Design of Thermo-Responsive Self-Assembly of PEGylated Fatty Acids: Switching Reversibly from Tubes or Vesicles to Micelles at Physiological Temperature — en

Maëva Almeidaa, b, Daniel Dudzinskib, Benoit Couturauda, Sylvain Prévostc, Viviane Lutz-Buenob,d, Najet Mahmoudie, Catherine Amiela, Fabrice Cousinb, Clémence Le Coeura, b

aInstitut Chimie et Matériaux Paris Est, Université Paris Est Créteil, CNRS, UMR 7182, 2 Rue Henri Dunant, 94320 Thiais, France
bLaboratoire Léon Brillouin, Université Paris-Saclay, CEA-CNRS UMR 12 CEA Saclay, 91191 Gif sur Yvette, France
cInstitut Laue Langevin, 71 avenue des Martyrs, CS 20156, CEDEX 9, 38042 Grenoble, France
dPSI Center for Neutron and Muon Sciences, 5232 Villigen PSI, Switzerland
eISIS Neutron and Muon Source, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Chilton, Oxfordshire OX11 0QX, UK

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The unmatched potential of nanoparticles for biomedical applications has fostered an intense research over these last decades, which stems from the fact that they can be used as active or passive nanocarriers for drug delivery to targeted cells. Depending on the type of drug and/or target cell, they need to meet numerous requirements such as colloidal stability, biocompatibility, low cytotoxicity, optimal tissue penetration, among others. This is achieved through the fine tuning of the nanocarrier morphology, size and physicochemical properties at the nanoparticle level, as well as of the control of the viscoelastic properties of the suspensions into which they are dispersed. Last but not least, the nanocarriers have to be made stealth to prevent protein adsorption from physiological fluids, otherwise a protein shell will grow rapidly around them, and this so-called “protein corona” will drive their in vivo biological fate. To this aim goal, the most widespread strategy consist in decorating the nanocarriers by a protective polymeric shell of poly(ethylene glycol) (PEG) chains, since PEG is known to suppress non-specific protein adsorption thanks to the high level of hydration of its hydrophilic polyether backbone. PEGylated nanocarriers show thus longer blood half-lives and less non-specific cellular uptake compared to their unmodified counterparts. With these specifications in mind, we propose a simple one-pot route that enables to design thermo-responsive PEGylated self-assemblies of fatty acids into two morphology types at room temperature, multi-lamellar tubes or vesicles. These morphologies transit reversibly upon heating into small ellipsoidal micelles around physiological temperature (Figure 1). This mechanism is based on the insertion of four types of 4k end-capped PEG chains, capped at one or both ends with either 12-hydroxy stearic acid (12-HSA) or stearic acid (SA), into self-assemblies of 12-HSA, a bio-based green surfactant. The choice of 12-HSA is driven by the peculiar morphology of their aqueous self-assemblies when dispersed with alkanolamine counterions at room temperature: they form multilamellar tubes with a length ranging between 10 and 50 μm, a temperature tunable diameter of the order of 0.6 μm, and a core made of a few stacked bilayers separated by water. The jamming between tubes makes the suspensions gel-like. Above a given threshold temperature these tubes melt reversibly into small ellipsoidal micelles which turns the gelled solution into a Newtonian fluid. Moreover, the threshold temperature can be adjusted continuously over a broad range of temperature by an appropriate choice of the type of alkanolamine counterion and/or fatty acid/counterion ratio and we chose the conditions to set it at ~37 °C.

Figure 1. Morphology of self-assembled nanostructures based on mixtures of 12-HSA molecules with different PEGylated fatty acids (SA-PEG-SA, mPEG-SA, mPEG-HSA or HSA-PEG-HSA) above and under 37 °C.

Small Angle Neutron Scattering (SANS) was used to determine the precise structure of all systems under scrutiny. Experiments in “full contrast”, i.e. where all species are hydrogenated and dispersed in D2O, below 37 °C, all systems show the structural characteristic features of tubes (figure 2.a), except in case of di-functionalized chains by 12-HSA where vesicles are produced. Contrast variation was further used with deuterated end-capped PEG chains to mask contribution of fatty acids in order to shed light on the contribution to the scattering of PEG chains and demonstrate that they homogeneously decorate the vesicle and keep a Gaussian behavior. For both types of mono-functionalized PEG, the chains insert therefore homogenously in the multi-lamellar tubes. The mixtures of di-functionalized chains by 12-HSA with 12-HSA molecules produce PEGylated vesicles because the change of packing parameter induced by insertion of the telechelic chains no longer allows the formation of tubes. Conversely, mixtures of di-functionalized chains by SA with 12-HSA molecules enable to keep multi-lamellar tubes, a specific behavior that likely comes from the fact that they only insert by one end within the 12-HSA bilayers. At temperatures above 37 °C, as expected, all systems transit reversibly into small PEGylated ellipsoidal micelles, as shown by SANS (Figure 2.c), which in turn enable to tune the rheological properties. This very elegant way of preparing PEGylated nanostructures of controlled morphology and temperature response would enable numerous applications in drug delivery.

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Figure 2. (a) SANS scattering profiles of the different systems at 20 °C in a 100% D2O solvent. The spectra are successively shifted by a factor of 10 in intensity for clarity (data of HSA on an absolute scale). (b) Comparison of the SANS scattering profiles of pure PEG solution in a 100% D2O solvent, HSA-PEG-HSA/HSA mixture in a 100% D2O “full contrast” solvent, and HSA-PEG-HSA/HSA mixture in a 87% H2O/13% D2O “matching HSA” solvent at 20 °C. (c) SANS scattering profiles of the different systems at 20 °C in a 100% D2O solvent. The spectra are successively shifted by a factor of 10 in intensity for clarity (data of HSA on an absolute scale). Solid lines represent the best fit to the data (see models and fitting parameters in the article).

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