Micelle Stabilization via Entropic Repulsion: Balance of Force

Jan 9, 2015 - effects of entropic repulsion associated with PEG chain conformation and the ... The directionality is related to the entropic repulsive...
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Micelle Stabilization via Entropic Repulsion: Balance of Force Directionality and Geometric Packing of Subunit He Dong,† Reidar Lund,†,‡ and Ting Xu*,†,‡,§ †

Department of Materials Science and Engineering, University of California, Berkeley, Berkeley, California 94720, United States Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States § Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, United States ‡

S Supporting Information *

ABSTRACT: Nanoparticles, 10−30 nm in size, have shown great prospects as nanocarriers for drug delivery. We designed amphiphiles based on 3-helix peptide-PEG conjugate forming 15 nm micelles (defined as “3-helix micelles”) with good in vivo stability. Here, we investigated the effect of the site of PEG conjugation on the kinetic stability and showed that the conjugation site affects the PEG chain conformation and the overall molecular architecture of the subunit. Compared to the original design where the PEG chain is located in the middle of the 3-helix bundle, micelle kinetic stability was reduced when the PEG chain was attached near the N-terminus (t1/2 = 35 h) but was enhanced when the PEG chain was attached near the C-terminus (t1/2 = 80 h). Quantitative structural and kinetic analysis suggest that the kinetic stability was largely dictated by the combined effects of entropic repulsion associated with PEG chain conformation and the geometric packing of the trimeric subunits. The modular design approach coupled with a variety of well-defined protein stucture and functional polymers will significantly expand the utility of these materials as nanocarriers to meet current demands in nanomedine.

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INTRODUCTION Micelles, 10−30 nm in size, can cross different biological barriers and undergo deep tissue penetration and are ideal for nanocarriers and vaccine formulation.1−5 However, micelles in this size range have often shown limited in vivo stability, serious cargo leakage and rapid disassembly mainly due to their poor kinetic stability.6 Synthetic stabilization strategies were developed to cross-link the head and/or tail group of the amphiphile but may limit the micelle degradation.7−9 Molecular engineering has also been extensively used to improve micelle stability, such as tailoring the length and hydrophobicity of the hydrophobic tail and increasing the intermolecular interactions and the steric hindrance between the head and the tail blocks.10−13 However, this generally leads to structures where increased stability is associated with a large particle size. For many biomedical applications, for example, in nanomedicine, there is a great need to develop tunable, stable small carriers,14 where the micelle’s kinetic stability and size are decoupled. A new family of amphiphilic peptide−polymer conjugates were recently developed that form stable micelles (called “3-helix micelles”, 3HM), 15−17 nm in size.15,16 The 3HMs demonstrated enhanced kinetic stability independent of the presence of competing interactions and a long blood circulation half-life of 29 h.15 Furthermore, micelles based on mixtures of designed amphiphiles and other surfactants also maintain high stability.16 The 3HM is based on amphiphiles consisting of a helical peptide (protein data bank ID: 1coi19,20) with double alkyl chains attached to the N-terminus via a (6)-amino-hexanoic acid linker and a poly(ethylene glycol) (PEG) chain attached to the middle of peptide helix. The peptides self-associate into 3-helix bundles, © XXXX American Chemical Society

