Materials
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was obtained from Avanti Polar Lipids (Alabaster, AL, USA), 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI dye, Mol.wt – 933.87, CAS Number – 41085-99-8), Poly(vinyl alcohol) [Mw 89,000–98,000, 99+% hydrolyzed] were obtained from Merck (Darmstadt, Germany),4-(2-Hydroxyethyl)piperazine-1-ethanesulfonicacid;N-(2-Hydroxyethyl)piperazineN′-(2 ethane sulfonic acid) was obtained from MP Biochemicals, and Sucrose was purchased from Merck. N,N,N′,N′-Tetramethyl ethylenediamine (TEMED), Ammonium persulfate (APS), 1,6-Diphenyl-1,3,5-hexatriene (DPH) [CAS Number: 1720-32-7] and N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl)phenylammonium p-toluenesulfonate (TMA-DPH) [CAS Number: 115534-33-3] were purchased from Sigma Aldrich. Microscope glass slides were purchased from Labtech Medico P Ltd. 3D-printed rings were printed with Acrylonitrile Butadiene Styrene (ABS) material using the Creater 3 Pro Machine (Flashforge). Random Positioning Machine (RPM), which simulates microgravity was custom built and modified in the lab.
GUV formation using PVA-assisted gel swelling method
The GUV were synthesized using PVA assisted method as described previously (Parigoris et al., 2020). The synthesis chamber with spherical ring with dimension of 20 mm diameter*1.5 cm height were 3D printed and attached to the piranha treated glass slide as shown in the Fig. 1. Briefly, 300 µL of 5% PVA solution (w/v), 1 µL of TEMED and 10 µL of 10% APS was prepared and drop casted into custom build synthesis chamber and heated for 1 h at 45–50°C in the hot plate for the gel solution to dry. Next 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid was resuspended with chloroform at a concentration of 1mg/ml. Then the stock solution was mixed with 10 µL of 10 µM 1,1′-Dioctadecyl-3,3,3′,3′-tetra methylindocarbo cyanine perchlorate (DiI) dye. Then, the labelled lipid solution was drop-casted on top of the dried PVA gel solution and vacuum dried for 30 minutes. After drying, the rehydration of the lipid film was performed using different buffers- Buffer A (600 mM sucrose) and Buffer B (500 mM sucrose and 50 mM HEPES). 800 µL of Buffer A and Buffer B were added to the dried films and stored at room temperature for 1 h. GUVs were stored at 4°C for around 30 mins before imaging. These can be stored at -20°C or lower for long term storage purposes.
Figure 1 Schematic of the PVA-assisted gel swelling process for GUV synthesis and the influence of microgravity on the artificial cells using a random positioning machine (RPM).
Simulation of microgravity
Studying the long-term effects of microgravity requires sending the samples in sounding rockets, or actual spaceflight for the stipulated number of days, which is not only an expensive ordeal, but also requires extensive resources and manpower. To overcome that, there are numerous instruments like clinostats, random positioning machine, etc. available to simulate microgravity in the laboratory. The Random Positioning Machine is a ground-based lab instrument to simulate the microgravity conditions in a controlled laboratory environment. The instrument works on the principle of averaging the gravity vector to zero, such that a state of weightlessness is developed at the center of the instrument [1]. It consists of two perpendicular axes which are programmed to rotate in a randomized direction, constantly reorienting and distributing the gravity vector in all directions. Thus, it does not allow the minimum time required for the loaded samples to adjust to any particular direction of the gravity vector, allowing them to experience a state of weightlessness [2]. In the current study, we used a desktop RPM machine to simulate the effects of microgravity on the GUV particles. The machine was run at 14 rpm (27°C), which simulates microgravity in the range of 10 − 3 to 10 − 4 [3]. The samples loaded in Eppendorf tubes and filled to the brim with 300 µL. They were kept at the center position of the stage in the RPM for 24 h, then taken out and observed for morphological changes.
