Interaction of methoxy-functionalized manganese(II)-oxide

The interaction with the cellular membrane is a crucial step in the further processing of nanomaterials by cells.(Vasir and Labhasetwar, 2008) Especially Janus particles, which represent anisotropic particles with at least two chemically or physically different domains, are supposed to show substantial membrane activity due to their amphiphilic nature. In first experiments, I use anisotropic Janus particles, which consist of a manganese(II)-oxide part with a size of 14.9 ± 1.1 nm and a gold part with a diameter of 6.7 ± 0.4 nm, to research the interaction of Janus particles with artificial membranes.

To verify successful synthesis of Janus type particles transmission electron microscopy experiments are conducted (TEM, experiments performed by Isabel Schick, Institute of Inorganic and Analytical Chemistry, Johannes-Gutenberg university, Mainz, Germany).

Representative TEM images can be found in Figure 4.1.2 A and B. The Au-domains of the Janus particles appear dark in TEM images due to their high electron density (see Figure 4.1.2 B). The MnO domain is brighter. Figure 4.1.2 B shows heterodimeric composites of MnO and Au in which both parts are exposed to the media implying that real anisotropic Janus particles are formed during synthesis. Further details about

synthesis of Janus particles can be found in chapter 3.1 or in the publication by Schick et al..(Schick et al., 2014) Schick and coworkers as well as Schladt and coworkers also demonstrated that the choice of the surfactant stabilizing the gold precursor particles during synthesis is crucial for the asymmetric growth of the metal oxide on the gold lattice.(Schladt et al., 2010) Using oleic acid and oleylamine as surfactants lead to epitaxial growth of MnO domains on the gold surface producing nanocomposites with a flowerlike structure. Here, 1-octadecanethiol was used to stabilize the Au-seed particles during the thermal decomposition of manganese(II)-oleat leading to anisotropic growth of a MnO-domain on the gold particle.

Figure 4.1.2: Representative TEM images of A spherical MnO-particles and B Au@MnO-Janus particles (Images have been measured by Isabel Schick, Institute of Inorganic and Analytical Chemistry, Johannes-Gutenberg University, Mainz, Germany). Scale bars: 20 nm C

Scheme of functionalization of the different domains. The metal oxide domain was functionalized with polyethylene glycol (PEG), which is attached to the nanoparticle surface via

a dopamine anchor. The other end of the PEG carried a methoxy-group. The gold part is functionalized with 1-octadecanethiol (ODT).

In the present study, the MnO-domain of the Janus particles is functionalized with polyethylene glycol with an average molecular weight of 600 g/mol (PEG600), which binds to the metal oxide via an attached dopamine anchor (DOPA) in a nearly covalent manner.(Xu et al., 2004) The other end of the PEG carries a methoxy-group. This functionalization renders the MnO-domain hydrophilic, whereas the gold-domain is hydrophobic due to functionalization with 1-octadecanethiol (ODT). A scheme of the surface functionalization can be found in Figure 4.1.2 C. For comparison, I use spherical MnO-particles with a diameter of 10.1 ± 0.7 nm. Again, the surface of the metal oxide is functionalized with DOPA-PEG-OMe.

To examine the interaction of amphiphilic Janus particles with artificial lipid bilayers I use giant unilamellar vesicles (GUVs) produced by electroformation. GUVs were

produced from 99.5 mol% 1,2-dioleoyl-s,n-glycerol-3-phosphatidylcholine (DOPC) and 0.5 mol% of TexasRed® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) to allow for fluorescence microscopic exploration of the membrane. DOPC is a natural lipid and its main phase transition temperature lies at -17°C (http://avantilipids.com, 14.03.2014). Thus, at room temperature it is in the liquid-crystalline (Lα-)phase.

Figure 4.1.3: Interaction of Janus particles with GUVs A, B, C epifluorescence images of DOPC/TR-DHPE GUVs treated with 1 µg/ml spherical MnO-DOPA-PEG-OMe nanoparticles in the equatorial focal plane. D, E, F epifluorescence images of DOPC/TR-DHPE GUVs treated

with 1 µg/ml ODT-Au@MnO-DOPA-PEG-OMe Janus-nanoparticles in the equatorial focal plane. Scale bar: 20 µm Inlet in F: upper half of the GUV shown in F treated with

ODT-Au@MnO-DOPA-PEG-OMe Janus-nanoparticles.

DOPC/TR-DHPE-GUVs filled with a 100 mM sucrose solution were added to a 90 mM solution of glucose, which allowed the particles to sediment and produced an osmotic gradient across the membrane, which prevented unintended deflation of vesicles.