forming a trimeric subunit where three PEG chains are anchored to the side of the bundle. When micelles form, the polymer chains confined in close proximity act as elastic springs that push against the neighboring subunit through directed entropic forces. The directionality is related to the entropic repulsive force by the compressed PEG chains. As demonstrated in our previous work,16 the entropic repulsion will push against the neighboring 3-helix bundles to prevent their dissociation from the micelles, analogous to the mechanism of Christmas tree stand where three screws push in to hold the tree in place. All previous studies are based on peptide-PEG conjugates where the PEG is attached in the middle of the 3-helix bundle, that is, position 14 (called “S-14”). This was mainly to take advantage of previous studies that the PEGylated 3-helix bundle demonstrated enhanced stability of protein secondary and tertiary structures.20 Varying the PEG conjugation site will maintain the molecular composition and amphiphilicity without compromising 3-helix bundle formation. However, it affects the architecture and packing parameter of the amphiphile as well as the cross-sectional area mismatch between the head and tail groups. Because the headgroup of the trimeric subunit has welldefined size and shape similar to that of capsomers in a virus particle, a balance between the entropic repulsion from the PEG chain compression and the geometric packing of the trimeric subunits should be critical to achieve good kinetic stability of the 3HM. Systematic studies on the structure and stability of Received: November 14, 2014 Revised: January 8, 2015

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DOI: 10.1021/bm501659w Biomacromolecules XXXX, XXX, XXX−XXX

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of PEGylated peptides from unconjugated peptides was achieved by using a linear AB gradient where solvent A consisted of water plus 0.1% (v/v) TFA and solvent B consisted of acetonitrile plus 0.1% (v/v) TFA. Elution started with 30% A and 70% B and the conjugates were eluted at ∼80−90% B. Small-Angle X-ray Scattering (SAXS). SAXS experiments were carried out at the SAXS/WAXS/GISAXS beamline 7.3.3. of the Advanced Light Source (ALS) at the Lawrence Berkeley National Laboratory (LBNL). The instrument was operated using an X-ray energy of 10 keV and a sample−detector length of 1.9 m, providing a Qrange of 0.001 to 0.4 Å−1, where Q = ((4π)/λ)sin(θ/2), θ = scattering angle, and λ = 1.24 Å. The instrument was equipped with a 1 M Pilatus detector. The samples were loaded in standard boron-quartz capillaries (Charles Supper) held in a customized “homemade” sample holder that permitted the sample to be filled and removed using a syringe. In this way, background subtraction could be made quantitatively and absolute intensity calibration was possible using water as a primary standard. All samples were dissolved in phosphate buffer (25 mM, pH = 7.4) at concentrations of ∼0.5−1 mM, unless otherwise stated. Samples were filtered through 0.2 μm nylon filters and were monitored closely to account for possible beam damage. It was found that no radiation damage was visible for 5 s acquisition times. The absolute scattering intensity was obtained using a calibrated glassy carbon sample kindly provided by Dr. Jan Ilavsky at APS, ANL, Illinois. Fluorescence Self-Quenching and Recovery. In the fluorescence recovery experiments, the concentration was ∼3.6 mM for nonlabeled peptide (acceptor) and 15 μM for labeled peptide (donor). After 16 h of incubation at RT, two solutions (5:1 volume ratio) were mixed, giving a donor: acceptor molar ratio of ∼1:40. Time-dependent fluorescence intensity was recorded every 30 s upon mixing with the excitation wavelength at 494 nm and emission at 527 nm. Data was fitted into equation I(t) = I(∞)+[I(0) − I(∞)][fe−k1t+(1-f)e‑k2t] where k2 represents the rate of monomer desorption from labeled micelles and can be used to compare the kinetics stability of different micelles. Differential Scanning Calorimetry (DSC). For the DSC experiments, samples were either incubated at RT for overnight or thermally annealed up to 70 °C as described. All micelle solutions were prepared in phosphate buffer (25 mM, pH = 7.4) at a final amphiphile concentration of ∼200 μM. Samples were degassed for 5 min before loading into the sample chamber. Approximately six hundred microliters of sample and buffer were loaded into two parallel stainless steel cells that were sealed tightly under the pressure of ∼27 psi to prevent water evaporation during the heating cycle. As DSC is highly sensitive to the heat capacity change of the bulk solvent, the exact same buffer used to dissolve and dilute peptides was loaded in the control chamber to avoid any variation in ionic strength from batch to batch. The temperature was increased from 5 to 85 °C at a rate of 1 °C/min with a 15 min equilibration time at 5 °C. DSC thermograms were obtained through concentration normalization and based correction using manufacture-provided MicroCal software.

