Sub-20 nm Stable Micelles Based on a Mixture of Coiled-Coils: A

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Sub-20 nm Stable Micelles Based on a Mixture of Coiledcoils: A Platform for Controlled Ligand Presentation JooChuan Ang, Dan Ma, Benson Thomas Jung, Sinan Keten, and Ting Xu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00917 • Publication Date (Web): 17 Aug 2017 Downloaded from http://pubs.acs.org on August 19, 2017

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Biomacromolecules

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Sub-20 nm Stable Micelles Based on a Mixture of Coiled-coils: A Platform for

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Controlled Ligand Presentation

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JooChuan Ang1, Dan Ma2, Benson T. Jung1, Sinan Keten2, Ting Xu*1,3,4

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1

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California 94720, United States

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2

8

Engineering, Northwestern University, Evanston, Illinois 60208, United States

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3

Department of Chemistry, University of California, Berkeley, California 94720, United States

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4

Material Science Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720,

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United States

Department of Materials Science and Engineering, University of California, Berkeley,

Department of Civil and Environmental Engineering and Department of Mechanical

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ACS Paragon Plus Environment

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Abstract

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Ligand-functionalized, multivalent nanoparticles have been extensively studied for

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biomedical applications from imaging agents to drug delivery vehicles. However, the ligand

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cluster size is usually heterogeneous and the local valency is ill-defined. Here, we present a

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mixed micelle platform hierarchically self-assembled from a mixture of two amphiphilic 3-helix

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and 4-helix peptide-PEG-lipid hybrid conjugates. We demonstrate that the local multivalent

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ligand cluster size on the micelle surface can be controlled based on the coiled-coil oligomeric

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state. The oligomeric states of mixed peptide bundles were found to be in their individual native

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states. Similarly, mixed micelles indicate the orthogonal self-association of coiled-coil

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amphiphiles. Using differential scanning calorimetry, fluorescence recovery spectroscopy, and

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coarse-grained molecular dynamics simulation, we studied the distribution of coiled-coil bundles

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within the mixed micelles and observed migration of coiled-coils into nanodomains within the

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sub-20 nm mixed micelle. This report provides important insights into the assembly and

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formation of nanophase-separated micelles with precise control over the local multivalent state

15

of ligands on the micelle surface.

16 17


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Biomacromolecules

Introduction

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Nanocarriers can exploit characteristics of tumor growth, such as leaky vasculature and

3

poor lymphatic drainage, for passive targeting via the enhanced permeation and retention (EPR)

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effect1, 2. In addition to the EPR effect, nanocarriers can also be actively targeted to up-regulated

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cellular receptors or components present on tumor cells by incorporating surface grafted

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recognition moieties, such as sugar moieties3, epidermal growth factor4, folate5, 6, antibodies7 and

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peptides8, 9, to improve the efficacy of treatment and minimize side effects. Active targeting is of

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particular importance since many drug carriers encounter difficulties in accessing cancer cells

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deep in the tumor tissues and to interact with target cells after accumulation10. A wide variety of

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nanocarriers can be readily prepared, including liposomes11-13, dendrimers14, 15, micelles16, 17, and

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virus-like particles18, 19 and targeting ligands can be conjugated to the nanocarrier surface20.

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Moreover, multiple copies of ligands on the nanocarrier surface can be a viable route to improve

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the targeting efficiency of nanocarriers; biological multivalent inhibitors can increase binding

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avidity by 10–109 fold21-24. Experimentally, a 30 nm nanoparticle with four RGD peptides

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showed 25-fold enhancement in endothelial cell binding25. Three folate groups led to 2,500-fold

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enhancement in dendrimers binding to a surface26. There has been a concerted effort to develop

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peptide-based nanomaterials towards higher complexity and enhanced structural control27, 28.

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However, in contrast to well-regulated structural control seen in viruses21, there is limited

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structural control in existing nanocarriers over the spatial distribution of ligands and the

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orientation of ligand relative to the particle surface, which determines the availability of ligand

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binding sites. To this end, we have developed a new class of 3-helix micelle (3HM) nanocarrier

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based on amphiphilic coiled-coil peptide-polymer-lipid conjugates29-35 that has the potential to

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exert control over the local multivalency of presented ligand clusters.