1 µg/ml nanoparticles were added to the GUV solution. The effects of Janus particle administration to DOPC-GUVs were followed for 30 minutes using epi-flourescence microscopy (see Figure 4.1.3). Figure 4.1.3 A, B and C show GUVs, which are exposed to spherical isotropic MnO particles. The GUVs do not show any irregularities

in shape or in the membrane over the whole time. In contrast, immediately after addition of ODT-Au@MnO-DOPA-PEG-OMe Janus particles, the membrane of the GUVs starts to form membrane tubes to the inside of the GUVs. These membrane tubes become even more pronounced after 15 minutes incubation and eventually lead to the formation of small vesicles inside the GUVs (see Figure 4.1.3). Notably, the smaller vesicles inside the GUVs can be exclusively found in the top half of the GUV indicating that they are taken up by the GUV and contain media with lower density (90 mM glucose) compared to media filling the GUV (100 mM sucrose). To ensure, that the observed effect does not result from a change in the osmotic gradient, I controlled the osmolalities of the used solutions. The observed osmolalities of the used solutions can be found in table 4.1.1.

Table 4.1.1: Osmolality of solutions used in GUV experiments.

osmolality / mosmol/kg

100 mM sucrose 119

1 µg/ml ODT-Au@MnO-DOPA-PEG-OMe

+ 90 mM glucose 96

1 µg/ml MnO-DOPA-PEG-OMe

+ 90 mM glucose 96

The measured osmolalities indicate that both glucose-nanoparticle solutions were hypoosmolar compared to the sucrose solution inside GUVs. Thus, the membrane of vesicles is set under tension, which also prevents thermal membrane undulations that are observed for tension-free membranes.(Boal, 2012) Apparently, attractive forces between Janus particles and DOPC/TR-DHPE GUVs are sufficient to induce membrane deformations, but are not high enough to promote complete membrane wrapping of single particles as demonstrated in simulations by Ding et al. for Janus particles or by Yue et al. for spherical particles.(Ding and Ma, 2012; Yue et al., 2013) This is conceivable since the hydrophobic Au-domain experiences repulsive forces from the polar head groups of the membrane lipids. However, as depicted in Figure 4.1.1 Janus particles could also insert into the lipid bilayer and form pores as proposed by Alexeev and coworkers.(Alexeev et al., 2008) Using coarse grain simulations, the authors could demonstrate that small Janus particles are able to produce pores in lipid bilayers in which the Janus particles line the pore rim. Thereby, the hydrophobic part of

the particles interacts with the hydrophobic chains of the lipids. Although I cannot completely rule out this way of interaction between the Janus particles and the lipid bilayer at this point, it appears to be very unlikely because pores in the membrane would presumably allow molecules, e.g. the sucrose to diffuse into the vesicles, and thus, to eliminate the osmotic gradient. Without the osmotic gradient one would observe membrane undulations, which did not appear in my measurements as the contour of the GUVs remains completely circular (Figure 4.1.3). Additionally, the Janus particles used in the simulations were small having a size comparable to the lipid bilayer thickness. Particles used in this study are larger with a diameter of the gold part of 6.7 nm and insertion into the lipid bilayer appears therefore less likely. Anyhow, vesicles treated with Janus particles exhibit massive membrane deformations and eventually vesicles form from tubulations. Energetically, deformation of a lipid bilayer can be described by the local bending energy per unit area, which amounts to 2κB/R², where κB denotes the bending modulus and R is the radius of curvature.(Boal, 2012) For lipid bilayers κB has a value of 10 – 20 kBT. Thus, formation of a spherical vesicle with a surface of 4πR² from a planar lipid bilayer costs approximately 500 kBT of energy and is independent of vesicle radius.(Reynwar et al., 2007) Zimmerberg and coworkers calculated that the interaction between a single curvature-inducing protein, e.g. BAR-domain protein, and the lipid bilayer is on the order of 10 kBT, which is one order of magnitude lower than the energy needed to form a vesicle.(Zimmerberg and McLaughlin, 2004) However, Reynwar et al. proposed a mechanism for cooperative membrane bending by proteins or Janus particles. (Reynwar et al., 2007) In coarse grain simulations, the authors showed that Janus particles placed on a lipid bilayer interact with each other via long-range interactions. A single particle on a lipid bilayer produces a local curvature in the membrane. When the local curvatures induced by two particles overlap, the particles experience a long-range attractive force due to energy minimization of the elastic energy stored in the membrane due to the induced local curvature. Thus, particle pairs form and produce a larger membrane tubule, which attracts more particles. Eventually, a particle filled vesicle pinches off, demonstrating a passive (not actively driven by the cell) way of endocytosis in this artificial lipid bilayer system. As I observe similar steps of membrane perturbation in my experiments, it is likely that the examined Janus particles follow the route described by Reynwar and coworkers. Nevertheless, the modes of interaction between particles and membrane remain unclear, as the particles are invisible in my experiments. I also performed experiments, in which the methoxy group was replaced by a fluorescein isothiocyanate (FITC), but vesicles treated with these particles did not show any tubulation (data not shown) suggesting a change in the interaction between membrane and particle. To

increase the biocompatibility of our MnO-based nanoparticles, the MnO-domain was coated with a thin silica layer, which is supposed to preserve leakage of Mn²⁺-ions. The effect of this functionalization on the nanoparticle-membrane interaction will be examined in the next chapter.

4.1.2.2 Interaction between silica-coated manganese(II)-oxide Janus particles