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Bioprinting of Synthetic Cell-like Lipid Vesicles to Augment the Functionality of Tissues after Manufacturing
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  • Ole Thaden
    Ole Thaden
    Bioprinting & Tissue Engineering Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
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  • Nicole Schneider
    Nicole Schneider
    Bioprinting & Tissue Engineering Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
    More by Nicole Schneider
  • Tobias Walther
    Tobias Walther
    Biophysical Engineering of Life Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
    Max Planck Institute for Medical Research, Heidelberg 69120, Germany
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  • Erin Spiller
    Erin Spiller
    Bioprinting & Tissue Engineering Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
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  • Alexandre Taoum
    Alexandre Taoum
    Bioprinting & Tissue Engineering Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
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  • Kerstin Göpfrich
    Kerstin Göpfrich
    Biophysical Engineering of Life Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
    Max Planck Institute for Medical Research, Heidelberg 69120, Germany
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    Orcidhttps://orcid.org/0000-0003-2115-3551
  • Daniela Duarte Campos*
    Daniela Duarte Campos
    Bioprinting & Tissue Engineering Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
    *E-mail: [email protected]
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    Orcidhttps://orcid.org/0000-0002-3194-8983
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ACS Synthetic Biology

Cite this: ACS Synth. Biol. 2024, 13, 8, 2436–2446
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https://doi.org/10.1021/acssynbio.4c00137
Published July 18, 2024

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Abstract

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Bioprinting is an automated bioassembly method that enables the formation of human tissue-like constructs to restore or replace damaged tissues. Regardless of the employed bioprinting method, cells undergo mechanical stress that can impact their survival and function postprinting. In this study, we investigate the use of a synthetic cell-like unit, giant unilamellar vesicles (GUVs), as adjuvants of the cellular function of human cells postprinting, or in future as the complete replacement of human cells. We analyzed the impact of two nozzle-based bioprinting methods (drop-on-demand and extrusion bioprinting) on the structure, stability, and function of GUVs. We showed that over 65% of the GUVs remain intact when printing at 0.5 bar, demonstrating the potential of using GUVs as a synthetic cell source. We further increased the stability of GUVs in a cell culture medium by introducing polyethylene glycol (PEG) into the GUV lipid membrane. The presence of PEG, however, diminished the structural properties of GUVs postprinting, and reduced the interaction of GUVs with human cells. Although the design of PEG-GUVs can still be modified in future studies for better cell–GUV interactions, we demonstrated that GUVs are functional postprinting. Chlorin e6-PEG-GUVs loaded with a fluorescent dye were bioprinted, and they released the dye postprinting only upon illumination. This is a new strategy to deliver carriers, such as growth factors, drugs, nutrients, or gases, inside large bioprinted specimens on a millimeter to centimeter scale. Overall, we showed that printed GUVs can augment the functionality of manufactured human tissues.

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1. Introduction

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Tissue engineering aims to create functional tissues for the repair or replacement of damaged tissues. (1) A combination of cells, biocompatible materials, and bioactive molecules is used to fabricate such tissues. (2) Bioprinting is a common approach to fabricate tissues, where a combination of biocompatible materials and cells (bioinks) is deposited in a spatially defined manner. (2−4) Various bioprinting methods are currently available for generating tissue-like constructs, including drop-on-demand (DOD), (5) extrusion, (6) and laser-assisted bioprinting. (7) Hydrogels are often used as the basis of bioinks to provide an extracellular matrix-like environment for the cells and to render the bioinks printable. (8) Over the past years, not only bioinks loaded with dissociated cells (9) but also bioinks containing spheroids (10) and microgels (11) were bioprinted. In recent years, the idea of encapsulating liposomes in hydrogels has emerged to achieve defined drug delivery in the field of tissue engineering. (12−14) Liposomes are small vesicles composed of phospholipids that form a lipid bilayer in aqueous solutions. (15,16) Liposomes with a diameter larger than 1 μm are known as giant unilamellar vesicles (GUVs) and have been used in biophysics as a synthetic cell model. (15,17) GUVs can be produced by electroformation, (18) gentle hydration, (19) and gel-assisted swelling (20) or with a microfluidic setup. (21,22) GUVs can be loaded with hydrophilic carriers (encapsulated in an aqueous solution in the vesicle’s interior) or hydrophobic parts (located in the bilayer). (13) Generally speaking, GUVs are a versatile resource that can be tailored for different biomedical applications, for example, by functionalizing the surface with site-specific ligands or increasing the stability of biomolecule carriers that are physically separated from the surrounding environment by a lipid bilayer. (13,14) Past studies have shown that various trigger mechanisms can control the release of GUV cargos including temperature, (23) pH shift, (24) and light. (25) As a result of these findings, GUVs can be currently used in several applications, such as in the development of synthetic cells by bottom-up bioassembly, and as synthetic bioreactors when loaded with mammalian cells. (17,26) Compared to smaller liposomes, GUVs enable direct visualization by fluorescence microscopy and the encapsulation of larger amounts of carriers. For these multiple exciting reasons, GUVs have the potential to be used as synthetic adjuvants of human cells for tissue engineering and regenerative medicine applications, due to their ability to deliver biochemical and biophysical cues, as well as to become a potential synthetic surrogate for human cells in future.
In this study, we bioprinted GUVs to explore their interaction with human cells and the potential to aid cellular function postprinting. A bioprinting protocol has been established herein consisting of GUV production, filtration, and the bioprinting process itself. We investigated the stability of GUVs under physiological conditions and assessed the portion of intact GUVs after the bioprinting process. Furthermore, we modified the lipid membrane of GUVs with the addition of PEGylated lipids to improve the stability of GUVs in cell media and bioinks. Additionally, we demonstrated the printability of GUVs together with human cells in different bioinks and the stability of GUVs for up to 7 days postprinting. Finally, we synthesized the GUVs with a photosensitizer and used them for the delivery of a membrane impermeable dye postprinting by illuminating the bioprinted constructs.

2. Results and Discussion

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2.1. Production of GUVs Compatible with Bioprinting

A new protocol was established to enable the use of GUVs in bioprinting, either as synthetic cell adjuvants or as a potential replacement for human cells. Our protocol for bioprinting GUVs using DOD and extrusion bioprinting methods was adapted from past bioprinting studies, which included mammalian cell expansion, encapsulation in hydrogels, and printing (Figure 1). (3,10,27−30) The first step is to produce GUVs composed of dipalmitoylphosphatidylcholine (DOPC) lipids using the electroformation method (Figure 1A). (18) The electroformation method is a simple and fast procedure used to generate a large number of GUVs (approximately 17 × 106 GUVs per production cycle) without the use of surfactants or mineral oils, which could potentially be harmful for human cells. (31) Given that GUVs are polydisperse, we needed to implement filtration and concentration steps (Figure 1B). (32) The GUV stock solution was filtered through a 10 μm filter membrane to exclude smaller GUVs and the remaining residues from the production process. The filtration process reduced the total number of GUVs with a diameter less than 8 μm by 90% (Figure S1). The average size of synthetic vesicles used in biomedical applications is about 1 μm, and even smaller vesicles can be designed for drug delivery to be taken up by cells. (33,34) In bioprinting, however, GUV sizes in the mammalian cell range are required (larger than 8 μm) to recapitulate the cellular architecture in natura and allow the loading of larger amounts of carriers to be delivered inside the bioprinted constructs. Moreover, GUV sizes above 8 μm allowed us to visualize them during different steps by fluorescence microscopy.

Figure 1

Figure 1. Bioprinting of giant unilamellar vesicles (GUVs). A) Production of GUVs with defined filling (Alexa Fluor 488 phalloidin, inset: scale bar of 10 μm) by electroformation. B) Filtration of GUVs by size with a 10 μm filter membrane to collect a stock solution with an average GUV diameter similar to human cells. C) Bioprinting GUVs by DOD or extrusion.

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After the filtration step, GUVs were imaged by fluorescence microscopy, and the total number of GUVs was counted. Next, a working solution with a defined concentration of GUVs was prepared and transferred to a bioprinting cartridge (Figure 1C). Based on the range of cell densities used in previous bioprinting works with human cells (105–107 cells/mL), we chose a density of 5 × 105 GUVs/mL for bioprinting experiments. (3,5,35)

2.2. Stability of GUVs Postprinting

A key requirement in bioprinting with human cells is the warranty of cell survival during the printing process. Similarly, in this study, GUVs should remain intact during bioprinting to render a functional 3D construct postprinting. To investigate the impact of the printing process on GUVs, we diluted the GUV solution in sucrose to exclude the effect of cell media or bioinks in this experiment and bioprinted the vesicles by DOD and extrusion bioprinting at different pressures (more images of GUVs postprinting at different pressures are available Figure S2a). The number of GUVs larger than 8 μm was counted from fluorescence images before and after the printing process, and the percentage of intact GUVs was calculated (Figure 2A).

Figure 2

Figure 2. GUV stability during bioprinting. A) Comparison of GUV stability after drop-on-demand (DOD) and extrusion bioprinting in GUV solution (iso-osmolar sucrose solution). A higher GUV density of 2 × 106 GUVs/mL and was used to visually count GUVs more easily. B) Incubation of DOPC-GUVs in the cell medium for 1 h. Scale bars represent 50 μm.

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The maximum percentage of intact GUVs was 65.7 ± 16.1% after DOD bioprinting and 50.2 ± 6.2% after extrusion bioprinting, both at 0.5 bar. This means that the number of GUVs larger than 8 μm was reduced by 34.3% and 49.8%, respectively, after DOD and extrusion bioprinting. During DOD bioprinting, the GUV solution was dispensed through a 300 μm nozzle, resulting in the formation of a shear force gradient in the kilopascal range, with the maximum force being detected at the edges of the nozzle. (36) This has been observed in our previous studies using human mesenchymal stromal cells (MSCs), as well as in this study, where we observed MSC viability postprinting above 90% under the same conditions as those for printing GUVs (Figure S3). Therefore, the impact of the shear stress during the printing process needs to be controlled when bioprinting GUVs. The shear stress experienced by GUVs during bioprinting can stretch the lipid membrane and increase the membrane tension. When the critical lysis tension is reached, pores are formed and the lipid bilayer ruptures. (16) Liquid-phase GUVs, like the GUVs used in this study, tend to close the pores rather quickly due to the hydrophobic properties of acyl chains and a low intermembrane viscosity. (16) Whether GUVs that ruptured during the bioprinting process and close the pores to form smaller GUVs, or are dispersed throughout the sample, requires further investigation.
During extrusion bioprinting, the GUVs are pushed through an additional 12.7 mm long stainless-steel nozzle with a 330 μm diameter. This additional element created an increased shear stress experienced by GUVs, which resulted in a lower, but not statistically significant, percentage of intact GUVs compared to DOD bioprinting. (37)
After the bioprinting process, GUVs should be ideally not only intact but also remain stable in their surrounding environment. So far in our study, GUVs have been prepared and bioprinted in an isoosmolar solution. GUVs, which have been used previously as a membrane model, have a melting point less than 37 °C. (16,38) Given our interest to use GUVs in direct contact with human cells, with hydrogels, and in future, with other physiological liquids, we investigated their behavior when suspended in a cell culture medium at 37 °C. Thus, we incubated GUVs in a cell medium at 37 °C and 5% CO2, i.e., under the exact same culture conditions used for the human cell culture. After 3h of incubation, GUVs agglomerated at the bottom of the culture plate (Figure 2B). It is known that multivalent ions in solution can increase the hemifusion upon the contact of lipid bilayers. (39,40) As the cell medium contains multiple ions, there is a high probability that this leads to the agglomeration of GUVs. Interestingly, incubation in DPBS and glucose solution with CaCl2 (1.8 mM) showed the same effect of agglomeration (Figure S4). Overall, GUVs were mostly stable during bioprinting, with an average of 65.7% GUVs staying intact. However, the formulation of GUVs containing only DOPC could not be used further in cell culture experiments. For this reason, we aimed to reduce GUV agglomeration by introducing the polymer chains of polyethylene glycol (PEG) into GUV membranes for follow-up experiments, as described in the results given in the next section.

2.3. Effect of PEGylation on GUV Stability Postprinting

One strategy to improve the stability of GUVs in the cell culture medium at 37 °C is to incorporate long PEG chains into the membrane, so that stealthy GUVs can be formed. (41) In past studies, PEGylated lipids have been used to increase the half-life of liposomes in vivo by sterically increasing their physiological stability. (42−44) On the down side, it is known that the addition of PEGylated lipids can reduce the interaction of GUVs with cells. (45) In this study, we modified the membrane of GUVs with 5% PEGylated lipids (PEG5-GUVs, Mw of 2000 g mol–1), and examined the changes in stability postprinting in the cell medium at 37 °C. The overall percentage of intact PEG5-GUVs postprinting was lower, but not statistically significant, than that of pure GUVs (Figure 3A). A maximum percentage of intact PEG5-GUVs of 52.6 ± 15.4% was observed for DOD printing, and 54.4 ± 20.9% for extrusion printing, both at 1 bar. A possible explanation to the reduced number of intact PEG5-GUVs postprinting compared to nonmodified GUVs is that the addition of PEGylated lipids resulted in a more loosely packed bilayer with higher bending rigidity. (46) This may have caused a reduction of the critical lysis tension, which reduced the ability to close pores after rupture. (46) Interestingly, extrusion bioprinting did not decrease the percentage of intact PEG5-GUVs compared to DOD. Shear stress in DOD and extrusion bioprinting can vary between 5 and 50 kilopascals (when using controlled printing parameters and bioinks compatible with human cell encapsulation), whereas in vivo cells can experience increased shear stress (up to 10 Pa in human microvessels). (36,37,47,48) Therefore, if GUVs could withstand increased bending rigidity without losing the ability to close pores, this could be potentially beneficial for withstanding the printing process.

Figure 3

Figure 3. PEG5-GUV stability during bioprinting. A) Comparison of PEG5-GUV stability after DOD and extrusion bioprinting of PEG5 GUVs in an isoosmolar sucrose solution, and DOD printing of PEG5 GUVs with a 20% cholesterol proportion. A density of 2 × 106 PEG5-GUV/mL was used. B) Incubation of PEG5-GUVs in a cell medium for 1 h. C) Agglomeration of DOPC- and PEG5-GUVs in DMEM after 1 h incubation. Segmented outline of agglomerates and GUVs (left) with the average agglomeration area normalized to the GUV area (right). Scale bars represent 50 μm. *p < 0.05 and ****p < 0.0001.