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3HM has many desirable attributes as an effective drug delivery nanocarrier such as long

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circulation, deep tumor penetration, minimal cargo leakage in serum, and renal clearance30-32, 34.

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We have previously described the hierarchical self-assembly pathway of 3-helix bundle

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amphiphiles at the air-water interface36 as well as the kinetic pathway of 3HM formation in

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solution37. 3HM is based on a common coiled-coil protein tertiary structure that is routinely used

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to present ligand clusters on cell surfaces38-40. Coiled-coil self-assembly has high fidelity and

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orthogonal coiled-coil sequences41 have been conjugated and used to assemble higher ordered


Biomacromolecules

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nanostructures such as protein-like nanoparticles42 and nanocages43, 44. Mixed micelles generated

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from two or more building blocks have been used to tune stability and drug loading capacity of

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conventional polymeric micelles for drug delivery45. This approach also allows for the

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incorporation of multiple functionalities, including ligand targeting, into our system. The use of

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mixed micelles based on coiled-coil peptides would allow the generation of ligand clusters with

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well-defined local multivalency determined by the oligomeric state of the coiled-coils within the

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mixed micelle. This approach also opens new possibilities to generate multi-compartment

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micelles with the two coiled-coil building blocks possessing different functionalities.

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The generation of micron-sized inorganic multi-compartment, Janus, or patchy particles

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has been well studied and optimized, whereas development of sub-100 nm patchy particles have

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been more challenging46. Patchy particles have been generated from linear dendritic polymers47,

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48

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roughness57, layer-by-layer deposition58, self-assembled monolayers59-61, host-guest binding62,

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metal coordination63, and polymer grafting64 to produce the patches. However, none of these

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approaches allow control over the precise ligand cluster size, or local multivalency on the surface

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of the nanoparticles. Ligand clustering with controlled cluster size observed in viruses is vital to

17

improve binding selectivity and accuracy, and yet, has not been systematically investigated due

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to the lack of a platform that can regulate the clustering of ligands.

, di- and tri-block copolymers49-55, polymersomes56, and other systems that exploit surface

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In this contribution, we present a micellar platform that utilizes hierarchical self-assembly

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of amphiphilic coiled-coil peptide-PEG-lipid hybrid conjugates to control the local multivalency

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of ligands on the micelle surface based on the coiled-coil oligomeric state. A mixture of 3-helix

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and 4-helix peptide bundles was shown to retain their individual native oligomeric states. Mixed

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micelles with excellent stability in the presence of bovine serum albumin can be generated from

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mixtures of 3-helix and 4-helix amphiphilic peptide-PEG-lipid conjugates at various ratios. At

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various ratios, mixed micelles showed similar stability compared to the individual micelles. At a

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1:1 mixing ratio of 3HM:4HM, the thermal transition associated with alkyl chain packing within

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the hydrophobic core of the mixed micelle was found to re-distribute from a single homogeneous

28

transition for an annealed sample to two distinct transitions indicating the formation of distinct

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domains as it was cooled down to room temperature. Fluorescence self-quenching and recovery

30

experiments further affirmed the formation of nanodomains within the mixed micelles. Coarse-


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grained MD simulations illustrated the self-organization of separate phases consisting of helix

2

peptide bundles with different oligomerization states. This study provides valuable insight into

3

the self-assembly of orthogonal coiled-coil peptide-PEG-lipid conjugate amphiphiles into patchy

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micelles and guidance to develop biomaterials with controlled local multivalency for ligand

5

cluster presentation.

6 7

Experimental methods

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Synthesis of amphiphilic peptide-polymer conjugates and fluorescein-labeled peptide-polymer

9

conjugates

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Synthesis of amphiphilic peptide-polymer conjugates using solid-phase peptide synthesis based

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on standard Fmoc-chemistry has been previously described in detail29, 32, 33. Briefly, the two

12

peptides

13

(EVEALEKKVAALECKVQALEKKVEALEHGW)

14

(GGGEIWKLHEEFLCKFEELLKLHEERLKKL). PAL-PEG-PS resin (0.17 mmol/g loading,

15

ThermoFisher Scientific) was used to conduct solid-phase synthesis of the peptides on a Prelude

16

peptide synthesizer (Protein Technologies). The N-termini of the peptides are modified with a