amphiphillic micelles as a function of PEGylation position can provide useful insights to understand the molecular origin of the unusual stability of 3HMs and to delineate the effects of the molecular packing of the alkyl tails, intermolecular interactions of the headgroups and the entropy associated with PEG chain deformation. Here, we evaluated the effects of PEGylation position on the micelle stability using two analogs where the PEG is attached to the peptide helix near its N- (position 7, called “S-7”) and Cterminus (position 28, called “S-28”), respectively. Figure 1

Figure 1. Schematic drawing of the designed amphiphilic trimeric subunits where the headgroup contains a 3-helix bundle with varying sites of PEG conjugation. The compression of PEG chain depends on the site of conjugation and work together with the geometric packing in tuning micelle stability.

shows the schematic drawings of the amphiphilic trimeric subunits where the headgroup contains a 3-helix bundle with varying sites of polymer conjugation. PEGylation close to the core/shell interface, that is, position 7, interferes with the molecular packing of the alkyl chains in the hydrophobic core and the micelle stability was compromised. As the PEGylation site moves away from the core/shell interface, the stability of the 3HM was improved. Interestingly, S-28 demonstrated slightly better stability than S-14. Solution small-angle X-ray scattering (SAXS) studies showed that for S-28, PEG localizes both within the shell layer of the micelle and on the micelle surface, potentially modifying the surface chemistry of micelles.

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EXPERIMENTAL METHODS

Synthesis of Amphiphilic Peptide−Polymer Conjugates and Fluorescein Labeled Peptide−Polymer Conjugates. The synthesis of amphiphilic peptide-PEG conjugates followed previous reported procedures.15,16,21 Cysteine mutation was made on the original peptide sequence (1coi: EVEALEKKVAALEKKVQALEKKVEALEHGW) at the position K7, K14, and H28 to attach PEG (MW = 2000 Da). Fluorescein was attached at the C-terminus by selective deprotection of the Alloc protected lysine residue followed by amide coupling with carboxy-terminated fluorescein. Specifically, the Alloc group on the lysine residue at the C-terminus was selectively removed by utilizing Pd(PPh3)4 catalyst and a radical trapping agent, PhSiH3 in DCM. The reaction was repeated five times. The resulting free amino groups of lysine were used for conjugating carboxy-terminated fluorescein using HBTU/DIPEA chemistry. The coupling reaction was performed at room temperature for 24 h and repeated twice. Ninhydrin test was performed to ensure the completion of the coupling reaction. Cleavage was carried out using a cocktail of 90:8:2 TFA/TIS/ water for 3 h. Crude peptides were precipitated in cold ether, isolated, and dried for the conjugation of polymers and subsequent purification by HPLC. The amphiphilic conjugates were purified by RP-HPLC (Beckman Coulter) on a C4 column (Vydac column 22 mm × 250 mm). The flow rate was 8 to 10 mL/min. Elution was monitored with a diode array detector at wavelengths of 220 and 280 nm. Optimized separation

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RESULTS AND DISCUSSION Upon dissolution in aqueous solution (pH = 7.4), all amphiphiles exhibited alpha helical structures as characterized by two minimum peaks at 208 and 222 nm (Supporting Information Figure S1). The molar ellipticity ratio (1.06) between the two peaks confirmed the formation of a tertiary structure of coiledcoil. The helicity of the three amphiphiles is comparable suggesting that the location of the PEG chains did not affect the peptide secondary and tertiary structures. All three amphiphilic peptide-PEG conjugates showed comparable CMC values at ∼4 μM suggesting the molecular architecture and geometric packing among the headgroups have minimum effect on the thermodynamic stability of the 3-helix micelles. The micelles were eluted as a single species with low polydispersity characterized by size exclusion chromatography (SEC) (Figure 2). B