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The addition of cholesterol, one of the main components in mammalian cell membranes, could help increasing the bending rigidity and potentially help withstanding the shear stress during the printing process. (49−51) PEG5-GUVs with a cholesterol content of 20% were printed by DOD printing, and the percentage of intact GUVs postprinting was evaluated. There was no significant improvement in withstanding the shear stress during the printing process by the addition of 20% cholesterol to the vesicles.
Another strategy to increase the bending rigidity of GUVs is to fill their core with a hydrogel, for example, agarose hydrogel, which helps close the pores in the lipid bilayer. (16,52) Moreover, the agarose hydrogel present in the lumen of GUVs could diffuse slower out of the pores after rupture, giving the bilayer more time to close the pores. A further alternative to reduce shear stress during printing is to use a larger nozzle. (36)
The incubation of PEG5-GUVs in the cell medium at 37 °C showed a reduced number of agglomerations compared to nonmodified GUVs, and most of the PEG5-GUVs stayed intact for at least 1 h of incubation (Figure 3B). The evaluation of the average area of DOPC and PEG5-GUVs after 1 h incubation by image analysis showed a significantly increased normalized area per agglomerate for DOPC-GUVs compared to the PEGylated GUVs (Figures 3C, S5, and S6). Therefore, we demonstrated that the addition of PEGylated lipids is a suitable strategy to increase the stability of GUVs under physiological conditions. Next, PEG5-GUVs were filled with a cell membrane impermeable dye (Alexa Fluor 488) and seeded with HeLa cells for over a period of 48 h. We observed no fusion of PEG5-GUVs or absorption during this time. Moreover, PEG5-GUVs were secluded from human cells as it has been reported in the literature (Figure S7). (45)

2.4. Impact of Different PEG Concentrations on the GUV Size, Production Quality, and Physiological Stability

Following the previous findings, we analyzed the impact of PEG concentration on the production of GUVs. PEG-GUVs with different concentrations of PEGylated lipids were produced by electroformation, filtered, and analyzed by fluorescence microscopy (Figure 4A). Increasing concentrations of PEGylated lipids in the GUVs significantly reduced the mean diameter. PEG5-GUVs showed a 29.4% smaller mean diameter compared to the nonmodified GUVs (Figure 4B). A possible explanation for the observed GUV reduced sizes with higher PEG concentrations is a looser packing and change of curvature of the lipid bilayer, given that PEG chains occupy a larger space and interact repulsively. (44,53−55) Moreover, it has been reported that the maximum shielding effect of PEG chains occurs at concentrations between 8% and 10%. (44,56) It is also known that higher PEG concentrations (e.g., >60% PEG with a Mw of 2000 g mol–1) can lead to the formation of micelles, thus making them unsuitable for bioprinting applications. (44) In our work, using higher PEG concentrations in GUV production resulted in worse production quality by electroformation and overall a lower GUV yield. We conclude that the addition of PEGylated lipids is a trade-off among an increased stability under physiological conditions, GUV size, and the production quality.

Figure 4

Figure 4. GUVs with different concentrations of PEGylated lipids after filtration and encapsulation in 1% agarose hydrogel. A) GUVs with different concentrations of PEGylated lipids after filtration. Scale bar represents 150 μm, and in insets, it is 50 μm. B) Mean diameter of GUVs after filtration with different concentrations of PEGylated lipids. n = 3 with more than 175 GUVs per image. C) Projection images of z-stacks using the hydrogel containing GUVs with 0% (DOPC) and 5% (PEG5) PEGylated lipids 1 h and 72 h after encapsulation. Scale bars represent 50 μm. D) Effect of different PEG concentrations on the number of GUVs for 1 h, 24 h, and 72 h after encapsulation. n = 3. *p < 0.05 and **p < 0.001.

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After accessing the stability of the GUVs in a 2D physiological environment, we investigated in a follow-up experiment the influence of PEGylation on the stability in a more realistic 3D environment. Therefore, we encapsulated PEG-GUVs with different PEG concentrations in 1% w/v agarose bioink and evaluated their stability over time. The hydrogel was incubated at 37 °C, and at different time points, the number of GUVs was counted by recording z-stacks (n = 3) using the hydrogel by fluorescence microscopy (Figure 4C). The projection images of the z-stacks were used to count the GUVs with a diameter of >8 μm over time (Figure 4D). No significant difference in the number of GUVs was observed in the sample 1 h after encapsulation. Note that in the DOPC sample, small agglomerations of GUVs were visible that had come into contact during mixing. After 24 h and 72 h of incubation, the number of GUVs in the DOPC sample was significantly lower than the number of GUVs in the PEG sample with 5% PEGylated lipids. More precisely, the GUV abundance decreased by 40% after 72 h of incubation. However, increasing the proportion of PEGylated lipids from 5% to 7% did not show an improved effect of steric stabilization during incubation. Additionally, after 72 h of incubation, more agglomeration and slight deformation of GUVs were observed. The hydrogel reduced the movement of GUVs preventing larger agglomerations as well as potential deformations due to high stiffness of the gel. (52) Deformation could have occurred during the encapsulation process due to shear forces while filling the hydrogel into the observation chambers. The encapsulation in a hydrogel showed that under physiological conditions the effect of agglomeration in the DOPC samples was reduced and single GUVs larger than 8 μm were visible. However, the addition of PEGylated lipids significantly increases the physiological stability of GUVs over time and is, therefore, beneficial for tissue engineering applications.

2.5. Bioprinting of PEG5-GUVs

PEG5-GUVs were encapsulated in two bioinks and bioprinted by DOD. The chosen bioinks, agarose-collagen (Ag–Col) and gelatin methacryloyl (GelMA), have been used previously in past studies and were chosen due to their compatibility with human cell bioprinting. (27−30,57) The printability of PEG5-GUVs in both bioinks was tested in three different geometrical shapes, including a ring, square, and triangle, as shown in Figure 5A,B. The Ag–Col and GelMA bioinks were mixed with PEG5-GUVs and printed at 0.2 and 0.3 bar, respectively. The shape and stability of PEG5-GUVs postprinting were evaluated by fluorescence microscopy. Several microscopic images were taken over a thickness of 290 μm, and a z-stack projection was made for each sample to visualize GUVs. GUVs with different sizes were observed in bioinks postprinting, which indicates that they were able to withstand not only DOD bioprinting but also the physical cross-linking of GelMA with UV-light.

Figure 5

Figure 5. Bioprinting of PEG5-GUVs in agarose–collagen (Ag–Col) and gelatin methacryloyl (GelMA) bioinks in different geometrical shapes. A) Photographs of bioprinted Ag–Col constructs, and microscopic z-stack projection of PEG5-GUVs in Ag–Col bioink postprinting. Scale bar represents 50 μm; in inserts, it is 10 μm;, in macroscopic images, it is 2 mm. B) Photographs of bioprinted GelMA constructs, and microscopic z-stack projection of PEG5-GUVs in GelMA bioinks postprinting. Scale bar represents 50 μm, in inserts, it is 10 μm, and in macroscopic images it is 2 mm. C) Normalized fluorescence intensity profile of FRAP measurements of PEG5-GUVs printed at 0.2 bar pressure in agarose hydrogels (right), and the diffusion coefficient of PEG5-GUVs postbioprinting (left). Scale bar represents 5 μm. D) Photographs of bioprinted Ag–Col bioink postprinting (insets, scale bar represents 2 mm), and microscopic z-stack projection of PEG5-GUVs (red) and MSCs (green, live cell cytoplasm staining) in the Ag–Col bioink after 72 h and 7 days of postprinting. Scale bar represents 50 μm; in macroscopic images, it is 2 mm.

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Additionally, the FRAP measurements of PEG5-GUVs printed at different pressures in agarose hydrogels revealed that membrane fluidity was not impacted by the printing process (Figures 5C and S8). As such, no significant differences in the measured diffusion coefficients of the analyzed GUV membranes were detected across all tested printing conditions, confirming that the bilayer architecture remained intact after printing. Furthermore, the values measured for the diffusion coefficients were well comparable with those found in the literature for similar DOPC-based GUV membranes. (58,59)
In a follow-up experiment, PEG5-GUVs were encapsulated with MSCs in the Ag–Col bioink, bioprinted in circular 3D shapes, and cultured in supplemented DMEM for 7 days (Figure 5D). PEG5-GUVs remained intact during the 7 days of incubation, and we did not observe signs of interaction with MSCs, similar to what has been demonstrated with HeLa cells in our 2D experiments (Figure S7). This was an expected outcome based on the findings described in previous reports. (45,60) In sum, we conclude that PEG5-GUVs can be bioprinted to fabricate tissue-like 3D structures useful for multiple tissue engineering applications.

2.6. Spatiotemporal Release of a Carrier by Ce6-PEG5-GUVs after Bioprinting

One application of PEG5-GUVs in bioprinting is the controlled delivery of biomolecules or supplements inside bioinks postprinting. In order to show the capability of controlling the delivery of substances loaded into PEG5-GUVs, we equipped our synthetic system with a light-controlled release mechanism. The photosensitizer chlorin e6 (Ce6) was loaded into PEG5-GUVs during electroformation by adding 125 μM Ce6 to the production buffer. The release mechanism was activated by illumination (357 nm and for up to 5 min duration) of the Ce6-PEG5-GUVs, which resulted in the oxidation of unsaturated lipids and consequently triggered the formation of pores in the lipid bilayer. (61,62) After incorporating Ce6 into the membranes of PEG5-GUVs, we detected a fluorescent signal in the far-red channel, indicating a successful incorporation of Ce6 into the lipid bilayer (Figure 6A). Furthermore, Ce6-PEG5-GUVs were mixed into a glucose solution (onto a 2D surface) and illuminated for 5 min with an LED set at a wavelength of 357 nm. By bright field microscopy, we observed a decrease in the contrast between the lumen of the vesicles and their surroundings (Figure 6B). This indicates a functional pore-forming mechanism that enables the influx of the surrounding glucose solution into the vesicles, and the efflux of sucrose solution out of the vesicles.

Figure 6

Figure 6. Studying the release of a fluorescent dye from bioprinted Ce6-PEG5-GUVs upon illumination. A) Fluorescence image of Ce6-PEG5-GUVs. B) Ce6-PEG5-GUVs loaded with sucrose and cultured in glucose solution before and after illumination with an LED set at a wavelength of 357 nm for 5 min. C) Fluorescence images showing bioprinted 1% w/v agarose constructs encapsulated with Alexa Fluor 488-loaded Ce6-PEG-GUVs before (upper images) and after (lower images) the release of the dye by illumination with an LED set at a wavelength of 357 nm for 5 min. Scale bars represent 50 μm; in insets, they are 10 μm; in macroscopic images, they are 2 mm.

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In a follow-up experiment, we loaded Ce6-PEG5-GUVs with a fluorescence dye (Alexa Fluor 488 phalloidin), encapsulated the vesicles in 1% w/v agarose bioinks, and bioprinted by DOD (Figure 6C). After illuminating the bioprinted constructs for 5 min at 357 nm, we observed the transformation of Ce6-PEG5-GUVs into smaller units, possibly due to vesicle rupture and reformation. In addition, there was no fluorescence signal detected within the lumen of Ce6-PEG5-GUVs postprinting, indicating the formation of pores in the lipid bilayer, and the release of Alexa Fluor 488 phalloidin into the surrounding bioink. Therefore, we herein show that PEG5-GUVs can be encapsulated and utilized in a 3D environment, such as inside of a bioprinted construct, and we can use such synthetic cell-like units to control the local delivery of biomolecules. Future studies may focus on the use of other molecules, such as growth factors, nutrients, or even gases, which can be released in close proximity to cells in bioprinted 3D constructs. Additionally, for achieving a precise controlled release, the kinetics of natural diffusion of biomolecules needs to be further characterized.

3. Conclusion

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In this work, we report the first successful attempt to bioprint GUVs for tissue engineering applications. We established a new bioprinting protocol that includes the production, filtration, and bioprinting of GUVs, which allowed us to bioprint large amounts of cell-sized GUVs while maintaining the integrity of their membranes. The addition of PEGylated lipids to the GUVs increased their physiological stability in cell media, as well as supporting the stability in different bioinks up to 7 days postprinting. Using GUVs with 5% PEGylated lipids increased the stability over time by 40% compared to nonmodified GUVs. The addition of PEGylated lipids to the GUVs resulted in a trade-off between stability under physiological conditions (i.e., in cell culture medium), during interaction with cells, and during the bioprinting process. Upon loading the PEG5-GUVs with Ce6, we demonstrated that it is feasible to externally control the release of biomolecules from the GUVs in the surrounding environment. In sum, the combination of bioprinting technologies, multiple techniques available to functionalize the bilayer of synthetic vesicles, and the ability to precisely deliver biomolecules is a powerful interdisciplinary approach in tissue engineering and regenerative medicine.

4. Methods

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4.1. Lipids

The lipids 1,2-di(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (18:1 DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Liss Rhod PE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (PEG2000 PE) were purchased from Avanti Polar Lipids, Alabaster, USA. All lipids were stored in chloroform (288306, Sigma-Aldrich, St. Louis, USA) at −20 °C.

4.2. GUV Production

GUVs were produced by electroformation using a Vesicle Prep Pro (Nanion Technologies, Munich, Germany). (18) GUVs with different lipid proportions (MW-based) were produced. Nonmodified GUVs were prepared with 99% DOPC, and for fluorescence imaging 1% Liss Rhod PE. PEG-GUVs were produced with a variable concentration of PEG PE lipids among 1%, 5%, and 7%. Cholesterol containing GUVs were produced with 74% DOPC, 1% Liss Rhod PE, 20% cholesterol (C8667, Sigma-Aldrich, St. Louis, USA), and 5% PEG PE. For the GUV production, the lipids were mixed (3 mM solution), and 50 μL spread onto an indium tin oxide (ITO)-coated glass slide using a coverslip. The coated glass slide was left in a fume hood for at least 30 min to allow chloroform to evaporate. Afterward, the slide was placed into the Vesicle Prep Pro with a rubber ring (18 mm diameter) on top, and filled with 270 μL of sucrose solution (300 mM, S0389, Sigma-Aldrich, St. Louis, USA; osmolarity in the range of DMEM). A second ITO glass slide was used to seal the chamber, and an alternating electrical field (3 VPP, 5 Hz) at 37 °C was applied for 128 min. After the production step, the GUV solution was collected from the chamber and stored at 4 °C for up to 7 days.

4.3. Filtering of GUVs

After electroformation, the GUV solution was filtered using the membrane filtering method. (32) Briefly, a nuclepore polycarbonate filter membrane (25 μm diameter, 10 μm pore size, Whatman plc, Maidstone, UK) was placed into a swinnex filter holder (25 μm, Merck Millipore, Billerica, USA) and connected to polypropylene tubing components, a stopcock, and two 5 mL syringes (BBraun SE, Melsungen, Germany) forming an apparatus in the u-shape. The apparatus was filled with glucose solution (300 mM, G7021, Sigma-Aldrich, St. Louis, USA), with all remaining air bubbles were removed, and it was connected to two peristaltic pumps (custom-made, consisting of two stepper motors). A flow rate of 1 mL min–1 was used to filter 50 μL GUV solution for 30 min at room temperature. After filtration, the supernatant was collected beneath the filter membrane and stopcock, and stored overnight at 4 °C. The GUVs in the remaining glucose solution settled down during storage.