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short C6 alkyl linker, Fmoc-6-aminohexanoic acid, followed by a lysine residue. This allowed

18

for conjugation of two palmitic acid molecules to the N-terminus. Both peptides contain a

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cysteine residue at position 14 to facilitate conjugation of a maleimide-PEG chain (MW 2000,

20

Rapp Polymere) to the exterior of the coiled-coil bundles. Fluorescein was attached to the C-

21

terminus of the 3-helix peptide by selective deprotection of alloc-protected lysine followed by

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coupling the amine with 5(6)-carboxy-fluorescein. Specifically, 0.2 eq. of Pd(PPh3)4 was used as

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a catalyst with 24 eq. of phenylsilane as an allyl trapper in dichloromethane. The reaction was

24

carried out for 30 mins each, repeated 5 times.

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The conjugates were purified by RP-HPLC (Beckman Coulter) using a C-4 column (Vydac,

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Phenomenex). Separation of the conjugates was facilitated by using a linear AB gradient where

27

solvent A is water plus 0.1% (v/v) TFA and solvent B is acetonitrile plus 0.1% (v/v) TFA.

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Elution of the conjugate was ~85% B. Purification of the FAM-labeled conjugate was achieved

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using solvent B as isopropanol plus 0.1% (v/v) TFA and the purified product eluted ~90% B.

studied

here

are

based

on

a

3-helix and

coiled-coil a


4-helix

peptide coiled-coil

sequence65 peptide66

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The masses of the purified conjugates were verified by MALDI-TOF mass spectrometry (SI

2

Figure 1).

3

Size-exclusion chromatography (SEC)

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The sizes of 3-helix peptide, 4-helix peptide, and a 1:1 mixture of the two peptides were studied

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using SEC (Agilent instruments) on a BioSep-SEC-S4000 column (Phenomenex) at a

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concentration of ~100 µM. Elution was carried out using a flowrate of 1 mL/min with phosphate

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buffer (pH 7.4, 25 mM) for 15 minutes. The elution profiles were monitored with a UV-Vis

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detector at wavelengths of 220 nm and 280 nm.

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UV-vis heme titration

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Titration experiments were performed with 1 mL of ~8 µM solutions of 4-helix peptide or 4HM

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dissolved in phosphate buffer (25 mM, pH 7.4) in the presence of excess 3-helix peptide or 3HM,

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respectively. Spectra from 250–600 nm were recorded on a Hewlett-Packard 8453

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spectrophotometer using a 1 cm pathlength quartz cuvette after addition of each 2 µL aliquot of a

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~800 µM stock hemin solution in DMSO.

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Fluorescence self-quenching and recovery

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Fluorescence spectroscopy was performed using a LS-55 fluorescence spectrometer (Perkin

17

Elmer). Briefly, 200 µM of fluorescein-labeled 3HM (donor) and 400 µM of unlabeled 4HM

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(acceptor) were independently annealed at 70°C for 45 min then allowed to cool to room

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temperature before mixing for self-quenching experiments. Annealing conditions were selected

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based on previous studies36, 37. The two solutions were then mixed in a 2:1 (donor:acceptor)

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volume ratio at a total volume of 400 µL, giving a donor:acceptor molar ratio of 1:1. The sample

22

was loaded into a 1 mm by 10 mm quartz cell (Starna Cells). Temporal evolution of fluorescence

23

intensity at 520 nm was recorded every 30 seconds using an excitation wavelength of 450 nm.

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Self-quenching and recovery experiments were conducted with the sample cell maintained at

25

25°C for 22 hrs, increased to 70°C for 2 hrs, and finally decreased to 25°C for a further 20 hrs.

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The fluorescence intensity at various stages of heating and cooling were normalized to the initial

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fluorescence intensity at time zero.

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Forster Resonance Energy Transfer (FRET)


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Biomacromolecules

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A pair of lipophilic FRET pair, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO, donor) and

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1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, acceptor), was dissolved

3

in methanol (1 wt%) along with various 3HM:4HM ratios. The methanol was evaporated under

4

vacuum at 60°C for 3 hrs to form a thin film in a glass vial. Phosphate buffer was added to

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rehydrate the film at a concentration of 1 mg/ml. The solutions were annealed at 70°C for 45

6

mins then allowed to equilibrate over 16 hrs at room temperature and spin dialyzed using a 3K

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MWCO filter (Amicon Ultra-4, Millipore) to remove any un-encapsulated free dye in solution.