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Table 1. Fit Parameters Deduced from SAXS Data Analysis with a Detailed Core−Shell Model Including a Graded Density profile Pa Rm [Å]b σm [Å]c σint [Å]d Rc [Å]e a

S-7

S-14

S-28

24 ± 3 54 ± 2 6 4 17 ± 1

43 ± 3 65 ± 3 6 3 20 ± 1

56 ± 6 63 ± 5 9 5 22 ± 1

Aggregation number. bShell thickness. cRoughness of core interface. Outer micellar (shell) roughness. eCore radius.

d

increases from ∼17 to 22 Å from S-7 to S-28 due to a better molecular packing with the reduced entropic repulsion. The difference in the packing parameter shows the important role of the site-specific polymer conjugation in controlling the molecular geometry of the designed amphiphilic subunits. The fitting of the SAXS data also yielded the overall radial density profile of the micelles. The density profile of the shell was parametrized in terms of the Fermi−Dirac function 1 n(r ) = (r − R m) 1 + exp σ

Figure 2. SEC trace of amphiphilic peptide−polymer conjugates with different headgroups suggesting all three peptides self-assemble into monodisperse micelles. Peptide concentration = 200 μM.

Detailed structural information on each micelle was deduced from the solution SAXS using spherical core−shell model fit on an absolute intensity scale. Figure 3 shows the experimental and

(

m

)

Where Rm is the micellar radius and σm is a width parameter describing the "fuzziness" of the outer micellar shell. The overall radial density profile of each micelle is shown in Figure 3. The apparent radial density of the micelle clearly differs upon varying the conjugation site. When comparing the average radial density profiles of S-14 and S-28, the shell thickness Rm stays almost constant of ∼65 Å. However, the S-28 micelles are “fussier”, that is, exhibits a broader corona−solution interface. This is reflected by an increase in the smearing parameter from about 10−15%. The fussier surfaces increase the thickness of the micellar shell and the diffusion time of monomers through the corona and should slow down the subunit desorption.25 On the basis of the Rg of free PEG2K (∼1.9 nm) in solution,26 the micelle composed of triblock copolymer-like amphiphile is expected to have a diameter over 3 nm higher than that of S-14. The similar micelle sizes in conjunction with the fuzzier surface suggest that the S-28 amphiphile does not adopt a spatial arrangement similar to a triblock copolymer.27,28 Rather, part of the PEG chain is localized on the side of the 3-helix bundle as schematically shown in Figure 1b. Thus, on one side the PEG chains fill the gaps between the peptide helix bundles to form a conelike subunit that renders better geometric packing in a micelle. On the other hand, the deformation of the PEG chain in S-28 is smaller than that in S-14 and should exert a lower repulsion. Although small-angle neutron scattering (SANS) combined with contrast variation is necessary to isolate and deduce the particular distribution of PEG in the core, the present data suggest a change in the apparent radial density of the micelles upon varying the conjugation site. This is expected to affect the kinetic stability as discussed below. The kinetic stability of the micelle was evaluated by monitoring the fluorescence recovery of self-quenched fluorophore covalently attached to the micelle surface. 6,29 Fluorescein was covalently linked at the C-terminus of the amphiphiles and does not change the size of the micelle based on the dynamic light scattering (DLS) results. (Supporting Information Figure S2) Self-quenched fluorescein-labeled micelles were mixed with a large excess of nonlabeled micelles at a molar ratio of 1:40. As the subunit exchanges, fluorescein-

Figure 3. Experimental and fitted SAXS profiles of micelles composed of PEG at different positions along the peptide backbone. Inset: the density profiles parametrized using the Fermi−Dirac function.