4.4. Encapsulation of GUVs in Agarose

For the encapsulation of GUVs in 1% w/v agarose hydrogels, a stock GUV solution was first prepared by concentrating and mixing of three filtration runs. Then, 10 μL of the stock solution was filled into a cell counting slide (Logos Biosystems, Anyang, South Korea), three images were taken with a fluorescence microscope (EVOS M5000, RFP lightcube, Thermo Fisher Scientific, Waltham, USA), and the overall number of GUVs in the stock solution was determined with a program GUVdetector (University Cologne). (63) A stock solution of 3% w/v low-temperature gelling agarose (A9045, Sigma-Aldrich, St. Louis, USA) was prepared by dissolving agarose powder in Milli-Q water and autoclaved at 120 °C for 2 h. The stock solution of GUVs was diluted with high glucose Dulbecco’s modified eagle medium (DMEM 11965092, Gibco, Thermo Fisher Scientific, Waltham, USA), supplemented with 10% fetal bovine serum (FBS, F0679, SAFC, Merck KGaA, Darmstadt, Germany) and 1% sodium pyruvate (100 mM, 11530396, Thermo Fisher Scientific, Waltham, USA). For bioink encapsulation experiments, the GUV stock solution was mixed with 3% w/v agarose using wide orifice pipet tips to dilute it to a 1% w/v agarose hydrogel, having a final concentration of 5 × 105 GUVs/mL. For long-term investigation, 20 μL of 1% w/v agarose hydrogel was filled into 37 °C preheated observation chambers and sealed with silicone (Twinsil, Picodent, Wipperfürth, Germany). The observation chambers consist of a bovine serum albumin (BSA, 8076, Carl Roth, Karlsruhe Germany)-coated surface (covered for 2 h with 2.5% BSA, afterward phosphate buffered saline (PBS, A0956, AppliChem, Darmstadt, Germany), washed in DI water, and stored overnight), double-sided adhesive, and a coverslip. During the experiment, the observation chambers were stored in an incubator at 37 °C. For each sample, three z-stacks were taken at different positions by fluorescence microscopy, and the projection image was analyzed.

4.5. Agglomeration of GUVs

500 μL of DOPC and PEG5-GUVs at a concentration of 5 × 105 GUVs/mL in supplemented DMEM were seeded into 24-well plates and incubated for 1 h. Fluorescence images were taken after the incubation period, and the area per agglomerate in the sample was evaluated by image analysis using IMARIS (10.1.1, Oxford Instruments, Abingdon, UK). Agglomerates/GUVs in each image were segmented (a surface grain size of 1 μm, background elimination active, and a threshold of 50 (DOPC) and 35 (PEG5)), and the area was measured. Only aggregates with an area of above 10 μm2 were considered GUVs or agglomerates. The average area per agglomerate/GUV was normalized to the total GUV area per image.

4.6. Bioprinting of GUVs

A stock solution of GUVs was prepared by concentrating and mixing of the GUV solutions from three filtration runs. 10 μL of the stock solution was filled into a cell counting slide, three images were taken with a fluorescence microscope, and the overall number of GUVs in the stock solution was determined with the program GUVdetector. (63) The GUV stock solution was then diluted in an isoosmolar sucrose solution (300 mM) to a final GUV concentration of 2 × 106 GUVs/mL (higher concentration of GUVs was used for evaluation postprinting) using wide orifice pipet tips. The control sample was taken and stored in a reaction tube. The rest of the solution was transferred into the reservoir of the printhead. The printhead of the bioprinter (SuperFill with custom design, Black Drop Biodrucker GmbH, Aachen, Germany) consists of an electromagnetic microvalve with a 300 μm orifice (Fritz Gyger AG, Gwatt, Switzerland). For extrusion bioprinting, an additional stainless-steel nozzle (7018314, 0.33 mm inner diameter, Nordson Corporation, Westlake, Ohio, US) was mounted to the printhead. The printing step was conducted by ejecting single droplets (500 droplets, an opening time of 450 μs) at 0.2, 0.5, and 1 bar into a reaction tube from a distance of 10 mm. After printing, the samples were diluted 1:5 in an isoosmolar glucose solution (300 mM), and 10 μL was loaded into a cell counting slide. After 5 min, the GUVs settled at the bottom of the slide, and three fluorescence images were taken. The percentage of intact GUVs was calculated as the ratio of the number of GUVs after and before the printing process:
PercentageofintactGUVs=NumberofGUVsafterprintingNumberofGUVsbeforeprinting×100%

4.7. FRAP Measurements

FRAP experiments were conducted using an LSM 900 Zeiss confocal fluorescence microscope equipped with a Plan-Apochromat 20× 0.8 Air M27 objective (Carl Zeiss AG, Oberkochen, Germany). A circular area with a diameter of 5 μm was chosen at the top plane of the GUV following their encapsulation into an agarose gel either by bioprinting (0.2, 0.5, and 1 bar conditions) or simple mixing (0.0 bar conditions). The chosen area was bleached at 100% laser power (λex = 561 nm for LissRhod) at 297 ms frame rate using 15× digital zoom for 2 iterations. After bleaching, 100 images were acquired to follow the recovery track of the fluorescent signal, while four images were acquired as a baseline prior to the bleaching event. Per condition, triplicates were measured by analyzing three GUVs per triplicate. A custom-written MatLab script was used to calculate the diffusion coefficients in μm2 s–1 using MatLab R2020a – Update 6 (Mathworks, Inc., Natick, Massachusetts, USA). (64)

4.8. Bioprinting of GUVs with MSCs

For printing GUVs with cells into different geometrical constructs, 0.5% w/v agarose–0.2% w/v collagen and gelatin methacrylol (GelMA, monomer concentration not disclosed by the manufacturer, TissueFab 905429, Sigma-Aldrich, St. Louis, USA) were used. The hydrogels were prepared as follows: first, a stock solution of agarose (3% w/v) and collagen (0.32 mg mL–1) hydrogels were prepared. For the collagen stock solution, 80% (v/v) of collagen type 1 (calf skin, 0.4% solution in HCl, L7213, Sigma-Aldrich) was mixed on ice with 10% (v/v) 10-fold DMEM (D2429, Sigma-Aldrich, St. Louis, USA) and 5% (v/v) HEPES buffer (1 M, 12509079, Fisher Scientific, Hampton, USA) and neutralized with 5% (v/v) sodium hydroxide (0.7 M, 1310–73–2, Sigma-Aldrich, St. Louis, USA).
Bone marrow-derived mesenchymal stem cells (MSCs, C-12975, PromoCell, Heidelberg, Germany) were expanded in DMEM, supplemented with 1% penicillin–streptomycin (Pen–Strep, P4333, Sigma-Aldrich, St. Louis, USA), 1% sodium pyruvate (100 mM), and 10% FBS. The GelMA hydrogel was handled as described in the manufacturer’s protocol. Prior to encapsulation, MSCs were stained with a live-cell cytoplasmic dye (0.3 mM, Cytolight Rapid dye green, 4705, Sartorius, Göttingen) according to the manufacturer’s protocol.
The PEG5-GUV stock solution was prepared as described in the GUV production section. The final Ag–Col bioink consists of 0.5% agarose, 0.2% collagen, and a cell and GUV concentration of 5 × 105/mL, respectively. The bioinks were transferred to the reservoir and printed by DOD bioprinting (Ag–Col: 0.2 bar, printhead temperature at 30 °C, printing platform temperature at 1 °C, and 450 μs valve opening time; GelMA: 0.3 bar, printhead temperature at 25 °C, printing platform temperature at 10 °C, and 450 μs valve opening time). GelMA bioinks were cross-linked after bioprinting with a UV-LED (M365L3) connected to a lightguide (LLG05–4H) for 2 min with the LED driver (LEDD1B) limited to 0.7 A, and at a distance of 30 mm (all purchased from Thorlabs, Dachau, Germany). The constructs were imaged by fluorescence microscopy directly after the printing process by recording images with a thickness of 290 μm through the sample. The printed Ag–Col constructs with GUVs and cells were covered in DMEM (supplemented with 10% FBS, 1% sodium pyruvate (100 mM) and 1% Pen–Strep) after printing, incubated for over a period of 7 days, and imaged after 72 h and 7 days.

4.9. Viability Evaluation of MSCs Postbioprinting

MSCs were cultured as described above and printed at a concentration of 2 × 106 cells/mL in a supplemented DMEM solution at different pressures into a 96-well plate. In one well, 10 000 cells were seeded by printing the according number of droplets into each well. After printing, 100 μL of supplemented DMEM was added to every well, and the cells were incubated for 24 h. After the incubation period, the cells were stained with calcein-AM and ethidium homodimer-1 (LIVE/DEAD viability kit L3224, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s protocol to assess the viability postbioprinting. In short, samples were washed with PBS and incubated for 30 min with PBS containing the dyes. Afterward, fluorescence microscopy images were taken, and the number of living and dead cells were counted. Cell viability was calculated using the following formula:
Cellviability=NumberoflivingcellsTotalnumberofcells×100%

4.10. Counting of GUV Numbers per Sample

The counting of GUVs per image was done using the tool GUVdetector, where round GUVs larger than 8 μm were detected by the circular Hough transformation. (63) By combining the automated algorithm to detect the GUVs and manually compared with the original image, the number and size of the GUVs were evaluated. For 10× images, a minimum radius of 8 pixels, and for 20× images a minimum radius of 13 pixels were set in GUVdetector. To calculate the number of GUVs per milliliter, the dimensions of one image combined with the height of the cell counting slide chamber (100 μm) were used.

4.11. Spatiotemporal Release of Ce6 from GUVs after Bioprinting

PEG5-GUVs were produced with a buffer consisting of sucrose (300 mM), Ce6 (125 μM), and the cell-membrane impermeable dye Alexa Fluor 488 phalloidin (2 μM, A12379, Thermo Fisher Scientific, Waltham, USA). After the production, 5 × 105 GUVs were mixed in agarose bioinks and transferred to the bioprinter. The bioinks were printed by DOD (0.2 bar, printhead temperature at 30 °C, printing platform temperature at 1 °C, and 450 μs valve opening time), and afterward, the samples were illuminated with a 357 nm wavelength LED (100% intensity, EVOS light cube DAPI 2.0 AMEP4950, Thermo Fisher Scientific, Waltham, USA) for 5 min to initiate pore formation and biomolecule release. After illumination, GUVs in 3D gels were imaged by fluorescence microscopy, and GUVs in 2D were imaged by light microscopy. After imaging the GUVs printed in agarose bioinks, the projections of z-stacks were created with 290 μm and a step size of 5 μm.

4.12. Statistical Analysis

All results were presented as mean ± standard deviation (SD) with the corresponding sample size described in the respective figure captions. Comparisons between multiple experimental groups with two factors were conducted using a two-way ANOVA with Tukey’s post hoc test (Prism 10.1.2, GraphPad Software, Boston, USA). Comparisons with multiple groups with one factor were conducted using a one-way ANOVA with Tukey’s post hoc test. For comparison between two groups, an unpaired, two-tailed student’́s t-test was conducted. Statistical significance was labeled as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.000.1.

Data Availability

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The data that support the findings of this study are available from the corresponding author upon a reasonable request.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.4c00137.

  • Efficiency of the GUV filtering process (Figure S1); GUV images after DOD and extrusion printing (Figure S2); MSC viability postbioprinting (Figure S3); effect of salts on the agglomeration of GUVs (Figure S4); agglomeration of DOPC-GUVs in DMEM (Figure S5); agglomeration of PEG5-GUVs in DMEM (Figure S6); interaction of PEGylated GUVs with cells (Figure S7); fluorescence intensity curves for FRAP measurements on printed PEG5-GUVs (Figure S8) (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Ole Thaden - Bioprinting & Tissue Engineering Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
    • Nicole Schneider - Bioprinting & Tissue Engineering Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
    • Tobias Walther - Biophysical Engineering of Life Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany;  Max Planck Institute for Medical Research, Heidelberg 69120, Germany
    • Erin Spiller - Bioprinting & Tissue Engineering Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
    • Alexandre Taoum - Bioprinting & Tissue Engineering Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany
    • Kerstin Göpfrich - Biophysical Engineering of Life Group, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg 69120, Germany;  Max Planck Institute for Medical Research, Heidelberg 69120, Germany;  Orcidhttps://orcid.org/0000-0003-2115-3551
  • Author Contributions

    O.T., K.G., and D.D.C conceived the study. O.T., N.S., and A.T. performed the experiments. T.W. performed FRAP measurements. O.T., E.S., and T.W. performed data analysis. O.T., K.G., and D.D.C wrote the manuscript with the input from all authors.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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O.T, K.G, and D.D.C acknowledge funding from Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy via the Excellence Cluster 3D Matter Made to Order (EXC-2082/1–390761711). Additionally, T.W. thanks the Studienstiftung des deutschen Volkes e.V.