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The samples were incubated with BSA (20 mg/mL) at 37°C and time-resolved fluorescence

9

emission intensity was monitored every 15 mins from 475–650 nm for 24 hrs using an excitation

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wavelength of 450 nm.

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Differential scanning calorimetry (DSC)

12

DSC thermograms probing the alkyl core thermal transitions were obtained using a VP-Microcal

13

calorimeter (GE). ~550 uL of sample and buffer solutions were degassed for 10 mins before

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being loaded into two hermetically sealed tantalum cells at a pressure of ~28 psi. The

15

temperature was increased from 5°C to 60°C at a scanrate of 1 °C/min with 15-minute

16

equilibration at 5°C. Baseline correction and concentration normalization were performed using

17

the Origin software provided by Microcal.

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Coarse-grained molecular dynamics (CGMD) simulation

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The molecular dynamics simulations presented in this work were coarse-grained (CG)

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simulations based on the dissipative particle dynamics (DPD) technique. Considering that the

21

length and time-scale limitations of all-atomistic simulations prohibit comprehensive evaluation

22

of micelle formation, we chose to utilize a DPD67, 68 CG simulation methodology for micelle

23

formation simulations. DPD is a Lagrangian thermostat technique that enables fast CG

24

simulations by employing hydrodynamics interactions to describe phase separation in materials.

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This method has been widely used in studying the behavior of lipid membranes, block

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copolymers, and a broad range of complex fluids 69-71.

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The DPD model presented here adopted the lipid model developed by Groot and Rabone72, PEG

28

model developed by Ying71, and the peptides are modeled based on our previous publications35,

29

73

. This approach has been previously utilized to efficiently study 3HM self-assembly and


Biomacromolecules

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35, 73

1

resulted in predictions that agree well with experimental data

. All CG simulations in this

2

work were performed using a variation of the DPD approach using the open source MD

3

simulation package LAMMPS74. A DPD thermostat procedure adopting an NVT ensemble with

4

a constant temperature 300 K was adopted for simulations, and periodic boundary conditions

5

were applied in three dimensions. Here the time scale is 𝜏 = 24.32 𝑝𝑠, length scale is 𝑅! =

6

0.8 𝑛𝑚. 69, 71 Each simulated system consists of 100 randomly distributed amphiphilic molecules

7

(with 3-helix and 4-helix components at a 1:1 ratio), and then solvated in explicit DPD water

8

beads. All CG simulations were carried out for approximately 1.8 − 2.5 𝜇𝑠, and tend to reach

9

equilibrium before 800 ns. The equilibration of the simulations was assured by checking that the

10

average micelle aggregation number was stable to ±3 in 200 ns. The last 150 ns of the

11

equilibrated simulations were chosen for data analysis.

12 13

Results and discussion

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A mixture of 3-helix and 4-helix peptides showed retention of their individual oligomeric

15

states as shown in Figure 1. Figure 1a shows that the SEC trace of a 1:1 mixture of 3-helix:4-

16

helix peptide (black) can be deconvoluted into two distinct peaks corresponding to the individual

17

3-helix and 4-helix peptides (blue and red, respectively). This is the first indication that mixture

18

of the peptides does not result in cross-oligomerization since they retain their original bundle

19

sizes. Next, Figure 1b shows the absorption at 412 nm due to binding of heme to the bis-histidyl

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ligation pockets of the 4-helix peptide75-77. The ratio at which the slope of the data changes

21

corresponds to the number of heme molecules bound to each 4-helix bundle. 4-helix peptide in

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presence of 5% 3-helix peptide (red) indicates ~3.8 hemes bound per 4-helix bundle, close to the

23

native state (in the absence of 3-helix peptides, shown in black) binding capacity of 4 heme

24

molecules per 4-helix bundle. Retention of the heme-binding pocket of the 4-helix peptide

25

suggests minimal perturbation of the 4-helix bundle oligomeric state in the presence of 3-helix

26

peptides. Absence of an inflection point when there is no 4-helix peptide available for heme to

27

bind is shown in blue. Figure 1c shows the fluorescence intensity change as a function of

28

unlabeled 4-helix peptide mixed with FAM-labeled 3-helix peptide. Minimal increase in the

29

fluorescence intensity of FAM-labeled 3-helix peptide when mixed with 4-helix peptide at

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various mixing fractions (red trace) indicates that there is a lack of cross-oligomerization


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Biomacromolecules

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between the two coiled-coil peptides. Furthermore, the similar behavior of the mixtures even in

2

the presence of thermal annealing (black trace) does not promote cross-oligomerization. This

3

further suggests that the self-assembly of the two peptides are orthogonal and there is an absence

4

of cross-oligomerization when the two peptides are mixed.

