biophys. J. 2009, 96, 4552-4560. (44) Liu, G.; Zhu, L.; Ji, W.; Feng, C.; Wei, Z. Inversion of the Supramolecular chirality of nanofibrous structures through co-assembly with achiral molecules. Angew. Chem. Int. Ed. 2016, 55, 2411-2415.


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For Table of Contents Use Only

Stereoselective Stabilization of Polymeric Vitamin E Conjugate Micelles Min Gao, 1,† Jian Deng, 1,† Huiying Chu, 2,† Yu Tang, 3 Zheng Wang, 1,4 Yanjun Zhao*,1,4 Guohui Li*,2

The poor stability of vitamin E TPGS micelles was addressed by multivalent conjugation of vitamin E to amphiphilic methoxy poly(ethylene glycol)-co-poly(glutamic acid) and stereoselective regulation of backbone and side chain. When both backbone and vitamin E are dextrorotatory, the conjugate micelles exhibited the best stability; the critical micelle concentration decreased over 100 times. This work provides a simple means to enhance TPGS micelle stability.


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Table of content 70x35mm (300 x 300 DPI)


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Scheme 1. Interplay of poly(glutamic acid) backbone chirality and side chain (vitamin E) chirality on the stability of polymer-vitamin E conjugate micelles. D: dextrorotatory; L: levorotatory; D,L: racemic, D + L: physical mixture of D and L (1:1, molar ratio). 158x72mm (300 x 300 DPI)


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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 (α-tocopherol), respectively. Detailed information of micelle composition was shown in Table S1. 84x64mm (300 x 300 DPI)


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Figure 2. The critical micelle concentration (CMC) of eight types of polymer conjugate micelles (n = 3). mPEG (10,000 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. 85x75mm (300 x 300 DPI)


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Figure 3. The hydrodynamic diameters of eight types of mPEG-PGlu-VE conjugate micelles with different chirality (n = 3). 85x85mm (300 x 300 DPI)


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Figure 4. The 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). 85x40mm (300 x 300 DPI)


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Figure 5. The 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 h, 4 h, 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 nm and 600-700 nm, respectively. Scale bar = 30 µm. 175x65mm (300 x 300 DPI)


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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) The curve of FRET ratio against time for 3 types of selected micelles; the slope was compared statistically (n = 3). * p < 0.05. 168x55mm (300 x 300 DPI)


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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. 123x35mm (300 x 300 DPI)


Stereoselective Stabilization of Polymeric Vitamin E Conjugate

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