References

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    Liposomes are vesicular structures made of lipids that are formed in aqueous solutions. Structurally, they resemble the lipid membrane of living cells. Therefore, they have been widely investigated, since the 1960s, as models to study the cell membrane, and as carriers for protection and/or delivery of bioactive agents. They have been used in different areas of research including vaccines, imaging, applications in cosmetics and tissue engineering. Tissue engineering is defined as a strategy for promoting the regeneration of tissues for the human body. This strategy may involve the coordinated application of defined cell types with structured biomaterial scaffolds to produce living structures. To create a new tissue, based on this strategy, a controlled stimulation of cultured cells is needed, through a systematic combination of bioactive agents and mechanical signals. In this review, we highlight the potential role of liposomes as a platform for the sustained and local delivery of bioactive agents for tissue engineering and regenerative medicine approaches.
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    Cheng, R.; Liu, L.; Xiang, Y.; Lu, Y; Deng, L.; Zhang, H.; Santos, H. A.; Cui, W. Advanced liposome-loaded scaffolds for therapeutic and tissue engineering applications. Biomaterials 2020, 232, 119706,  DOI: 10.1016/j.biomaterials.2019.119706
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    Advanced liposome-loaded scaffolds for therapeutic and tissue engineering applications
    Cheng, Ruoyu; Liu, Lili; Xiang, Yi; Lu, Yong; Deng, Lianfu; Zhang, Hongbo; Santos, Helder A.; Cui, Wenguo
    Biomaterials (2020), 232 (), 119706CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    A review. Liposome is one of the most commonly used drug delivery systems in the world, due to its excellent biocompatibility, satisfactory ability in controlling drug release, and passive targeting capability. However, some drawbacks limit the application of liposomes in clin., such as problems in transporting, storing, and difficulties in maintaining the drug concn. in the local area. Scaffolds usually are used as implants to supply certain mech. supporting to the defective area or utilized as diagnosis and imaging methods. But, in general, unmodified scaffolds show limited abilities in promoting tissue regeneration and treating diseases. Therefore, liposome-scaffold composite systems are designed to take advantages of both liposomes' biocompatibility and scaffolds' strength to provide a novel system that is more suitable for clin. applications. This review introduces and discusses different types of liposomes and scaffolds, and also the application of liposome-scaffold composite systems in different diseases, such as cancer, diabetes, skin-related diseases, infection and human immunodeficiency virus, and in tissue regeneration like bone, teeth, spinal cord and wound healing.
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    Jesorka, A.; Orwar, O. Liposomes: Technologies and analytical applications. Annu. Rev. Anal. Chem. 2008, 1 (1), 801– 832,  DOI: 10.1146/annurev.anchem.1.031207.112747
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    Liposomes: Technologies and analytical applications
    Jesorka, Aldo; Orwar, Owe
    Annual Review of Analytical Chemistry (2008), 1 (), 801-832CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews Inc.)
    A review. Liposomes are structurally and functionally some of the most versatile supramol. assemblies in existence. Since the beginning of active research on lipid vesicles in 1965, the field has progressed enormously and applications are well established in several areas, such as drug and gene delivery. In the anal. sciences, liposomes serve a dual purpose: Either they are analytes, typically in quality-assessment procedures of liposome prepns., or they are functional components in a variety of new anal. systems. Liposome immunoassays, for example, benefit greatly from the amplification provided by encapsulated markers, and nanotube-interconnected liposome networks have emerged as ultrasmall-scale anal. devices. This review provides information about new developments in some of the most actively researched liposome-related topics.
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    Rideau, E.; Dimova, R.; Schwille, P.; Wurm, F. R.; Landfester, K. Liposomes and polymersomes: a comparative review towards cell mimicking. Chem. Soc. Rev. 2018, 47 (23), 8572– 8610,  DOI: 10.1039/C8CS00162F
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    Liposomes and polymersomes: a comparative review towards cell mimicking
    Rideau, Emeline; Dimova, Rumiana; Schwille, Petra; Wurm, Frederik R.; Landfester, Katharina
    Chemical Society Reviews (2018), 47 (23), 8572-8610CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    Cells are integral to all forms of life due to their compartmentalization by the plasma membrane. However, living organisms are immensely complex. Thus there is a need for simplified and controllable models of life for a deeper understanding of fundamental biol. processes and man-made applications. This is where the bottom-up approach of synthetic biol. comes from: a stepwise assembly of biomimetic functionalities ultimately into a protocell. A fundamental feature of such an endeavor is the generation and control of model membranes such as liposomes and polymersomes. We compare and contrast liposomes and polymersomes for a better a priori choice and design of vesicles and try to understand the advantages and shortcomings assocd. with using one or the other in many different aspects (properties, synthesis, self-assembly, applications) and which aspects have been studied and developed with each type and update the current development in the field.
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    Göpfrich, K.; Platzman, I.; Spatz, J. P. Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells. Trends Biotechnol. 2018, 36 (9), 938– 951,  DOI: 10.1016/j.tibtech.2018.03.008
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    Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells
    Gopfrich Kerstin; Platzman Ilia; Spatz Joachim P
    Trends in biotechnology (2018), 36 (9), 938-951 ISSN:.
    With the ultimate aim to construct a living cell, bottom-up synthetic biology strives to reconstitute cellular phenomena in vitro - disentangled from the complex environment of a cell. Recent work towards this ambitious goal has provided new insights into the mechanisms governing life. With the fast-growing library of functional modules for synthetic cells, their classification and integration become increasingly important. We discuss strategies to reverse-engineer and recombine functional parts for synthetic eukaryotes, mimicking the characteristics of nature's own prototype. Particularly, we focus on large outer compartments, complex endomembrane systems with organelles, and versatile cytoskeletons as hallmarks of eukaryotic life. Moreover, we identify microfluidics and DNA nanotechnology as two technologies that can integrate these functional modules into sophisticated multifunctional synthetic cells.
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    Angelova, M. I.; Dimitrov, D. S. Liposome electroformation. Faraday Discuss. Chem. Soc. 1986, 81, 303– 311,  DOI: 10.1039/dc9868100303
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    Liposome electroformation
    Angelova, M.; Dimitrov, D.
    Faraday Discussions of the Chemical Society (1986), 81 (1), 303-11CODEN: FDCSB7; ISSN:0301-7249.
    Liposome formation and lipid swelling on Pt electrodes in distd. water and water solns. in d.c. elec. fields were investigated for different amts. of a neg. charged lipid (mixt. from 71% phosphatidylcholines, 21.5% phosphatidylethanolamines and 7.5% phosphatidylserines), and a neutral lipid (dimyristoylphosphatidylcholine, DMPC). Neg. charged lipids do not form liposomes without fields when the thickness of the dried lipid layer is ≤90 bilayers. The rate and extent of swelling of layers thicker than 90 bilayers is largest on the cathode, smaller without fields and smallest on the anode. The theory, based on the assumption that osmotic and electrostatic forces drive lipid swelling and liposome formation. is in semi-quant. agreement with the exptl. data; in particular, it gives the obsd. linear dependence of the rate of swelling on the inverse lipid layer thickness. To induce liposome formation for layers thinner than 90 bilayers it was necessary to apply a neg. potential which is proportional to the logarithm of the inverse layer thickness. The characteristic crit. potential is proportional to RTk/F; R being the gas const., Tk the abs. temp., and F the Faraday const. This indicates that redistribution of counterions may be the cause which increases the repulsive electrostatic intermembrane forces to overcome van der Waals attraction. For thicknesses <10 bilayers, formation of very thin-walled liposomes of narrow size distribution and mean diam. of ∼30 μm was obsd. These liposomes grow in size before detachment, and a formula for the kinetics of growth was derived, which is in very good agreement with the exptl. data. The effects of d.c. field on DMPC swelling are smaller and lead to formation of liposome-like structures of different appearance. Bilayer sepn. and bending are prerequisites for liposome formation from hydrating lipids. Therefore, a possible mol. mechanism is that membranes should be destabilized to bend and fuse to form liposomes. This requires the right proportion between structured regions, in the form of bilayers, and defects and (or) nonbilayer structures, and in many cases external constraints, in particular, elec. fields.
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    Reeves, J. P.; Dowben, R. M. Formation and properties of thin-walled phospholipid vesicles. J. Cell. Physiol. 1969, 73 (1), 49– 60,  DOI: 10.1002/jcp.1040730108
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    Formation and properties of thin-walled phospholipid vesicles
    Reeves, John P.; Dowben, Robert M.
    Journal of Cellular Physiology (1969), 73 (1), 49-60CODEN: JCLLAX; ISSN:0021-9541.
    Large nos. of thin-walled vesicles, 0.5-10 μ in diam., can be formed by permitting a thinly spread layer of hydrated phospholipids to swell slowly in distd. H2O or in an aq. nonelectrolyte soln. Electron micrographs of phospholipid analyses indicated that the walls consist of a single or a few bilayers. The vesicles can be centrifuged and resuspended in another medium to make them a useful system for studying permeability. The osmolarity of the soln. in the interior of the vesicles can be estd. by immersion refractometry and the osmolarity of the internal aq. phase is linearly related to that of the external medium.
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    Weinberger, A.; Tsai, F.; Koenderink, G. Gel-Assisted Formation of Giant Unilamellar Vesicles. Biophys. J. 2013, 105 (1), 154– 164,  DOI: 10.1016/j.bpj.2013.05.024
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    Gel-Assisted Formation of Giant Unilamellar Vesicles
    Weinberger, Andreas; Tsai, Feng-Ching; Koenderink, Gijsje H.; Schmidt, Thais F.; Itri, Rosangela; Meier, Wolfgang; Schmatko, Tatiana; Schroder, Andre; Marques, Carlos
    Biophysical Journal (2013), 105 (1), 154-164CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Giant unilamellar vesicles or GUVs are systems of choice as biomimetic models of cellular membranes. Although a variety of procedures exist for making single walled vesicles of tens of microns in size, the range of lipid compns. that can be used to grow GUVs by the conventional methods is quite limited, and many of the available methods involve energy input that can damage the lipids or other mols. present in the growing soln. for embedment in the membrane or in the vesicle interior. Here, we show that a wide variety of lipids or lipid mixts. can grow into GUVs by swelling lipid precursor films on top of a dried polyvinyl alc. gel surface in a swelling buffer that can contain diverse biorelevant mols. Moreover, we show that the encapsulation potential of this method can be enhanced by combining polyvinyl alc.-mediated growth with inverse-phase methods, which allow (bio)mol. complexation with the lipids.
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    Weiss, M. Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics. Nat. Mater. 2018, 17 (1), 89– 95,  DOI: 10.1038/nmat5005
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    Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics
    Weiss, Marian; Frohnmayer, Johannes Patrick; Benk, Lucia Theresa; Haller, Barbara; Janiesch, Jan-Willi; Heitkamp, Thomas; Boersch, Michael; Lira, Rafael B.; Dimova, Rumiana; Lipowsky, Reinhard; Bodenschatz, Eberhard; Baret, Jean-Christophe; Vidakovic-Koch, Tanja; Sundmacher, Kai; Platzman, Ilia; Spatz, Joachim P.
    Nature Materials (2018), 17 (1), 89-96CODEN: NMAACR; ISSN:1476-1122. (Nature Research)
    Compartments for the spatially and temporally controlled assembly of biol. processes are essential towards cellular life. Synthetic mimics of cellular compartments based on lipid-based protocells lack the mech. and chem. stability to allow their manipulation into a complex and fully functional synthetic cell. Here, the authors present a high-throughput microfluidic method to generate stable, defined sized liposomes termed 'droplet-stabilized giant unilamellar vesicles (dsGUVs)'. The enhanced stability of dsGUVs enables the sequential loading of these compartments with biomols., namely purified transmembrane and cytoskeleton proteins by microfluidic pico-injection technol. This constitutes an exptl. demonstration of a successful bottom-up assembly of a compartment with contents that would not self-assemble to full functionality when simply mixed together. Following assembly, the stabilizing oil phase and droplet shells are removed to release functional self-supporting protocells to an aq. phase, enabling them to interact with physiol. relevant matrixes.
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    Haller, B.; Göpfrich, K.; Schröter, M.; Janiesch, J.-W.; Platzman, I.; Spatz, J. P. Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems. Lab Chip. 2018, 18 (17), 2665– 2674,  DOI: 10.1039/C8LC00582F
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    Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems
    Haller, Barbara; Goepfrich, Kerstin; Schroeter, Martin; Janiesch, Jan-Willi; Platzman, Ilia; Spatz, Joachim P.
    Lab on a Chip (2018), 18 (17), 2665-2674CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    In this manuscript, we introduce a simple, off-the-shelf approach for the on-demand creation of giant unilamellar vesicles (GUVs) or multicompartment synthetic cell model systems in a high-throughput manner. To achieve this, we use microfluidics to encapsulate small unilamellar vesicles in block-copolymer surfactant-stabilized water-in-oil droplets. By tuning the charge of the inner droplet interface, adsorption of lipids can be either inhibited, leading to multicompartment systems, or induced, leading to the formation of droplet-stabilized GUVs. To control the charge d., we formed droplets using different molar ratios of an uncharged PEG-based fluorosurfactant and a neg.-charged PFPE carboxylic acid fluorosurfactant (Krytox). We systematically studied the transition from a multicompartment system to 3D-supported lipid bilayers as a function of lipid charge and Krytox concn. using confocal fluorescence microscopy, cryo-SEM and interfacial tension measurements. Moreover, we demonstrate a simple method to release GUVs from the surfactant shell and the oil phase into a physiol. buffer - providing a remarkably high-yield approach for GUV formation. This widely applicable microfluidics-based technol. will increase the scope of GUVs as adaptable cell-like compartments in bottom-up synthetic biol. applications and beyond.
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    Karamdad, K. Engineering thermoresponsive phase separated vesicles formed: Via emulsion phase transfer as a content-release platform. Chem. Sci. 2018, 9 (21), 4851– 4858,  DOI: 10.1039/C7SC04309K
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    Zong, W.; Shao, X.; Chai, Y.; Wang, X.; Han, S.; Chu, H.; Zhu, C.; Zhang, X. Controllable drug release of pH-sensitive liposomes encapsulating artificial cytosol system. bioRxiv 2021, 2021– 05,  DOI: 10.1101/2021.05.24.445400
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    Dreher, Y.; Jahnke, K.; Schröter, M.; Göpfrich, K. Light-Triggered Cargo Loading and Division of DNA-Containing Giant Unilamellar Lipid Vesicles. Nano Lett. 2021, 21 (14), 5952– 5957,  DOI: 10.1021/acs.nanolett.1c00822
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    Light-Triggered Cargo Loading and Division of DNA-Containing Giant Unilamellar Lipid Vesicles
    Dreher, Yannik; Jahnke, Kevin; Schroeter, Martin; Goepfrich, Kerstin
    Nano Letters (2021), 21 (14), 5952-5957CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    A minimal synthetic cell should contain a substrate for information storage and have the capability to divide. Notable efforts were made to assemble functional synthetic cells from the bottom up, however often lacking the capability to reproduce. Here, we develop a mechanism to fully control reversible cargo loading and division of DNA-contg. giant unilamellar vesicles (GUVs) with light. We make use of the photosensitizer Chlorin e6 (Ce6) which self-assembles into lipid bilayers and leads to local lipid peroxidn. upon illumination. On the time scale of minutes, illumination induces the formation of transient pores, which we exploit for cargo encapsulation or controlled release. In combination with osmosis, complete division of two daughter GUVs can be triggered within seconds of illumination due to a spontaneous curvature increase. We ultimately demonstrate the division of a selected DNA-contg. GUV with full spatiotemporal control-proving the relevance of the division mechanism for bottom-up synthetic biol.
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    Elani, Y. Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules. Sci. Rep. 2018, 8 (1), 4564,  DOI: 10.1038/s41598-018-22263-3
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    Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules
    Elani Yuval; Trantidou Tatiana; Wylie Douglas; Law Robert V; Ces Oscar; Elani Yuval; Wylie Douglas; Ces Oscar; Dekker Linda; Polizzi Karen
    Scientific reports (2018), 8 (1), 4564 ISSN:.
    There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.
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    Duarte Campos, D. F.; Blaeser, A.; Buellesbach, K. Bioprinting Organotypic Hydrogels with Improved Mesenchymal Stem Cell Remodeling and Mineralization Properties for Bone Tissue Engineering. Adv. Healthc. Mater. 2016, 5 (11), 1336– 1345,  DOI: 10.1002/adhm.201501033
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    Duarte Campos, D. F.; Blaeser, A.; Weber, M. Three-dimensional printing of stem cell-laden hydrogels submerged in a hydrophobic high-density fluid. Biofabrication 2013, 5 (1), 015003,  DOI: 10.1088/1758-5082/5/1/015003
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    Three-dimensional printing of stem cell-laden hydrogels submerged in a hydrophobic high-density fluid
    Duarte Campos Daniela F; Blaeser Andreas; Weber Michael; Jakel Jorg; Neuss Sabine; Jahnen-Dechent Wilhelm; Fischer Horst
    Biofabrication (2013), 5 (1), 015003 ISSN:.