5

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Figure 1: Mixtures of 3-helix and 4-helix peptides. (a) SEC traces of 4-helix peptide (blue), 3-

8

helix peptide (red), and a 1:1 mixture of 3-helix and 4-helix peptides (black) measured at 280 nm.

9

(b) UV titration of heme bound to 4-helix peptide bundles in the absence of 4-helix peptide

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(purple), 5% 4-helix peptide (red), and 100% 4-helix peptide (black). (c) Fluorescence recovery

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of various ratios of 3-helix to 4-helix mixtures. Freshly prepared mixtures and annealed samples

12

show no significant difference in fluorescence intensity.

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We seek to understand the oligomeric state of the coiled-coil bundles upon conjugation of

15

alkyl tails and self-assembled into micelles. 3-helix peptide-polymer-lipid conjugate amphiphiles

16

have previously been observed to retain its native trimeric state at the air-water interface36.


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Varying ratios of 3-helix and 4-helix peptide amphiphiles were found to form 15-20 nm particles

2

in aqueous solution by DLS (SI Figure 2). Since amphiphiles of both 3-helix and 4-helix peptides

3

form micelles of similar sizes, SEC is not a viable method to study the individual oligomeric

4

states of the peptides within mixed micelles. However, cofactor binding of heme to the coiled-

5

coil bundle headgroup of 4-helix amphiphiles, as elucidated for the peptide mixtures in Figure 1b,

6

allows probing of the oligomeric state when mixed micelles are formed in the presence of 3-helix

7

amphiphiles. Figure 2a shows the UV-vis heme titration into an aqueous solution of ~8 µM 4HM.

8

Incorporation of heme into the hydrophobic pocket of the 4-helix peptide is evident from the

9

increase in the Soret peak at 412 nm and the poorly resolved Qα and Qβ bands at 560 and 529 nm,

10

respectively75, 78, 79. As more heme is added, the absorption maxima experienced a blue shift, due

11

to the absorbance of free heme in solution. Figure 2b plots the absorbance at 412 nm as a

12

function of heme per 4-helix amphiphile. In the absence of 3HM, the tertiary structure of 4-helix

13

bundle within 4HM (black) retained native-like heme binding capability75, indicating that there is

14

no substantial deleterious effects of di-alkyl conjugation to the N-terminus and PEGylation of the

15

coiled-coil exterior. More importantly, in the presence of excess 3-helix amphiphiles (red), the

16

number of heme molecules bound to the 4-helix amphiphiles was not compromised indicating

17

that the tertiary structure is likely to be preserved. Once again, no change in slope was observed

18

for a solution of 3HM in the absence of 4HM for heme binding (blue). The orthogonality and

19

peptide native oligomeric states were preserved when the peptides were conjugated with two

20

hexadecanoic acid tails at the peptide N-terminus and the resulting amphiphiles mixed.

21

22


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Biomacromolecules

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Figure 2: (a) UV-vis spectra of heme titrated into a ~8 µM solution of 4HM upon addition of 0,

2

0.4, 1.2, 2, 3, 4, 4.8, 6, 7.2, and 8 eq. of heme per 4-helix bundle. The vertical black line indicates

3

the peak at 412 nm which corresponds to the absorbance of heme within the hydrophobic interior

4

of the 4-helix bundle. (b) Absorbance at 412 nm monitored as a function of heme molecules per

5

4-helix bundle ratio for pure 4HM (black), pure 3HM (blue), and at 4HM:3HM ratio of 5:95

6

(red). The ratio at which the slope changes indicates the number of heme molecules bound to

7

each 4-helix bundle.