Characterization of GUV under normal gravity and microgravity conditions
Optical microscopy and Slide Preparation
The synthesized GUVs were imaged under brightfield and fluorescence microscopy (Infinity, inverted Culture Microscope iOX 600). Before imaging, glass slides were treated with a 1% BSA solution for 30 minutes to prevent non-specific adhesion of lipid structures to the glass surface. Further analysis on GUV size and shape characterization were carried out using Image J software. Yield was analyzed by counting the number of GUVs present in a 5 µL GUV solution droplet and calculating the count per µL.
Fluorescence Anisotropy Measurements
The membrane fluidity of GUVs was investigated by the fluorescence anisotropy assay as described previously (Cheng & London, 2011). The DPH and TMA-DPH were used as a fluorescence probe. In the cuvettes, 0.5 µM of DPH / TMA-DPH was added to 100 µM of GUVs obtained for both normal and microgravity condition. The fluorescence anisotropy values of GUVs were measured under microgravity for different time periods ranging from 0 h to 50 h at room temperature using multimode plate reader (BioTek Synergy H1 Multimode Reader, Agilent). Autopolarizers with slit widths with a nominal band-pass of 5 nm were used for both excitation and emission. DPH and TMA-DPH fluorescence anisotropy values were measured at the excitation wavelength of 358 nm and emission wavelength of 410 nm. The anisotropy 〈r〉 values were calculated as shown in the below Eq. 1
$$\langle r\rangle = \frac{{I}_{\parallel }- {GI}_{\perp }}{{I}_{\parallel }+ {2GI}_{\perp }}$$
1
…..
where, I∥ and I⊥ are the emission intensities with polarizers parallel and perpendicular to the direction of the polarized exciting light, respectively. The values of the G-factor (ratio of the sensitivities of the detection system for vertically [IHV] and horizontally [IHH] polarized light) were determined separately for each sample. Applying Eq. 2 the lipid-order parameter S values were calculated from the anisotropy values using the following expression (Pottel et al., 1983):
$$S= \frac{\sqrt{(1- \frac{2r}{{r}_{0}} + \frac{5r}{{r}_{0}})} - 1 + \frac{r}{{r}_{0}}}{2\frac{r}{{r}_{0}}}$$
2
…..
where r0 is the fluorescence anisotropy of DPH/TMA DPH in the absence of any rotational motion of the probe. In this case r0 is taken to be 0.4.
Calculation of Membrane microviscosity and Rotational Diffusion Coefficient
Microviscosity helps in the correlation of classical hydrodynamic expressions to lipid membranes in biological cells. In DPH-labeled systems, we can obtain the microviscosity by simply measuring fluorescence polarization, using Shinitzky's equation (Lee, 2003) for the approximate evaluation of microviscosity \(\stackrel{-}{\eta }\). This equation is obtained by rearranging and approximating the well-known Perrin equation as follows (Eq. 3) for an r0 of 0.4 for DPH.
$$\stackrel{-}{\eta }= \frac{2.4r}{0.4 -r}$$
3
…..
Fluorescent depolarization is caused by the rotational diffusion of fluorophores, which corresponds to the diffusivity of the biomolecule to which it binds. We have calculated the rotational membrane diffusivity which will provide information on the fluidity of the membrane as described by using the Eq. (4) (Lakowicz, 1999);
$$\frac{r}{{r}_{0}}=1+ 6D\tau$$
4
…..
Where r0 and r are the fluorescence anisotropy of DPH in the absence and presence of any rotational motion of the probe respectively, D is the rotational diffusion coefficient of the fluorophore, and τ the fluorescence lifetime of the fluorophore.
Statistical Analysis
All the results are represented as Mean ± SEM. Statistical comparison has been conducted using the ONE-WAY ANOVA in Graph Pad prism software. The Tukey Test was used as Post Hoc test for intergroup comparison. The difference between different groups was considered statistically significant if P < 0.05.