    Over the last decade, bioprinting technologies have begun providing important tissue engineering strategies for regenerative medicine and organ transplantation. The major drawback of past approaches has been poor or inadequate material-printing device and substrate combinations, as well as the relatively small size of the printed construct. Here, we hypothesise that cell-laden hydrogels can be printed when submerged in perfluorotributylamine (C(12)F(27)N), a hydrophobic high-density fluid, and that these cells placed within three-dimensional constructs remain viable allowing for cell proliferation and production of extracellular matrix. Human mesenchymal stem cells and MG-63 cells were encapsulated into agarose hydrogels, and subsequently printed in high aspect ratio in three dimensional structures that were supported in high density fluorocarbon. Three-dimensional structures with various shapes and sizes were manufactured and remained stable for more than six months. Live/dead and DAPI stainings showed viable cells 24 h after the printing process, as well as after 21 days in culture. Histological and immunohistochemical analyses after 14 and 21 days revealed viable cells with marked matrix production and signs of proliferation. The compressive strength values of the printed gels consequently increased during the two weeks in culture, revealing encouraging results for future applications in regenerative medicine.
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    Betsch, M.; Cristian, C.; Liu, Y.; Blaeser, A.; Schöneberg, J.; Vogt, M.; Buhl, E. M.; Fischer, H.; Duarte Campos, D. F. Incorporating 4D into Bioprinting: Real-Time Magnetically Directed Collagen Fiber Alignment for Generating Complex Multilayered Tissues. Adv. Healthc. Mater. 2018, 7 (21), 1800894,  DOI: 10.1002/adhm.201800894
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    Duarte Campos, D. F.; Rohde, M.; Ross, M. Corneal bioprinting utilizing collagen-based bioinks and primary human keratocytes. J. Biomed Mater. Res. A 2019, 107 (9), 1945– 1953,  DOI: 10.1002/jbm.a.36702
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    Stein, H.; Spindler, S.; Bonakdar, N.; Wang, C.; Sandoghdar, V. Production of isolated giant unilamellar vesicles under high salt concentrations. Front. Physiol. 2017, 8, 63,  DOI: 10.3389/fphys.2017.00063
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    Production of Isolated Giant Unilamellar Vesicles under High Salt Concentrations
    Stein Hannah; Spindler Susann; Sandoghdar Vahid; Bonakdar Navid; Wang Chun
    Frontiers in physiology (2017), 8 (), 63 ISSN:1664-042X.
    The cell membrane forms a dynamic and complex barrier between the living cell and its environment. However, its in vivo studies are difficult because it consists of a high variety of lipids and proteins and is continuously reorganized by the cell. Therefore, membrane model systems with precisely controlled composition are used to investigate fundamental interactions of membrane components under well-defined conditions. Giant unilamellar vesicles (GUVs) offer a powerful model system for the cell membrane, but many previous studies have been performed in unphysiologically low ionic strength solutions which might lead to altered membrane properties, protein stability and lipid-protein interaction. In the present work, we give an overview of the existing methods for GUV production and present our efforts on forming single, free floating vesicles up to several tens of μm in diameter and at high yield in various buffer solutions with physiological ionic strength and pH.
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    Tamba, Y.; Terashima, H.; Yamazaki, M. A membrane filtering method for the purification of giant unilamellar vesicles. Chem. Phys. Lipids 2011, 164 (5), 351– 358,  DOI: 10.1016/j.chemphyslip.2011.04.003
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    Banerjee, R. Liposomes: Applications in Medicine. J. Biomater Appl. 2001, 16 (1), 3– 21,  DOI: 10.1106/RA7U-1V9C-RV7C-8QXL
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    Walde, P.; Cosentino, K.; Engel, H.; Stano, P. Giant Vesicles: Preparations and Applications. ChemBiochem 2010, 11 (7), 848– 865,  DOI: 10.1002/cbic.201000010
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    Giant Vesicles: preparations and Applications
    Walde, Peter; Cosentino, Katia; Engel, Helen; Stano, Pasquale
    ChemBioChem (2010), 11 (7), 848-865CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. There is considerable interest in prepg. cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biol. cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chem. compds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible prepn. of giant vesicles are available. The key concepts of currently known methods for prepg. giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.
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    Lee, W.; Debasitis, J.; Lee, V. Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication. Biomaterials 2009, 30 (8), 1587– 1595,  DOI: 10.1016/j.biomaterials.2008.12.009
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    Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication
    Lee, Wonhye; Debasitis, Jason Cushing; Lee, Vivian Kim; Lee, Jong-Hwan; Fischer, Krisztina; Edminster, Karl; Park, Je-Kyun; Yoo, Seung-Schik
    Biomaterials (2009), 30 (8), 1587-1595CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    The authors present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-contg. collagen was crosslinked by coating the layer with nebulized aq. sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in 2 distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell-hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was obsd. on both planar and non-planar surfaces. The authors' results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing.
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    Blaeser, A.; Duarte Campos, D. F.; Puster, U.; Richtering, W.; Stevens, M. M.; Fischer, H. Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity. Adv. Healthc. Mater. 2016, 5 (3), 326– 333,  DOI: 10.1002/adhm.201500677
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    Lucas, L.; Aravind, A.; Emma, P.; Marquette, M.; Courtial, C. Rheology, simulation and data analysis toward bioprinting cell viability awareness. Bioprinting 2021, 21, e00119  DOI: 10.1016/j.bprint.2020.e00119
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    Bhatia, T.; Husen, P.; Brewer, J. Preparing giant unilamellar vesicles (GUVs) of complex lipid mixtures on demand: Mixing small unilamellar vesicles of compositionally heterogeneous mixtures. Biochim. Biophys. Acta, Biomembr. 2015, 1848 (12), 3175– 3180,  DOI: 10.1016/j.bbamem.2015.09.020
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    Preparing giant unilamellar vesicles (GUVs) of complex lipid mixtures on demand: Mixing small unilamellar vesicles of compositionally heterogeneous mixtures
    Bhatia, Tripta; Husen, Peter; Brewer, Jonathan; Bagatolli, Luis A.; Hansen, Per L.; Ipsen, John H.; Mouritsen, Ole G.
    Biochimica et Biophysica Acta, Biomembranes (2015), 1848 (12), 3175-3180CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    Giant unilamellar vesicles (GUVs) are simple model membrane systems of cell-size, which are instrumental to study the function of more complex biol. membranes involving heterogeneities in lipid compn., shape, mech. properties, and chem. properties. The authors have devised a method that makes it possible to prep. a uniform sample of ternary GUVs of a prescribed compn. and heterogeneity by mixing different populations of small unilamellar vesicles (SUVs). The validity of the protocol has been demonstrated by applying it to ternary lipid mixt. of DOPC, DPPC, and cholesterol by mixing small unilamellar vesicles (SUVs) of two different populations and with different lipid compns. The compositional homogeneity among GUVs resulting from SUV mixing is quantified by measuring the area fraction of the liq. ordered-liq. disordered phases in giant vesicles and is comparable to that in GUVs of the prescribed compn. produced from hydration of dried lipids mixed in org. solvent. The authors' method opens up the possibility to quickly increase and manipulate the complexity of GUV membranes in a controlled manner at physiol. buffer and temp. conditions. The new protocol will permit quant. biophys. studies of a whole new class of well-defined model membrane systems of a complexity that resembles biol. membranes with rafts.
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    Lira, R. B.; Dimova, R. Fusion assays for model membranes: a critical review. Adv. Biomembr. Lipid Self-Assem. 2019, 30, 229– 270,  DOI: 10.1016/bs.abl.2019.09.003
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    Banquy, X.; Kristiansen, K.; Lee, D. W.; Israelachvili, J. N. Adhesion and hemifusion of cytoplasmic myelin lipid membranes are highly dependent on the lipid composition. Biochim. Biophys. Acta, Biomembr. 2012, 1818 (3), 402– 410,  DOI: 10.1016/j.bbamem.2011.10.015
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    Adhesion and hemifusion of cytoplasmic myelin lipid membranes are highly dependent on the lipid composition
    Banquy, Xavier; Kristiansen, Kai; Lee, Dong Woog; Israelachvili, Jacob N.
    Biochimica et Biophysica Acta, Biomembranes (2012), 1818 (3), 402-410CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    We report the effects of calcium ions on the adhesion and hemifusion mechanisms of model supported myelin lipid bilayer membranes of differing lipid compn. As in our previous studies the lipid compns. used mimic "healthy" and "diseased-like" (exptl. autoimmune encephalomyelitis, EAE) membranes. Our results show that the interaction forces as a function of membrane sepn. distance are well described by a generic model that also (and in particular) includes the hydrophobic interaction arising from the hydrophobically exposed (interior) parts of the bilayers. The model is able to capture the mech. instability that triggers the onset of the hemifusion event, and highlights the primary role of the hydrophobic interaction in membrane fusion. The effects of lipid compn. on the fusion mechanism, and the adhesion forces between myelin lipid bilayers, can be summarized as follows: in calcium-free buffer, healthy membranes do not present any signs of adhesion or hemifusion, while diseased membranes hemifuse easily. Addn. of 2 mM calcium favors adhesion and hemifusion of the membranes independently of their compn., but the mechanisms involved in the two processes were different: healthy bilayers systematically presented stronger adhesion forces and lower energy barriers to fusion compared to diseased bilayers. These results are of particular relevance for understanding lesion development (demyelination, swelling, vacuolization and/or vesiculation) in myelin assocd. diseases such as multiple sclerosis and its relationship to lipid domain formation in myelin membranes.
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    Kowalska, M.; Broniatowski, M.; Płachta, Ł; Wydro, P.; Wydro, P. The effect of the polyethylene glycol chain length of a lipopolymer (DSPE-PEGn) on the properties of DPPC monolayers and bilayers. J. Mol. Liq. 2021, 335, 116529,  DOI: 10.1016/j.molliq.2021.116529
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    The effect of the polyethylene glycol chain length of a lipopolymer (DSPE-PEGn) on the properties of DPPC monolayers and bilayers
    Kowalska, Magdalena; Broniatowski, Marcin; Mach, Marzena; Plachta, Lukasz; Wydro, Pawel
    Journal of Molecular Liquids (2021), 335 (), 116529CODEN: JMLIDT; ISSN:0167-7322. (Elsevier B.V.)
    Due to the growing importance of controlled drug delivery systems (DDS), the main task of nanotechnol. is to develop stable, effective and non-toxic nanocarriers in which the drug can be encapsulated and delivered to a specific diseased site in the patient's body. Currently, one of the most popular ways to improve the pharmacokinetic and physicochem. properties of liposomes is introducing into their structure poly(ethylene glycol) chains conjugated with 1,2-disteroil-sn-glycero-3-phosphoethanolamine (DSPE) mols. Because the research so far does not give an unequivocal answer which length of PEG chains is more beneficial for liposomes properties, the aim of this work was to investigate the influence of this parameter (DSPE-PEG350, DSPE-PEG750 and DSPE-PEG2000) on model DPPC membrane. The studies were performed on monolayer and bilayer systems and were related to the surface pressure measurements, Brewster angle microscopy expts., Grazing Incidence X-ray Diffraction studies, dynamic light scattering and zeta potential measurements and the expts. with the calcein release and steady-state fluorescence anisotropy of DPH. The obtained results proved that the mol. organization of the DPPC membrane strongly depends on the length of poly(ethylene glycol) chains conjugated with DSPE. Moreover, the addn. of different lengths of polymer chains changes the properties of formulated liposomes, esp. their stability, permeability, size and surface charge.
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    Allen, T. M.; Hansen, C.; Martin, F.; Redemann, C.; Yau-Young, A. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim. Biophys. Acta, Biomembr. 1991, 1066, 29– 36,  DOI: 10.1016/0005-2736(91)90246-5
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    Liposomes containing synthetic lipid derivatives of polyethylene glycol show prolonged circulation half-lives in vivo
    Allen, T. M.; Hansen, C.; Martin, F.; Redemann, C.; Yau-Young, A.
    Biochimica et Biophysica Acta, Biomembranes (1991), 1066 (1), 29-36CODEN: BBBMBS; ISSN:0005-2736.
    Novel synthetic lipid derivs. of polyethylene glycol (PEG) were synthesized and tested for their ability to decrease uptake of liposomes into the mononuclear phagocyte system (MPS, reticuloendothelial system) in mice and to prolong circulation half-lives of liposomes. A carbamate deriv. of PEG-1900 with distearoylphosphatidylethanolamine (PEG-DSPE) had the greatest ability to decrease MPS uptake of liposomes, at optimum concns. of 5-7 mol% in liposomes composed of sphingomyelin/egg phosphatidylcholine/cholesterol (SM/PC/Chol, 1:1:1, molar ratio). Results obtained with this compd. were equiv. to results previously obtained with 10 mol% monosialoganglioside GM1 in liposomes of similar compns. (Allen, T. M. and Chonn, A., 1987). Non-derivatized Me PEG or PEG-stearic acid (PEG-SA) were incapable of decreasing MPS uptake of liposomes. PEG-Chol and PEG-dipalmitoylglycerol (PEG-DPG) were intermediate in their effects on MPS uptake. Altering liposome size for liposomes contg. PEG-DSPE resulted in only minor changes in blood levels of liposomes. Half-lives of 0.1 μm liposomes of SM/PC/Chol/PEG-DSPE (1:1:1:0.2, molar ratio) in circulation was in excess of 20 h following either i.v. or i.p. injection. Liver plus spleen liposome levels for these liposomes was below 15% of injected label at 48 h following i.v. liposome injection and below 10% following i.p. injection. The major site of liposome uptake was in carcass tissues, with over 50% of label remaining in vivo at 48 h post-injections, either i.v. or i.p., in the carcass.
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    Allen, C.; Dos Santos, N.; Gallagher, R.; Chiu, G. N. C.; Shu, Y.; Li, W. M.; Johnstone, S. A.; Janoff, A. S.; Mayer, L. D.; Webb, M. S.; Bally, M. B. Controlling the Physical Behavior and Biological Performance of Liposome Formulations through Use of Surface Grafted Poly(ethylene Glycol). Biosci. Rep. 2002, 22, 225– 250,  DOI: 10.1023/A:1020186505848
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    Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol)
    Allen, C.; Dos Santos, N.; Gallagher, R.; Chiu, G. N. C.; Shu, Y.; Li, W. M.; Johnstone, S. A.; Janoff, A. S.; Mayer, L. D.; Webb, M. S.; Bally, M. B.
    Bioscience Reports (2002), 22 (2), 225-250CODEN: BRPTDT; ISSN:0144-8463. (Kluwer Academic/Plenum Publishers)
    A review. The presence of poly(ethylene glycol) (PEG) at the surface of a liposomal carrier has been clearly shown to extend the circulation lifetime of the vehicle. To this point, the extended circulation lifetime that the polymer affords has been attributed to the redn. or prevention of protein adsorption. However, there is little evidence that the presence of PEG at the surface of a vehicle actually reduces total serum protein binding. In this review we examine all aspects of PEG in order to gain a better understanding of how the polymer fulfills its biol. role. The phys. and chem. properties of the polymer are explored and compared to properties of other hydrophilic polymers. An evidence based assessment of several in vitro protein binding studies as well as in vivo pharmacokinetics studies involving PEG is included. The ability of PEG to prevent the self-aggregation of liposomes is considered as a possible means by which it extends circulation longevity. Also, a "dysopsonization" phenomenon where PEG actually promotes binding of certain proteins that then mask the vehicle is discussed.
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    Kenworthy, A. K.; Simon, S. A.; McIntosh, T. J. Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 1995, 68 (5), 1903– 1920,  DOI: 10.1016/S0006-3495(95)80368-1
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    Staufer, O.; Antona, S.; Zhang, D. Microfluidic production and characterization of biofunctionalized giant unilamellar vesicles for targeted intracellular cargo delivery. Biomaterials 2021, 264, 120203,  DOI: 10.1016/j.biomaterials.2020.120203
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    Microfluidic production and characterization of biofunctionalized giant unilamellar vesicles for targeted intracellular cargo delivery
    Staufer, Oskar; Antona, Silvia; Zhang, Dennis; Csatari, Julia; Schroeter, Martin; Janiesch, Jan-Willi; Fabritz, Sebastian; Berger, Imre; Platzman, Ilia; Spatz, Joachim P.
    Biomaterials (2021), 264 (), 120203CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    Lipid-based vesicles have found widespread applications in the life sciences, allowing for fundamental insights into membrane-based processes in cell biol. and as carrier systems for drug delivery purposes. So far, mostly small unilamellar vesicles (SUVs) with diams. of ∼100 nm have been applied as carrier systems for biomedical applications. Despite this progress, several systematic limitations have arisen due to SUV dimensions, e.g., the size and total amt. of applicable cargo is limited. Giant unilamellar vesicles (GUVs) might offer a pragmatic alternative for efficient cargo delivery. However, due to the lack of reliable high-throughput prodn. technologies for GUV-carrier systems, only little is known about their interaction with cells. Here we present a microfluidic-based mech. droplet-splitting pipeline for the prodn. of carrier-GUVs with diams. of ∼2 μm. The technol. developed allows for highly efficient cargo loading and unprecedented control over the biol. and physicochem. properties of GUV membranes. By generating differently charged (between -31 and + 28 mV), bioligand-conjugated (e.g. with E-cadherin, NrCam and antibodies) and PEG-conjugated GUVs, we performed a detailed investigation of attractive and repulsive GUV-cell interactions. Fine-tuning of these interactions allowed for targeted cellular GUV delivery. Moreover, we evaluated strategies for intracellular GUV cargo release by lysosomal escape mediated by the pH sensitive lipid DOBAQ, enabling cytoplasmic transmission. The presented GUV delivery technol. and the systematic characterization of assocd. GUV-cell interactions could provide a means for more efficient drug administration and will pave the way for hitherto impossible approaches towards a targeted delivery of advanced cargo such as microparticles, viruses or macromol. DNA-robots.
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    Mahendra, A.; James, H. P.; Jadhav, S. PEG-grafted phospholipids in vesicles: Effect of PEG chain length and concentration on mechanical properties. Chem. Phys. Lipids 2019, 218, 47– 56,  DOI: 10.1016/j.chemphyslip.2018.12.001
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    PEG-grafted phospholipids in vesicles: Effect of PEG chain length and concentration on mechanical properties
    Mahendra, Amit; James, Honey Priya; Jadhav, Sameer
    Chemistry and Physics of Lipids (2019), 218 (), 47-56CODEN: CPLIA4; ISSN:0009-3084. (Elsevier Ireland Ltd.)