8 9

To assess if mixed micelles were created and to quantify their stability80-82, the change in

10

emission of an encapsulated FRET pair at various 3HM:4HM ratios was measured in the

11

presence of bovine serum albumin (BSA). When both FRET molecules are encapsulated within

12

the same micelle, appropriate excitation of the donor molecule can lead to energy transfer to the

13

acceptor molecule due to the close proximity between the two molecules. As a result, excitation

14

at 450 nm (donor excitation) leads to a strong emission at 565 nm (acceptor emission). If the

15

mixed micelles dissemble, the encapsulated FRET molecules are released and diffuse apart,

16

eliminating the energy transfer. As such, a shift from 565 to 505 nm would be observed. Figure

17

3a shows the change in normalized FRET ratio over a 24-hour period. All micelles created from

18

various 3HM:4HM ratios displayed good stability over the time period studied with FRET ratios

19

above 0.9. Two formulations in particular, 3HM:4HM ratios of 50:50 and 0:100, displayed

20

higher stability than the remaining of the micellar ratios with FRET ratios maintained ~ 0.99

21

over 24 hrs. The good stability presumably stems from the unique architecture of the coiled-coil

22

peptide-polymer headgroup that can direct polymer entropic repulsion between subunits of the

23

micelle29. To further illustrate the high kinetic stability of 3HM and 4HM, the FRET pair, DiO

24

and DiI, was independently encapsulated within each of 3HM and 4HM and mixed in equal

25

molar ratios at room temperature as shown in Figure 3b. The emission spectra over 16 hrs

26

essentially remained constant, indicating no cargo leakage and slow subunit kinetic exchange of

27

the two micellar populations. The high kinetic stability of micellar formulations at various

28

3HM:4HM ratios indicates that they do not undergo significant disassembly in the presence of

29

serum albumin. The stability of the mixed micelles demonstrated in the presence of BSA


Biomacromolecules

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suggests that these mixed micelles may be compatible in vivo, which is critical for intravenous

2

drug delivery applications.

3 4

Figure 3: (a) Plot of normalized FRET ratio as a function of time for mixed micelles at various

5

ratios of 3HM:4HM. (b) Time-resolved fluorescence showing a 1:1 mixture of 3HM and 4HM,

6

each independently encapsulated with DiO and DiI, respectively.

7 8

Since the 50:50 ratio of 3HM:4HM showed higher stability than mixed micelles

9

composed of other ratios, it was further investigated. Initially, to probe the distribution of the two

10

coiled-coil amphiphiles within the mixed micelle, micelles composed of the individual

11

components – pristine 3HM and pristine 4HM were studied. Figure 4a shows DSC

12

measurements of the individual pristine micelles having very distinct phase transition

13

temperatures at 17°C and 40°C for 3HM and 4HM, respectively. Two separate micelle solutions

14

of 3HM and 4HM mixed in a 1:1 ratio displayed two prominent transitions at 17°C and 40°C,

15

indicative of discrete 3HM and 4HM populations in solution. However, a single homogeneous

16

transition ~ 21°C is apparent in Figure 4b upon thermal annealing of this mixture and subjected

17

to immediate DSC measurement (t = 0 hr). As the 1:1 mixture was allowed to equilibrate at room

18

temperature after thermal treatment, a separate transition at a higher temperature of ~33°C

19

appears and the enthalpy associated with the 33°C transition continued to increase from 1 hr to

20

16 hrs while the transition at 20°C decreased in enthalpy over the same time period. This

21

suggests that returning the thermally treated mixed micelles that are phase-mixed back to

22

ambient temperature led to the formation of distinct 3-helix and 4-helix domains within the

23

mixed micelles over the span of several to tens of hours.


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Biomacromolecules

1 2

Figure 4: DSC measurements of (a) pristine 3HM, 4HM, and 1:1 mixture of separately prepared

3

3HM and 4HM, (b) time evolution of 1:1 ratio of 3HM:4HM mixed micelle at times 0, 1, 16, 26,

4

and 40 hrs after thermal annealing. Solid red lines are fits to the data while dotted red lines

5

indicate the deconvoluted peaks.