fitted SAXS profiles using the radial density profile shown in the inset. The scattering profile was fitted using spherical core−shell model with a constant and a graded density profile of the core and corona, respectively. By convoluting the density profiles with a Gaussian density profile, a finite (fuzzy) interface between core and corona was included. In this simple approach, the radial density distribution of the headgroup was assumed to be the same and no lateral correlations were included.22,23 From the fits, we extracted aggregation number, average sizes of core and corona, and the density profiles. On the basis of the fitting results, all amphiphiles form spherical core−shell micelles with low polydispersity, which is consistent with the SEC results. An increased curvature of the subunit increases the repulsion in the corona and lowers aggregation numbers.24 The aggregation number in Table 1 increases from 24 to 46 to 56 as the PEGylation site moves toward the exterior of micelle. The core radius correspondingly C

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Figure 4. Fluorescence recovery of self-quenched fluorescein labeled micelles upon the addition of nonlabeled micelles. The exchange rate constants for different conjugates were determined via fitting of fluorescence recovery data into first-order exchange kinetics.

labeled amphiphiles are desorbed from the self-quenched micelles and inserted into nonlabeled micelles. This leads to an increase in fluorescence intensity over time. The relative fluorescence intensity change as a function of time is indicative of the rate of exchange kinetics.30 Figure 4 shows the results of the relative fluorescence intensity change of the three micelles at 20 °C post addition of the nonlabeled micelle solution. For S-7, 23% fluorescence intensity increase was observed over a 4 h period while only ∼7−8% increase was seen for S-14 and S28. Assuming that the rate-limiting step is the subunit desorption, the fluorescence intensity change is fitted using eq 1 with two first-order disassociation rate constants25,31−33 I(t ) = I(∞) + [I(0) − I(∞)][fe

−k1t

+ (1 − f )e

−k 2t

Figure 5. DSC thermograms of amphiphilic conjugates with different site of PEG conjugation and thermal history. (a) Samples of direct powder dissolution. (b) Annealed samples.

] (1)

The fast rate constant, k1 is incorporated to address the dilution effect of labeled micelles upon the addition of nonlabeled micelles. The slower rate constant, k2 represents the rate of the monomer desorption from labeled micelles and is used to evaluate the kinetic stability of three families of micelles. Experimentally, measurement of I(∞) at 20 °C is practically impossible due to the extremely slow exchange kinetics of the 3HMs. I(∞) was approximated and experimentally determined by measuring the fluorescence intensity of the fluorescein-labeled headgroup, that is, 1coi-PEG. The fitting results gave a rate constant, k2, of 3.25 × 10−4, 2.05 × 10−4, and 1.45 × 10−5 min−1 at 20 °C and a first order half-life of 35, 56, and 80 h for S-7, 14, and 28, respectively. This is in direct contrast to typical micelles with similar particle size that typically ranges from seconds to a few hours.6,13,29 Given the high kinetic stability, all micelles have great potential as nanocarriers. There are a few parameters that affect the overall kinetic stability of micelle, that is, the crystallinity of alkyl chains, the entropy to compress PEG chains, the directionalities of entropic repulsion and the geometric packing of headgroups. Effects from each parameter are interconnected. The alkyl chain packing can be influenced by the PEG chain conformation and headgroup packing and, in turn, will affect directionalities of entropic repulsion. DSC thermograms of all micelles exhibited endothermic phase transitions associated with the packing of alkyl chains. For micelle solutions without thermal treatment (Figure 5a), two main endothermic peaks were observed. The S7 micelle has the lowest Tm and S-28 has slightly higher Tm than that of S-14. Thermal annealing is routinely used to improve the local ordering of the lipid chain packing in a micelle. A single endothermic peak with decreased Tm was seen for all three micelles post thermal annealing (Figure 5b). The difference in alkyl chain packing could reflect headgroup arrangement and will be discussed in a future publication. Nevertheless, the lipid chains