    Incorporation of low mol. wt. poly-ethylene glycol (PEG) - grafted phospholipids in vesicle bilayers is known to increase the circulation time of liposomal drug delivery vehicles. Mech. properties of giant unilamellar DPPC vesicles contg. varying concns. of DSPE-PEG (PEG MW: 550, 1000 and 2000) were measured by micropipette aspiration assay or osmotic swelling. While the area compressibility modulus did not change significantly, the bending modulus and water permeability of the bilayer was found to increase with increasing mole fraction of DSPE-PEG. This increase was more pronounced for higher mol. wt. PEG. The measured bending modulus agreed with that predicted by scaling theory only at low mole fractions of DSPE-PEG. The water permeability was also measured as a function of the increase in area per lipid (due to steric repulsion between PEG chains), and for the same area per lipid, the PEG chain with MW 550 provided a greater resistance to water transport across the vesicle membrane compared to PEG 1000 and 2000. Lysis tension of the membrane, detd. by osmotic lysis method at different loading rates showed a decrease in membrane strength on inclusion of the polymer lipid. These results suggest that liposome lifetime in the circulation and the rate of drug delivery are affected by the mol. wt. and concn. of PEG in the bilayer.
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    Papaioannou, T. G.; Karatzis, E. N.; Vavuranakis, M.; Lekakis, J. P.; Stefanadis, C. Assessment of vascular wall shear stress and implications for atherosclerotic disease. Int. J. Cardiol. 2006, 113 (1), 12– 18,  DOI: 10.1016/j.ijcard.2006.03.035
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    Koutsiaris, A. G.; Tachmitzi, S.; Batis, N.; Kotoula, M. G.; Karabatsas, C. H.; Tsironi, E.; Chatzoulis, D. Z. Volume flow and wall shear stress quantification in the human conjunctival capillaries and post-capillary venules in vivo. Biorheology 2007, 44 (5–6), 375– 386
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    Volume flow and wall shear stress quantification in the human conjunctival capillaries and post-capillary venules in vivo
    Koutsiaris Aristotle G; Tachmitzi Sophia V; Batis Nick; Kotoula Maria G; Karabatsas Constantinos H; Tsironi Evagelia; Chatzoulis Dimitrios Z
    Biorheology (2007), 44 (5-6), 375-86 ISSN:0006-355X.
    Understanding the mathematical relationships of volume blood flow and wall shear stress with respect to microvessel diameter is necessary for the study of vascular design. Here, for the first time, volume flow and wall shear stress were quantified from axial red blood cell velocity measurements in 104 conjunctival microvessels of 17 normal human volunteers. Measurements were taken with a slit lamp based imaging system from the post capillary side of the bulbar conjunctiva in microvessel diameters ranging from 4 to 24 micrometers. The variation of the velocity profile with diameter was taken into account by using a profile factor function. Volume flow ranged from 5 to 462 pl/s with a mean value of 102 pl/s and gave a second power law best fitting line (r=0.97) deviating significantly from the third power law relation with diameter. The estimated wall shear stress declined hyperbolically (r=0.93) from a maximum of 9.55 N/m(2) at the smallest capillaries, down to a minimum of 0.28 N/m(2) at the higher diameter post capillary venules. The mean wall shear stress value for all microvessels was 1.54 N/m(2).
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    Lorent, J. H.; Levental, K.; Ganesan, L. Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape. Nat. Chem. Biol. 2020, 16 (6), 644– 652,  DOI: 10.1038/s41589-020-0529-6
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    Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape
    Lorent, J. H.; Levental, K. R.; Ganesan, L.; Rivera-Longsworth, G.; Sezgin, E.; Doktorova, M. D.; Lyman, E.; Levental, I.
    Nature Chemical Biology (2020), 16 (6), 644-652CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
    A fundamental feature of cellular plasma membranes (PMs) is an asym. lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compns. of individual PM leaflets nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophys. properties of both monolayers in living mammalian PMs. Phospholipid unsatn. is dramatically asym., with the cytoplasmic leaflet being approx. twofold more unsatd. than the exoplasmic leaflet. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophys. asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in the asym. structures of protein transmembrane domains. These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.
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    Doktorova, M.; LeVine, M. V.; Khelashvili, G.; Weinstein, H. A New Computational Method for Membrane Compressibility: Bilayer Mechanical Thickness Revisited. Biophys. J. 2019, 116 (3), 487– 502,  DOI: 10.1016/j.bpj.2018.12.016
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    A New Computational Method for Membrane Compressibility: Bilayer Mechanical Thickness Revisited
    Doktorova, Milka; Le Vine, Michael V.; Khelashvili, George; Weinstein, Harel
    Biophysical Journal (2019), 116 (3), 487-502CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, phys. models of bilayer deformation have utilized mech. consts. such as the moduli for bending rigidity (κC) and area compressibility (KA). However, the use of these models to quantify the energetics of membrane deformation assocd. with protein-membrane interactions, and the membrane response to stress is often hampered by the shortage of exptl. data suitable for the estn. of the mech. consts. of various lipid mixts. Although computational tools such as mol. dynamics simulations can provide alternative means to est. KA values, current approaches suffer significant tech. limitations. Here, we present a novel, to our knowledge, computational framework that allows for a direct estn. of KA values for individual bilayer leaflets. The theory is based on the concept of elasticity and derives KA from real-space anal. of local thickness fluctuations sampled in mol. dynamics simulations. We explore and validate the model on a large set of single and multicomponent bilayers of different lipid compns. and sizes, simulated at different temps. The calcd. bilayer compressibility moduli agree with values estd. previously from expts. and those obtained from a std. computational method based on a series of constrained tension simulations. We further validate our framework in a comparison with an existing polymer brush model and confirm the polymer brush model's predicted linear relationship with proportionality coeff. of 24, using elastic parameters calcd. from the simulation trajectories. The robustness of the results that emerge from the method allows us to revisit the origins of the bilayer mech. (compressible) thickness and in particular its dependence on acyl-chain unsatn. and the presence of cholesterol.
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    Karal, M. A. S.; Mokta, N.; Levadny, V. Effects of cholesterol on the size distribution and bending modulus of lipid vesicles. PLoS One 2022, 17 (1), e0263119  DOI: 10.1371/journal.pone.0263119
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    Effects of cholesterol on the size distribution and bending modulus of lipid vesicles
    Karal, Mohammad Abu Sayem; Mokta, Nadia Akter; Levadny, Victor; Belaya, Marina; Ahmed, Marzuk; Ahamed, Md. Kabir; Ahammed, Shareef
    PLoS One (2022), 17 (1), e0263119CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    The influence of cholesterol fraction in the membranes of giant unilamellar vesicles (GUVs) on their size distributions and bending moduli has been investigated. The membranes of GUVs were synthesized by a mixt. of two elements: elec. neutral lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol and also a mixt. of three elements: elec. charged lipid 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), DOPC and cholesterol. The size distributions of GUVs have been presented by a set of histograms. The classical lognormal distribution is well fitted to the histograms, from where the av. size of vesicle is obtained. The increase of cholesterol content in the membranes of GUVs increases the av. size of vesicles in the population. Using the framework of Helmholtz free energy of the system, the theory developed by us is extended to explain the exptl. results. The theory dets. the influence of cholesterol on the bending modulus of membranes from the fitting of the proper histograms. The increase of cholesterol in GUVs increases both the av. size of vesicles in population and the bending modulus of membranes.
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    Lira, R. B.; Steinkühler, J.; Knorr, R. L.; Dimova, R.; Riske, K. A. Posing for a picture: Vesicle immobilization in agarose gel. Sci. Rep. 2016, 6, 25254,  DOI: 10.1038/srep25254
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    Posing for a picture: vesicle immobilization in agarose gel
    Lira, Rafael B.; Steinkuhler, Jan; Knorr, Roland L.; Dimova, Rumiana; Riske, Karin A.
    Scientific Reports (2016), 6 (), 25254CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Taking a photo typically requires the object of interest to stand still. In science, imaging is potentiated by optical and electron microscopy. However, living and soft matter are not still. Thus, biol. prepns. for microscopy usually include a fixation step. Similarly, immobilization strategies are required for or substantially facilitate imaging of cells or lipid vesicles, and even more so for acquiring high-quality data via fluorescence-based techniques. Here, we describe a simple yet efficient method to immobilize objects such as lipid vesicles with sizes between 0.1 and 100 μm using agarose gel. We show that while large and giant unilamellar vesicles (LUVs and GUVs) can be caged in the pockets of the gel meshwork, small mols., proteins and micelles remain free to diffuse through the gel and interact with membranes as in agarose-free solns., and complex biochem. reactions involving several proteins can proceed in the gel. At the same time, immobilization in agarose has no adverse effect on the GUV size and stability. By applying techniques such as FRAP and FCS, we show that the lateral diffusion of lipids is not affected by the gel. Finally, our immobilization strategy allows capturing high-resoln. 3D images of GUVs.
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    Sandström, M. C.; Johansson, E.; Edwards, K. Structure of mixed micelles formed in PEG-lipid/lipid dispersions. Langmuir 2007, 23 (8), 4192– 4198,  DOI: 10.1021/la063501s
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    Structure of mixed micelles formed in PEG-lipid/lipid dispersions
    Sandstrom Maria C; Johansson Emma; Edwards Katarina
    Langmuir : the ACS journal of surfaces and colloids (2007), 23 (8), 4192-8 ISSN:0743-7463.
    Polyethylene glycol (PEG)-conjugated lipids are commonly employed for steric stabilization of liposomes. When added in high concentrations PEG-lipids induce formation of mixed micelles, and depending on the lipid composition of the sample, these may adapt either a discoidal or a long threadlike shape. The factors governing the type of micellar aggregate formed have so far not been investigated in detail. In this study we have systematically varied the lipid composition in lipid/PEG-lipid mixtures and characterized the aggregate structure by means of cryo-transmission electron microscopy (cryo-TEM). The effects caused by adding sterols, phosphatidylethanolamines, and phospholipids with saturated acyl chains to egg phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000 (EPC/DSPE-PEG2000) mixtures with a fixed amount (25 mol %) of DSPE-PEG2000 was studied. Further, the aggregate structure in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPC/DMPE-PEG2000) samples above and below the gel to liquid crystalline phase transition temperature (TC) was investigated. Our results revealed that lipid components, as well as environmental conditions, that reduce the lipid spontaneous curvature and increase the monolayer bending modulus tend to promote formation of discoidal micelles. At temperatures below the gel-to-liquid crystalline phase transition temperature reduced lipid/PEG-lipid miscibility, furthermore, likely contribute to the observed formation of discoidal rather than threadlike micelles.
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    Garbuzenko, O.; Barenholz, Y.; Priev, A. Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer. Chem. Phys. Lipids 2005, 135 (2), 117– 129,  DOI: 10.1016/j.chemphyslip.2005.02.003
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    Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer
    Garbuzenko, Olga; Barenholz, Yechezkel; Priev, Aba
    Chemistry and Physics of Lipids (2005), 135 (2), 117-129CODEN: CPLIA4; ISSN:0009-3084. (Elsevier B.V.)
    The aim of this study was to elucidate the effect of various mole percentages (0-25 mol%) of 2000 Da polyethylene glycol-disteroylphosphoethanolamine (PEG-DSPE) in the presence or absence of 40 mol% cholesterol and the effect of degree of satn. of phosphatidylcholine (PC) on the size and the lipid bilayer packing of large unilamellar vesicles (LUV). Egg PC (EPC, unsatd.) LUV and fully hydrogenated soy PC (HSPC, satd.) LUV partial sp. vol., specific compressibility, size, and packing parameter (PP) of lipids were characterized by measurements of d., ultrasonic velocity, specific turbidity, and dynamic light scattering. Liposome size and specific turbidity decreased with increase in temp. and PEG-DSPE mol%, except at 7 ± 2 mol%. At this PEG-DSPE mol%, an anomalous peak in liposome size of 15 ± 5 nm was obsd. We attribute this effect mainly to the change in the spatial structure of the PEG-DSPE mol., depending on whether the grafted PEG is in the mushroom or brush configuration. In the mushroom regime, i.e., when the grafted PEG is up to 4 mol% in LUV, the PEG moiety did not affect the additive PP of the lipids in the bilayer, and the PP value of PEG-DSPE is 1.044; while in the brush regime, i.e., when the grafted PEG is higher than 4 mol%, the PP of PEG-DSPE decreases exponentially, reaching the value of 0.487 at 30 mol% of grafted lipopolymer. The specific compressibility and additive PP values for the mixt. of matrix lipid (EPC or HSPC), cholesterol, and PEG-DSPE for all liposome compns. investigated reached their max. at 7 ± 2 mol% PEG-DSPE, the concn. of PEG-DSPE at which the highest biol. stability of the LUV is achieved.
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    Holthuis, J. C. M. Regulating membrane curvature. In Regulatory mechanisms of intracellular membrane transport Springer: 2004, 39 64.  DOI: 10.1007/b98566 .
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    Tirosh, O.; Barenholz, Y.; Katzhendler, J.; Priev, A. Hydration of polyethylene glycol-grafted liposomes. Biophys. J. 1998, 74 (3), 1371– 1379,  DOI: 10.1016/S0006-3495(98)77849-X
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    Hydration of polyethylene glycol-grafted liposomes
    Tirosh, Oren; Barenholz, Yechezkel; Katzhendler, Jehoshua; Priev, Aba
    Biophysical Journal (1998), 74 (3), 1371-1379CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    This study aimed to characterize the effect of polyethylene glycol of 2000 mol. wt. (PEG2000) attached to a dialkylphosphatidic acid (dihexadecylphosphatidyl (DHP)-PEG2000) on the hydration and thermodn. stability of lipid assemblies. Differential scanning calorimetry, densitometry, and ultrasound velocity and absorption measurements were used for thermodn. and hydrational characterization. Using a differential scanning calorimetry technique we showed that each mol. of PEG2000 binds 136±4 mols. of water. For PEG2000 covalently attached to the lipid mols. organized in micelles, the water binding increases to 210±6 water mols. This demonstrates that the two different structural configurations of the PEG2000, a random coil in the case of the free PEG and a brush in the case of DHP-PEG2000 micelles, differ in their hydration level. Ultrasound absorption changes in liposomes reflect mainly the heterophase fluctuations and packing defects in the lipid bilayer. The PEG-induced excess ultrasound absorption of the lipid bilayer at 7.7 MHz for PEG-lipid concns. over 5 mol % indicates the increase in the relaxation time of the headgroup rotation due to PEG-PEG interactions. The adiabatic compressibility (calcd. from ultrasound velocity and d.) of the lipid bilayer of the liposome increases monotonically with PEG-lipid concn. up to ∼7 mol %, reflecting release of water from the lipid headgroup region. Elimination of this water, induced by grafted PEG, leads to a decrease in bilayer defects and enhanced lateral packing of the phospholipid acyl chains. We assume that the dehydration of the lipid headgroup region in conjunction with the increase of the hydration of the outer layer by grafting PEG in brush configuration are responsible for increasing thermodn. stability of the liposomes at 5-7 mol % of PEG-lipid. At higher PEG-lipid concns., compressibility and partial vol. of the lipid phase of the samples decrease. This reflects the increase in hydration of the lipid headgroup region (up to five addnl. water mols. per lipid mol. for 12 mol % PEG-lipid) and the weakening of the bilayer packing due to the lateral repulsion of PEG chains.
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    Pepelanova, I.; Kruppa, K.; Scheper, T.; Lavrentieva, A. Gelatin-methacryloyl (GelMA) hydrogels with defined degree of functionalization as a versatile toolkit for 3D cell culture and extrusion bioprinting. Bioengineering 2018, 5 (3), 55,  DOI: 10.3390/bioengineering5030055
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    Gelatin-methacryloyl (GelMA) hydrogels with defined degree of functionalization as a versatile toolkit for 3D cell culture and extrusion bioprinting
    Pepelanova, Iliyana; Kruppa, Katharina; Scheper, Thomas; Lavrentieva, Antonina
    Bioengineering (2018), 5 (3), 55CODEN: BIOEBG; ISSN:2306-5354. (MDPI AG)
    Gelatin-methacryloyl (GelMA) is a semi-synthetic hydrogel which consists of gelatin derivatized with methacrylamide and methacrylate groups. These hydrogels provide cells with an optimal biol. environment (e.g., RGD motifs for adhesion) and can be quickly photo-crosslinked, which provides shape fidelity and stability at physiol. temp. In the present work, we demonstrated how GelMA hydrogels can be synthesized with a specific degree of functionalization (DoF) and adjusted to the intended application as a three-dimensional (3D) cell culture platform. The focus of this work lays on producing hydrogel scaffolds which provide a cell promoting microenvironment for human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) and are conductive to their adhesion, spreading, and proliferation. The control of mech. GelMA properties by variation of concn., DoF, and UV polymn. conditions is described. Moreover, hAD-MSC cell viability and morphol. in GelMA of different stiffness was evaluated and compared. Polymd. hydrogels with and without cells could be digested in order to release encapsulated cells without loss of viability. We also demonstrated how hydrogel viscosity can be increased by the use of biocompatible additives, in order to enable the extrusion bioprinting of these materials. Taken together, we demonstrated how GelMA hydrogels can be used as a versatile tool for 3D cell cultivation.
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    Guo, L.; Har, J. Y.; Sankaran, J.; Hong, Y.; Kannan, B.; Wohland, T. Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study. ChemPhyschem 2008, 9 (5), 721– 728,  DOI: 10.1002/cphc.200700611
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  1. Anna Burgstaller, Sara Madureira, Oskar Staufer. Synthetic cells in tissue engineering. Current Opinion in Biotechnology 2025, 92 , 103252. https://doi.org/10.1016/j.copbio.2024.103252
  2. Alexis Cooper, Anand Bala Subramaniam. Ultrahigh yields of giant vesicles obtained through mesophase evolution and breakup. Soft Matter 2024, 20 (48) , 9547-9561. https://doi.org/10.1039/D4SM01109K
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  • Abstract

