Personal web page : http://iramis.cea.fr/Pisp/patrick.guenoun/index.html
Laboratory link : http://iramis.cea.fr/nimbe/lions/
Cubosomes are nanometric particles formulated from lipid bicontinuous cubic phases. Water-lipid bicontinuous cubic phases [Luzzati, 1962] are made from a single continuous lipid bilayer, where the bilayer midplane is coincident with an infinite periodic minimal surface and subdivides the space into two interpenetrating, but not connected water channels [Scriven, 1976]. The large surface area of the lipid-water interface (400 m2g-1) makes these nano-objects suitable for entrapment of proteins, peptides, and other biomolecules. Also their polar-apolar continuous domains allow for the encapsulation of a broad range of hydrophilic-hydrophobic molecules and for a slow release of cargo, maintaining the therapeutic concentration range over a longer period of time. Cubosomes suffer however from limited possibilities to control load and delivery of potential cargo. The size of the water channels, one of the few available structural parameters, can only be varied in a relatively narrow size range and the chemical nature of the lipids(here monoolein named MO), defining also the lipid-water interface, cannot be significantly modified without compromising the integrity of the structure.
In this PhD project we aim at bringing the control of uptake and release in cubosomes to new possibilities by engineering reaction-ready cubosomes and achieving flexibility in cubosome functionalization by allowing for in-situ multi-dimensional orthogonal reactions combined with the power of inclusion complexation provided by cyclodextrins (CDs). In practice, standard structure-forming lipids will be functionalized to allow for orthogonal click-chemistry reactions to occur at the lipidwater interface in the channels of these lipid mazes. This will open the pathway for devising sophisticated strategies for the kinetics of charging and discharging the nanoparticles with a variety of relevant payloads.
The structure of cubosomes will be characterized by several techniques, such as small angle X-ray scattering (SAXS), cryo-electron microscopy (cryo-EM), NMR and differential scanning calorimetry (DSC) [Angelova, 2015; Li, 2015].
Despite extensive work on cubosomes, outstanding questions still hamper progress in the understanding and control of the encapsulation and release mechanisms. How many of the channels actually communicate with the solution, are all cubosomes open, what is the role of exact shape and size on the exchange processes, why are cubosomes most often decorated with single bilayer handles and what role do they play … these questions require designing advanced experimental geometries to inter-relate structure and properties. We will address them by performing kinetic experiments on single cubosomes under a fluorescence microscope. Although for many therapeutic applications cubosomes are brought into contact with cell cultures, experiments on model systems are required to assess the fundamental parameters controlling how cubosomes behave at oil/water or lipid bilayer interfaces [Falchi, 2015]. We will apply synthetic chemistry following robust pathways [Osornio, 2012] to achieve in-situ functionalization summarised. Pathways are flexible enough to accommodate efficient labelling requirements (deuterium or fluorescence)or modifications required during the course of the project such as adjusting a spacer between lipids and the desired functional groups. Functional groups are chosen to be consistent with modern ligation methods in order to allow further access to large libraries of compounds and high throughput methods. They have also to allow selective orthogonal reactions according to Sharpless click chemistry paradigm. Of particular interest, the Huisgen reaction AAC (Alkyne-azidecycloaddition), usually referred as the first click reaction, will be performed using different sets of parameters (Strain, Copper catalysis, Ruthenium catalysis…). Functional lipids are designed to react after cubosome formation but could also enter the preparation of cubosomes in a one-pot process. Multicompartment nanocarrier cubosomes were recently demonstrated through incorporation of amphiphilic cyclodextrins (CD) that could carry water insoluble substances [Zerkoune, 2016]. In this project, the channels surfaces will be functionalised with CDs by in-situ reactions. Cyclodextrins will be considered as:
i) model molecules to link to the internal cubosome membrane in the aqueous channel (e.g. common Azido-cyclodextrin)
ii) carrier of hidden molecules to the membrane
iii)cages to control release and modulate affinity constants, typically of moderately hydrophilic compound with known affinity toward cyclodextrin cavity.
Importantly, according to their ring size, Cds and interaction capability, CDs can offer multiple specific behaviour with pluronic stabilizers. With little chemical effort, inclusion compounds could be favoured or hampered. Structure determination. In addition to X-ray and other standard techniques, deuterated species will be synthesized for structure determination by contrast matching in neutron scattering. Deuterated MO will be prepared in a multistep yet simple synthesis from deuterated oleic acid [Darwish, 2013]. Mixtures of deuterated unmodified MO and chemically modified MO will allow controlling the degree of mixing of the modified molecules with bare ones. Deuterated compounds will also allow NMR experiments to inspect diffusion phenomena, reaction kinetics, and membrane organization.
[Angelova, 2015] Angelov, B., Angelova, A., Drechsler, M., Garamus, V.M., Mutafchieva, R. and Lesieur, S. (2015) Identification of large channels in cationic PEGylated cu- bosome nanoparticles by synchrotron radiation SAXS and Cryo-TEM imaging. Soft Matter, 11, 3686–3692, 2015.