6 7

Table 1: Phase transition temperatures and enthalpy for the corresponding data in Figure 4. Sample

Tm (°C)

ΔH (kJ/mol)

Total ΔH (kJ/mol)

3HM

17

25.1 ± 0.1

25.1 ± 0.1

4HM

40

33.5 ± 0.2

33.5 ± 0.2

1:1 t = 0hr

21

11.5 ± 0.04

11.5 ± 0.04

20

7.84 ± 0.06

33

1.28 ± 0.05

17

1.85 ± 0.02

33

7.35 ± 0.02

20

1.56 ± 0.02

37

7.60 ± 0.02

1:1 t = 1hr

1:1 t = 16hr

1:1 t = 40hr 8


9.12 ± 0.11

9.20 ± 0.04

9.11 ± 0.06

Biomacromolecules

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1

To further examine the phase separation behavior of nanodomains within the 1:1 ratio of

2

3-helix and 4-helix mixed micelles, the formation process of mixed micelles from 3HM and

3

4HM was studied using fluorescence self-quenching and recovery. Figure 5a shows the

4

fluorescence spectra of unlabeled 4HM added to a solution of FAM-labeled 3HM in a 1:1

5

mixture as a function of time at three temperatures – 25°C (red), heated to 70°C (black), then

6

cooled down to 25°C (blue). Figure 5b plots the normalized fluorescence intensity as a function

7

of time and the data at the three temperatures are in the same color scheme as Figure 5a. The

8

mixture of two separate micellar populations of 3HM and 4HM at the initial temperature of 25°C

9

experienced a slight ~3% increase in fluorescence intensity over a period of 22 hrs. This modest

10

increase in fluorescence is due to the excellent kinetic stability of 3HM and 4HM with minimal

11

subunit exchange between the two micelle populations33. However, when the temperature of the

12

mixture was raised to 70°C, the fluorescence intensity greatly increased by 58% in 2 hrs. This

13

large increase in fluorescence intensity is due to subunit exchange as the FAM-labeled 3-helix

14

amphiphiles are desorbed from the self-quenched 3HM-FAM and inserted into non-labeled 4HM.

15

As the sample cell was cooled back down to 25°C, the fluorescence intensity decreased 10%

16

over ~10 hrs. The FAM-labeled 3-helix amphiphiles reorganized themselves closer to each other,

17

thereby self-quenching the fluorescence and resulting in a decrease in fluorescence intensity.

18

This sequence of results show that 3HM and 4HM are kinetically stable at room temperature and

19

minimal subunit exchange occurs in the presence of the other micellar species. Addition of

20

thermal energy promotes the rapid exchange of amphiphiles between 3HM and 4HM, blending

21

to form mixed micelles. However, cooling down to ambient temperature resulted in spatial

22

rearrangement of the 3-helix and 4-helix amphiphiles within the mixed micelles to form 3-helix

23

rich and 4-helix rich nanodomains within the same micelle.

24


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Biomacromolecules

1 2

Figure 5: (a) Fluorescence spectra of 3HM-FAM mixed with 4HM at a 1:1 ratio as a function of

3

temperature. (b) Normalized fluorescence intensity at 520 nm plotted as a function of incubation

4

time at the different temperatures. Data points for 25°C, 70°C, and 25°C (post-heating) in red,

5

black, and blue, respectively.

6 7

To further augment our experimental results, CGMD simulations provided insight into the

8

arrangement of the peptides within the mixed micelles. Figure 6 maps the spatial distribution of

9

the 3-helix and 4-helix peptide headgroups and corresponding alkyl tails in spherical coordinates

10

in a single micelle. Clustering of 3-helix and 4-helix head groups is observed at equilibrium after

11

self-assembly. This supports our heme titration and SEC results (Figure 1), indicating an absence

12

of cross-oligomerization between the 3-helix and 4-helix amphiphiles upon assembly.

13

Additionally, simulation supports the experimental observation of intra-micellar nanophase

14

separation of 3-helix and 4 –helix amphiphiles from DSC and fluorescence recovery results.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Figure 6: Coarse-grained molecular dynamics simulation of 3HM mixed with 4HM at a 1:1 ratio.

3

Packing plots of (a) headgroup and (b) alkyl chains in the mixed micelle. Schematic on the left

4

illustrates the spherical coordinates used.