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    Figure 1

    Figure 1. Bioprinting of giant unilamellar vesicles (GUVs). A) Production of GUVs with defined filling (Alexa Fluor 488 phalloidin, inset: scale bar of 10 μm) by electroformation. B) Filtration of GUVs by size with a 10 μm filter membrane to collect a stock solution with an average GUV diameter similar to human cells. C) Bioprinting GUVs by DOD or extrusion.

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    Figure 2

    Figure 2. GUV stability during bioprinting. A) Comparison of GUV stability after drop-on-demand (DOD) and extrusion bioprinting in GUV solution (iso-osmolar sucrose solution). A higher GUV density of 2 × 106 GUVs/mL and was used to visually count GUVs more easily. B) Incubation of DOPC-GUVs in the cell medium for 1 h. Scale bars represent 50 μm.

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    Figure 3

    Figure 3. PEG5-GUV stability during bioprinting. A) Comparison of PEG5-GUV stability after DOD and extrusion bioprinting of PEG5 GUVs in an isoosmolar sucrose solution, and DOD printing of PEG5 GUVs with a 20% cholesterol proportion. A density of 2 × 106 PEG5-GUV/mL was used. B) Incubation of PEG5-GUVs in a cell medium for 1 h. C) Agglomeration of DOPC- and PEG5-GUVs in DMEM after 1 h incubation. Segmented outline of agglomerates and GUVs (left) with the average agglomeration area normalized to the GUV area (right). Scale bars represent 50 μm. *p < 0.05 and ****p < 0.0001.

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    Figure 4

    Figure 4. GUVs with different concentrations of PEGylated lipids after filtration and encapsulation in 1% agarose hydrogel. A) GUVs with different concentrations of PEGylated lipids after filtration. Scale bar represents 150 μm, and in insets, it is 50 μm. B) Mean diameter of GUVs after filtration with different concentrations of PEGylated lipids. n = 3 with more than 175 GUVs per image. C) Projection images of z-stacks using the hydrogel containing GUVs with 0% (DOPC) and 5% (PEG5) PEGylated lipids 1 h and 72 h after encapsulation. Scale bars represent 50 μm. D) Effect of different PEG concentrations on the number of GUVs for 1 h, 24 h, and 72 h after encapsulation. n = 3. *p < 0.05 and **p < 0.001.

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    Figure 5

    Figure 5. Bioprinting of PEG5-GUVs in agarose–collagen (Ag–Col) and gelatin methacryloyl (GelMA) bioinks in different geometrical shapes. A) Photographs of bioprinted Ag–Col constructs, and microscopic z-stack projection of PEG5-GUVs in Ag–Col bioink postprinting. Scale bar represents 50 μm; in inserts, it is 10 μm;, in macroscopic images, it is 2 mm. B) Photographs of bioprinted GelMA constructs, and microscopic z-stack projection of PEG5-GUVs in GelMA bioinks postprinting. Scale bar represents 50 μm, in inserts, it is 10 μm, and in macroscopic images it is 2 mm. C) Normalized fluorescence intensity profile of FRAP measurements of PEG5-GUVs printed at 0.2 bar pressure in agarose hydrogels (right), and the diffusion coefficient of PEG5-GUVs postbioprinting (left). Scale bar represents 5 μm. D) Photographs of bioprinted Ag–Col bioink postprinting (insets, scale bar represents 2 mm), and microscopic z-stack projection of PEG5-GUVs (red) and MSCs (green, live cell cytoplasm staining) in the Ag–Col bioink after 72 h and 7 days of postprinting. Scale bar represents 50 μm; in macroscopic images, it is 2 mm.

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    Figure 6

    Figure 6. Studying the release of a fluorescent dye from bioprinted Ce6-PEG5-GUVs upon illumination. A) Fluorescence image of Ce6-PEG5-GUVs. B) Ce6-PEG5-GUVs loaded with sucrose and cultured in glucose solution before and after illumination with an LED set at a wavelength of 357 nm for 5 min. C) Fluorescence images showing bioprinted 1% w/v agarose constructs encapsulated with Alexa Fluor 488-loaded Ce6-PEG-GUVs before (upper images) and after (lower images) the release of the dye by illumination with an LED set at a wavelength of 357 nm for 5 min. Scale bars represent 50 μm; in insets, they are 10 μm; in macroscopic images, they are 2 mm.