[Astolfi, 2017] Astolfi, P.; Giorgini, E.; Gambin, V.; … Vita, F.; Francescangeli, O.; Marchini, C. & Pisani, M. (2017), Lyotropic Liquid- Crystalline Nanosystems as Drug Delivery Agents for 5-Fluorouracil: Structure and Cytotoxicity, Langmuir 33, 12369-12378.
[Barriga, 2018] Barriga H.M.G., Holme M.N. and Stevens M.M. (2018), Cubosomes: the next generation of smart lipid nanoparticles, Angew. Chemie Int. Ed. 57, 2.
[Chong, 2011] Chong, Y.T.J., Mulet, X., Waddington, L.J., Boyd, B.J. and Drummond, C.J. (2011), Steric stabilisation of selfassembled cubic lyotropic liquid crystalline nanoparticles: high throughput evaluation of triblock polyethylene oxide-polypropylene
oxide-polyethylene oxide copolymers. Soft Matter, 7:4768.
[Darwish, 2013] Darwish, T.A., Luks, E., Moraes, G., Yepuri, N.R., Holden, P.J., James, M., (2013), Synthesis of deuterated oleic acid and its phospholipid derivative [D64]dioleoyl-sn-glycero-3-phosphocholine. J. Label. Compd. Radiopharm. 56, 520–529.
[Elamari, 2010] Elamari, H., Jlalia, I., Louet, C., Herscovici, J., Meganem, F., Girard, C., (2010), On the reactivity of activated alkynes
in copper and solvent-free Huisgen’s reaction. Tetrahedron Asymmetry, Henri Kagan: An 80th Birthday Celebration Special Issue – Part 1 21, 1179–1183.
[Falchi, 2015] Falchi, A.M., Rosa, A., Atzeri, A., Incani, A., Lampis, S., Meli, V., Caltagirone, C. and Murgia, S., (2015), Effects of
monoolein-based cubosome formulations on lipid droplets and mitochondria of HeLa cells. Toxicology Research, 4, 1025-1036.
[Garg, 2006] Garg, G.; Singh, D.; Saraf, S. & Saraf, S. (2006), Management of benign prostate hyperplasia: An overview of alphaadrenergic
antagonist, Biological & Pharmaceutical Bulletin. 29, 1554-1558.
[Hyde, 1984] Hyde, S.T. and Andersson S.A (1984) Cubic structure consisting of a lipid bilayer forming an infinite periodic minimum
surface of the gyroid type in the glycerol monooleate-water system. Crystalline Materials, 1984.
[Kluzek, 2017] Kluzek, M., Tyler, A.I., Wang, S., Chen, R., Marques, C.M., Thalmann, F., Seddon, J.M. and Schmutz, M., 2017. Influence of a pH-sensitive polymer on the structure of monoolein cubosomes. Soft Matter, 13, 7571-7577.
[La, 2014] La, Y.; Park, C.; Shin, T. J.; Joo, S. H.; Kang, S. & Kim, K. T. (2014), Colloidal inverse bicontinuous cubic membranes of block copolymers with tunable surface functional groups, Nat. Chem. 6(6), 534-541.
[Landh, 1994] Landh, T. (1994), Phase behavior in the system pine needle oil Monoglycerides-Poloxamer 407-Water at 20ºC. J. Phys. Chem., 98, 8453.
[Li, 2015] Li, J.C., Zhu, N., Zhu, J.X., Zhang, W.J, Zhang, H.M., Wang, Q.Q, Wu, X.X., Wang, X., Zhang, J. and Hao, J.F. (2015) Self- Assembled cubic liquid crystalline nanoparticles for transdermal delivery of paeonol. Med Sci Monit., 21, 3298–3310.
[Luzzati, 1962] Luzzati V . and Husson F. (1962) The structure of the liquid-crystalline phases of lipid-water systems., J Cell Biol, 12(2):207–219.
[Osornio, 2012] Osornio, Y. M.; Uebelhart, P.; Bosshard, S.; Konrad, F.; Siegel, J. S. & Landau, E. M. (2012), Design and Synthesis
of Lipids for the Fabrication of Functional Lipidic Cubic-Phase Biomaterials, JOC 77, 10583-10595.
[Scriven, 1976] Scriven, L.E.(1976) Equilibrium bicontinuous structure. Nature, 263(5573):123–125
[Zerkoune, 2016] Zerkoune, L., Lesieur, S., Putaux, J.-L., Choisnard, L., Gèze, A., Wouessidjewe, D., Angelov, B., Vebert-Nardin, C., Doutch, J., Angelova, A. (2016), Mesoporous self-assembled nanoparticles of biotransesterified cyclodextrins and nonlamellar lipids as carriers of water-insoluble substances. Soft Matter 12, 7539–7550.