have better molecular packing in the hydrophobic core as PEG chains move toward the exterior of the micelles. To decouple the effect of alkyl chain packing and other parameters on micelle stability, the fluorescence recovery experiments were performed using S-14 with different thermal treatments to vary Tm. At 20 °C, similar kinetic stability (Supporting Information Figure 3a) (k = 1.88 × 10−4 min−1, t1/2 = 61 h vs k = 2.05 × 10−4 min−1, t1/2 = 56 h) were observed despite the lower Tm of the annealed sample and thus less ordered alkyl chain packing. However, at 37 °C, well above the Tm of both S-14 samples, the difference in exchange kinetics is more dramatic (Supporting Information Figure 3b). Despite a lower Tm, the annealed micelles have much slower exchange rate (k = 3.34 × 10−4 min−1, t1/2 = 35 h) than that of the nonannealed sample (k = 6.15 × 10−4 min−1, t1/2 = 19 h). Thus, the hydrophobic chain packing as characterized by Tm cannot be simply used to account for the kinetic stability observed in different micelles or micelles with different thermal treatments. On the basis of the results of structure and kinetic study described above, the difference in terms of kinetic stability observed for different micelles is attributed to the combined effect of headgroup geometric packing and directional entropic repulsion. For S-7, the decrease in Tm clearly reflects the competition between the molecular packing in the hydrophobic core and the entropy associated with PEG chain deformation. The PEG chains close to the interface need to be compressed to accommodate molecular packing of alkyl chains. From the SAXS data, the area per molecule at the core interface is 151 Å2 for S7 and 117 and 105 Å2 for S14 and S28, respectively. Thus, S-7 makes smaller micelles with a lower aggregation number to reduce the entropic penalty associated with the compression of PEG chains. This leads to larger curvature and disrupts the lipid packing, which in turn compromises the directionalities of entropic forces. The reduced micelle stability indicated that even D

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though the entropic repulsion in this case may be designed to be high, the micelle stabilization cannot be achieved if it is not well balanced. As the PEGylation site moves outward (S-14 and S28), the required chain compression is reduced. Solution SAXS showed that the PEG chain in S-28 adopts a conformation that positions PEG on the side of the helix bundle similar to S-14 as well as at the micelle surface. In comparison to S-14, S-28 has less entropic repulsion forces since the PEG chain compression is reduced but shows better geometric packing of the subunit as evidenced by the improved molecular packing of alkyl chains. Although further neutron scattering studies are needed to delineate the exact spatial arrangements of PEG chains as a function of PEGylation site, we speculate that the PEG chains are compressed just the right amount to render entropic repulsion to stabilize micelle without disrupting subunit packing, similar to that seen in virus capsomers. The observed enhancement in micelle kinetic stability in S-28 further confirmed the importance of engineering synergy between the entropic repulsion and the geometric packing of the subunit. This is in essence similar to the assembly of capsomers into viruses although there the repulsion is often electrostatic origin. An additional benefit of these findings is that the presentation of partial PEG chain on the micelle surface could potentially offer stealth properties, which are currently being validated in in vivo testing.34 Synthetically, this eliminates the need for additional surface PEGylation and leaves the peptide C-terminus available for other orthogonal modification.

CONCLUSIONS In summary, by varying the location of the PEG chain along the peptide backbone, micelles with tunable kinetic stability were achieved. In terms of amphiphile design, present studies showed that tailoring the shape of headgroup to optimize geometric packing in the shell of micelle can be effective for micelle stabilization in addition to engineering directional entropic repulsion. These new families of 3HMs are well suited to develop nanocarriers with tunable pharmacokinetics to meet current demands in nanomedicine. S-28 has the potential to simultaneously achieve micelle stability and surface PEGylation and warrants further investigation. ASSOCIATED CONTENT

S Supporting Information *

Peptide synthesis, purification and characterization by MALDI, CD, DLS, and SEC is available. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study is supported by National Institutes of Health under contracts 1R21EB016947-01A1. Use of the Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. E

DOI: 10.1021/bm501659w Biomacromolecules XXXX, XXX, XXX−XXX