5 6

The results shown here strongly suggest that the lack of cross-oligomerization between two

7

orthogonal 3-helix and 4-helix coiled-coil bundles, even when modified with di-alkyl chains and

8

PEG, can be an effective platform to specify the local multivalency of ligand clusters. Highly

9

stable sub-20 nm mixed micelles can be generated from a pair of orthogonal coiled-coil peptide-

10

PEG-lipid conjugates. Furthermore, the 3-helix and 4-helix amphiphiles were observed to phase

11

segregate within the mixed micelles, forming ‘patches’ of 3-helix and 4-helix rich domains.

12

Further work is needed to quantify the spatial distribution and domain size of 3-helix and 4-helix

13

regions within the mixed micelles. The phase separation is likely driven by incompatible

14

geometric packing of the coiled-coil headgroups in the micelle corona and alkyl tails in the

15

hydrophobic core. 4-helix amphiphiles have a higher density of alkyl chains attached to each


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Biomacromolecules

1

bundle compared to 3-helix amphiphiles. The mismatch in alkyl chain length could cause phase

2

separation in mixed micelles. The domain size of nanodomains of lipids, also known as lipid

3

rafts, in membranes is directly correlated to the mismatch in degree of alkyl unsaturation and

4

subsequently bilayer thickness83,

5

minimization between 3-helix and 4-helix amphiphiles contributes to the phase separation

6

phenomenon described here. This study is an important first step to elucidate the properties of

7

these new hybrid self-assembled mixed micellar structures, and to allow their optimization

8

regarding different fields of application, in particular the design of drug delivery nanocarriers

9

with targeting capability using clustered ligands with well-defined oligomeric states using either

10

the 3-helix or 4-helix domain.

11

Conclusion

84

. We hypothesize a similar effect of interfacial energy

12

In this report, we show the retention of coiled-coil bundle oligomeric states and the

13

orthogonality of 3-helix and 4-helix peptide-PEG-lipid amphiphiles in mixed micelles with

14

potential utility for control over the local multivalency of ligands. Additionally, the mixed

15

micelles displayed high stability in serum albumin, which is crucial for intravenous drug delivery

16

applications. Temporal evolution of alkyl packing and tracking the relative distribution of FAM-

17

tagged 3-helix amphiphiles within mixed micelles indicated partitioning of 3-helix and 4-helix

18

rich domains to form self-assembled sub-20 nm patchy micelles. The proposed approach

19

described here for generating mixed micelles with well-defined ligand cluster sizes is extremely

20

versatile due to the ability to engineer a variety of coiled-coils with different oligomeric states.

21

Coiled-coil based peptide-PEG-lipid conjugates are a promising class of materials to specify the

22

oligomeric state of ligands on surfaces for a wide range of diagnostic and therapeutic

23

applications.

24 25

ASSOCIATED CONTENT

26

Supporting Information.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

MALDI-TOF, DLS, and time-resolved fluorescence spectroscopy of mixed micelles are

2

provided. This material is available free of charge via the Internet at http://pubs.acs.org.

3

AUTHOR INFORMATION

4

Corresponding Author

5

*[email protected]

6

Author Contributions

7

The manuscript was written through contributions of all authors. All authors have given approval

8

to the final version of the manuscript.

9

Notes

Page 18 of 24

10

The authors declare no competing financial interest.

11

ACKNOWLEDGMENT

12

This study was supported by National Institutes of Health (Contract 5R21EB016947-02). Prof.

13

Xu is a core-principal investigator of Tsinghua-Berkeley Shenzhen Institute (TBSI). We

14

acknowledge the funding support of TBSI. B.T.J was supported by a NSF Graduate Fellowship

15

(DGE 1106400).

16

References

17 18 19 20 21 22 23 24 25

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For table of contents only

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Sub-20 nm Stable Micelles Based on a Mixture of Coiled-coils: A Platform for

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Controlled Ligand Presentation

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JooChuan Ang1, Dan Ma2, Benson T. Jung1, Sinan Keten2, Ting Xu*1,3,4

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California 94720, United States

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Engineering, Northwestern University, Evanston, Illinois 60208, United States

Department of Materials Science and Engineering, University of California, Berkeley,

Department of Civil and Environmental Engineering and Department of Mechanical

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Department of Chemistry, University of California, Berkeley, California 94720, United States

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Material Science Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720,

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United States

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Sub-20 nm Stable Micelles Based on a Mixture of Coiled-Coils: A

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