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      Rideau, Emeline; Dimova, Rumiana; Schwille, Petra; Wurm, Frederik R.; Landfester, Katharina
      Chemical Society Reviews (2018), 47 (23), 8572-8610CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      Cells are integral to all forms of life due to their compartmentalization by the plasma membrane. However, living organisms are immensely complex. Thus there is a need for simplified and controllable models of life for a deeper understanding of fundamental biol. processes and man-made applications. This is where the bottom-up approach of synthetic biol. comes from: a stepwise assembly of biomimetic functionalities ultimately into a protocell. A fundamental feature of such an endeavor is the generation and control of model membranes such as liposomes and polymersomes. We compare and contrast liposomes and polymersomes for a better a priori choice and design of vesicles and try to understand the advantages and shortcomings assocd. with using one or the other in many different aspects (properties, synthesis, self-assembly, applications) and which aspects have been studied and developed with each type and update the current development in the field.
    17. 17
      Göpfrich, K.; Platzman, I.; Spatz, J. P. Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells. Trends Biotechnol. 2018, 36 (9), 938– 951,  DOI: 10.1016/j.tibtech.2018.03.008
      17
      Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells
      Gopfrich Kerstin; Platzman Ilia; Spatz Joachim P
      Trends in biotechnology (2018), 36 (9), 938-951 ISSN:.
      With the ultimate aim to construct a living cell, bottom-up synthetic biology strives to reconstitute cellular phenomena in vitro - disentangled from the complex environment of a cell. Recent work towards this ambitious goal has provided new insights into the mechanisms governing life. With the fast-growing library of functional modules for synthetic cells, their classification and integration become increasingly important. We discuss strategies to reverse-engineer and recombine functional parts for synthetic eukaryotes, mimicking the characteristics of nature's own prototype. Particularly, we focus on large outer compartments, complex endomembrane systems with organelles, and versatile cytoskeletons as hallmarks of eukaryotic life. Moreover, we identify microfluidics and DNA nanotechnology as two technologies that can integrate these functional modules into sophisticated multifunctional synthetic cells.
    18. 18
      Angelova, M. I.; Dimitrov, D. S. Liposome electroformation. Faraday Discuss. Chem. Soc. 1986, 81, 303– 311,  DOI: 10.1039/dc9868100303
      18
      Liposome electroformation
      Angelova, M.; Dimitrov, D.
      Faraday Discussions of the Chemical Society (1986), 81 (1), 303-11CODEN: FDCSB7; ISSN:0301-7249.
      Liposome formation and lipid swelling on Pt electrodes in distd. water and water solns. in d.c. elec. fields were investigated for different amts. of a neg. charged lipid (mixt. from 71% phosphatidylcholines, 21.5% phosphatidylethanolamines and 7.5% phosphatidylserines), and a neutral lipid (dimyristoylphosphatidylcholine, DMPC). Neg. charged lipids do not form liposomes without fields when the thickness of the dried lipid layer is ≤90 bilayers. The rate and extent of swelling of layers thicker than 90 bilayers is largest on the cathode, smaller without fields and smallest on the anode. The theory, based on the assumption that osmotic and electrostatic forces drive lipid swelling and liposome formation. is in semi-quant. agreement with the exptl. data; in particular, it gives the obsd. linear dependence of the rate of swelling on the inverse lipid layer thickness. To induce liposome formation for layers thinner than 90 bilayers it was necessary to apply a neg. potential which is proportional to the logarithm of the inverse layer thickness. The characteristic crit. potential is proportional to RTk/F; R being the gas const., Tk the abs. temp., and F the Faraday const. This indicates that redistribution of counterions may be the cause which increases the repulsive electrostatic intermembrane forces to overcome van der Waals attraction. For thicknesses <10 bilayers, formation of very thin-walled liposomes of narrow size distribution and mean diam. of ∼30 μm was obsd. These liposomes grow in size before detachment, and a formula for the kinetics of growth was derived, which is in very good agreement with the exptl. data. The effects of d.c. field on DMPC swelling are smaller and lead to formation of liposome-like structures of different appearance. Bilayer sepn. and bending are prerequisites for liposome formation from hydrating lipids. Therefore, a possible mol. mechanism is that membranes should be destabilized to bend and fuse to form liposomes. This requires the right proportion between structured regions, in the form of bilayers, and defects and (or) nonbilayer structures, and in many cases external constraints, in particular, elec. fields.
    19. 19
      Reeves, J. P.; Dowben, R. M. Formation and properties of thin-walled phospholipid vesicles. J. Cell. Physiol. 1969, 73 (1), 49– 60,  DOI: 10.1002/jcp.1040730108
      19
      Formation and properties of thin-walled phospholipid vesicles
      Reeves, John P.; Dowben, Robert M.
      Journal of Cellular Physiology (1969), 73 (1), 49-60CODEN: JCLLAX; ISSN:0021-9541.
      Large nos. of thin-walled vesicles, 0.5-10 μ in diam., can be formed by permitting a thinly spread layer of hydrated phospholipids to swell slowly in distd. H2O or in an aq. nonelectrolyte soln. Electron micrographs of phospholipid analyses indicated that the walls consist of a single or a few bilayers. The vesicles can be centrifuged and resuspended in another medium to make them a useful system for studying permeability. The osmolarity of the soln. in the interior of the vesicles can be estd. by immersion refractometry and the osmolarity of the internal aq. phase is linearly related to that of the external medium.
    20. 20
      Weinberger, A.; Tsai, F.; Koenderink, G. Gel-Assisted Formation of Giant Unilamellar Vesicles. Biophys. J. 2013, 105 (1), 154– 164,  DOI: 10.1016/j.bpj.2013.05.024
      20
      Gel-Assisted Formation of Giant Unilamellar Vesicles
      Weinberger, Andreas; Tsai, Feng-Ching; Koenderink, Gijsje H.; Schmidt, Thais F.; Itri, Rosangela; Meier, Wolfgang; Schmatko, Tatiana; Schroder, Andre; Marques, Carlos
      Biophysical Journal (2013), 105 (1), 154-164CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Giant unilamellar vesicles or GUVs are systems of choice as biomimetic models of cellular membranes. Although a variety of procedures exist for making single walled vesicles of tens of microns in size, the range of lipid compns. that can be used to grow GUVs by the conventional methods is quite limited, and many of the available methods involve energy input that can damage the lipids or other mols. present in the growing soln. for embedment in the membrane or in the vesicle interior. Here, we show that a wide variety of lipids or lipid mixts. can grow into GUVs by swelling lipid precursor films on top of a dried polyvinyl alc. gel surface in a swelling buffer that can contain diverse biorelevant mols. Moreover, we show that the encapsulation potential of this method can be enhanced by combining polyvinyl alc.-mediated growth with inverse-phase methods, which allow (bio)mol. complexation with the lipids.
    21. 21
      Weiss, M. Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics. Nat. Mater. 2018, 17 (1), 89– 95,  DOI: 10.1038/nmat5005
      21
      Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics
      Weiss, Marian; Frohnmayer, Johannes Patrick; Benk, Lucia Theresa; Haller, Barbara; Janiesch, Jan-Willi; Heitkamp, Thomas; Boersch, Michael; Lira, Rafael B.; Dimova, Rumiana; Lipowsky, Reinhard; Bodenschatz, Eberhard; Baret, Jean-Christophe; Vidakovic-Koch, Tanja; Sundmacher, Kai; Platzman, Ilia; Spatz, Joachim P.
      Nature Materials (2018), 17 (1), 89-96CODEN: NMAACR; ISSN:1476-1122. (Nature Research)
      Compartments for the spatially and temporally controlled assembly of biol. processes are essential towards cellular life. Synthetic mimics of cellular compartments based on lipid-based protocells lack the mech. and chem. stability to allow their manipulation into a complex and fully functional synthetic cell. Here, the authors present a high-throughput microfluidic method to generate stable, defined sized liposomes termed 'droplet-stabilized giant unilamellar vesicles (dsGUVs)'. The enhanced stability of dsGUVs enables the sequential loading of these compartments with biomols., namely purified transmembrane and cytoskeleton proteins by microfluidic pico-injection technol. This constitutes an exptl. demonstration of a successful bottom-up assembly of a compartment with contents that would not self-assemble to full functionality when simply mixed together. Following assembly, the stabilizing oil phase and droplet shells are removed to release functional self-supporting protocells to an aq. phase, enabling them to interact with physiol. relevant matrixes.
    22. 22
      Haller, B.; Göpfrich, K.; Schröter, M.; Janiesch, J.-W.; Platzman, I.; Spatz, J. P. Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems. Lab Chip. 2018, 18 (17), 2665– 2674,  DOI: 10.1039/C8LC00582F
      22
      Charge-controlled microfluidic formation of lipid-based single- and multicompartment systems
      Haller, Barbara; Goepfrich, Kerstin; Schroeter, Martin; Janiesch, Jan-Willi; Platzman, Ilia; Spatz, Joachim P.
      Lab on a Chip (2018), 18 (17), 2665-2674CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
      In this manuscript, we introduce a simple, off-the-shelf approach for the on-demand creation of giant unilamellar vesicles (GUVs) or multicompartment synthetic cell model systems in a high-throughput manner. To achieve this, we use microfluidics to encapsulate small unilamellar vesicles in block-copolymer surfactant-stabilized water-in-oil droplets. By tuning the charge of the inner droplet interface, adsorption of lipids can be either inhibited, leading to multicompartment systems, or induced, leading to the formation of droplet-stabilized GUVs. To control the charge d., we formed droplets using different molar ratios of an uncharged PEG-based fluorosurfactant and a neg.-charged PFPE carboxylic acid fluorosurfactant (Krytox). We systematically studied the transition from a multicompartment system to 3D-supported lipid bilayers as a function of lipid charge and Krytox concn. using confocal fluorescence microscopy, cryo-SEM and interfacial tension measurements. Moreover, we demonstrate a simple method to release GUVs from the surfactant shell and the oil phase into a physiol. buffer - providing a remarkably high-yield approach for GUV formation. This widely applicable microfluidics-based technol. will increase the scope of GUVs as adaptable cell-like compartments in bottom-up synthetic biol. applications and beyond.
    23. 23
      Karamdad, K. Engineering thermoresponsive phase separated vesicles formed: Via emulsion phase transfer as a content-release platform. Chem. Sci. 2018, 9 (21), 4851– 4858,  DOI: 10.1039/C7SC04309K
      There is no corresponding record for this reference.
    24. 24
      Zong, W.; Shao, X.; Chai, Y.; Wang, X.; Han, S.; Chu, H.; Zhu, C.; Zhang, X. Controllable drug release of pH-sensitive liposomes encapsulating artificial cytosol system. bioRxiv 2021, 2021– 05,  DOI: 10.1101/2021.05.24.445400
      There is no corresponding record for this reference.
    25. 25
      Dreher, Y.; Jahnke, K.; Schröter, M.; Göpfrich, K. Light-Triggered Cargo Loading and Division of DNA-Containing Giant Unilamellar Lipid Vesicles. Nano Lett. 2021, 21 (14), 5952– 5957,  DOI: 10.1021/acs.nanolett.1c00822
      25
      Light-Triggered Cargo Loading and Division of DNA-Containing Giant Unilamellar Lipid Vesicles
      Dreher, Yannik; Jahnke, Kevin; Schroeter, Martin; Goepfrich, Kerstin
      Nano Letters (2021), 21 (14), 5952-5957CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      A minimal synthetic cell should contain a substrate for information storage and have the capability to divide. Notable efforts were made to assemble functional synthetic cells from the bottom up, however often lacking the capability to reproduce. Here, we develop a mechanism to fully control reversible cargo loading and division of DNA-contg. giant unilamellar vesicles (GUVs) with light. We make use of the photosensitizer Chlorin e6 (Ce6) which self-assembles into lipid bilayers and leads to local lipid peroxidn. upon illumination. On the time scale of minutes, illumination induces the formation of transient pores, which we exploit for cargo encapsulation or controlled release. In combination with osmosis, complete division of two daughter GUVs can be triggered within seconds of illumination due to a spontaneous curvature increase. We ultimately demonstrate the division of a selected DNA-contg. GUV with full spatiotemporal control-proving the relevance of the division mechanism for bottom-up synthetic biol.
    26. 26
      Elani, Y. Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules. Sci. Rep. 2018, 8 (1), 4564,  DOI: 10.1038/s41598-018-22263-3
      26
      Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules
      Elani Yuval; Trantidou Tatiana; Wylie Douglas; Law Robert V; Ces Oscar; Elani Yuval; Wylie Douglas; Ces Oscar; Dekker Linda; Polizzi Karen
      Scientific reports (2018), 8 (1), 4564 ISSN:.
      There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.
    27. 27
      Duarte Campos, D. F.; Blaeser, A.; Buellesbach, K. Bioprinting Organotypic Hydrogels with Improved Mesenchymal Stem Cell Remodeling and Mineralization Properties for Bone Tissue Engineering. Adv. Healthc. Mater. 2016, 5 (11), 1336– 1345,  DOI: 10.1002/adhm.201501033
      There is no corresponding record for this reference.
    28. 28
      Duarte Campos, D. F.; Blaeser, A.; Weber, M. Three-dimensional printing of stem cell-laden hydrogels submerged in a hydrophobic high-density fluid. Biofabrication 2013, 5 (1), 015003,  DOI: 10.1088/1758-5082/5/1/015003
      28
      Three-dimensional printing of stem cell-laden hydrogels submerged in a hydrophobic high-density fluid
      Duarte Campos Daniela F; Blaeser Andreas; Weber Michael; Jakel Jorg; Neuss Sabine; Jahnen-Dechent Wilhelm; Fischer Horst
      Biofabrication (2013), 5 (1), 015003 ISSN:.
      Over the last decade, bioprinting technologies have begun providing important tissue engineering strategies for regenerative medicine and organ transplantation. The major drawback of past approaches has been poor or inadequate material-printing device and substrate combinations, as well as the relatively small size of the printed construct. Here, we hypothesise that cell-laden hydrogels can be printed when submerged in perfluorotributylamine (C(12)F(27)N), a hydrophobic high-density fluid, and that these cells placed within three-dimensional constructs remain viable allowing for cell proliferation and production of extracellular matrix. Human mesenchymal stem cells and MG-63 cells were encapsulated into agarose hydrogels, and subsequently printed in high aspect ratio in three dimensional structures that were supported in high density fluorocarbon. Three-dimensional structures with various shapes and sizes were manufactured and remained stable for more than six months. Live/dead and DAPI stainings showed viable cells 24 h after the printing process, as well as after 21 days in culture. Histological and immunohistochemical analyses after 14 and 21 days revealed viable cells with marked matrix production and signs of proliferation. The compressive strength values of the printed gels consequently increased during the two weeks in culture, revealing encouraging results for future applications in regenerative medicine.
    29. 29
      Betsch, M.; Cristian, C.; Liu, Y.; Blaeser, A.; Schöneberg, J.; Vogt, M.; Buhl, E. M.; Fischer, H.; Duarte Campos, D. F. Incorporating 4D into Bioprinting: Real-Time Magnetically Directed Collagen Fiber Alignment for Generating Complex Multilayered Tissues. Adv. Healthc. Mater. 2018, 7 (21), 1800894,  DOI: 10.1002/adhm.201800894
      There is no corresponding record for this reference.
    30. 30
      Duarte Campos, D. F.; Rohde, M.; Ross, M. Corneal bioprinting utilizing collagen-based bioinks and primary human keratocytes. J. Biomed Mater. Res. A 2019, 107 (9), 1945– 1953,  DOI: 10.1002/jbm.a.36702
      There is no corresponding record for this reference.
    31. 31
      Stein, H.; Spindler, S.; Bonakdar, N.; Wang, C.; Sandoghdar, V. Production of isolated giant unilamellar vesicles under high salt concentrations. Front. Physiol. 2017, 8, 63,  DOI: 10.3389/fphys.2017.00063
      31
      Production of Isolated Giant Unilamellar Vesicles under High Salt Concentrations
      Stein Hannah; Spindler Susann; Sandoghdar Vahid; Bonakdar Navid; Wang Chun
      Frontiers in physiology (2017), 8 (), 63 ISSN:1664-042X.
      The cell membrane forms a dynamic and complex barrier between the living cell and its environment. However, its in vivo studies are difficult because it consists of a high variety of lipids and proteins and is continuously reorganized by the cell. Therefore, membrane model systems with precisely controlled composition are used to investigate fundamental interactions of membrane components under well-defined conditions. Giant unilamellar vesicles (GUVs) offer a powerful model system for the cell membrane, but many previous studies have been performed in unphysiologically low ionic strength solutions which might lead to altered membrane properties, protein stability and lipid-protein interaction. In the present work, we give an overview of the existing methods for GUV production and present our efforts on forming single, free floating vesicles up to several tens of μm in diameter and at high yield in various buffer solutions with physiological ionic strength and pH.
    32. 32
      Tamba, Y.; Terashima, H.; Yamazaki, M. A membrane filtering method for the purification of giant unilamellar vesicles. Chem. Phys. Lipids 2011, 164 (5), 351– 358,  DOI: 10.1016/j.chemphyslip.2011.04.003
      There is no corresponding record for this reference.
    33. 33
      Banerjee, R. Liposomes: Applications in Medicine. J. Biomater Appl. 2001, 16 (1), 3– 21,  DOI: 10.1106/RA7U-1V9C-RV7C-8QXL
      There is no corresponding record for this reference.
    34. 34
      Walde, P.; Cosentino, K.; Engel, H.; Stano, P. Giant Vesicles: Preparations and Applications. ChemBiochem 2010, 11 (7), 848– 865,  DOI: 10.1002/cbic.201000010
      34
      Giant Vesicles: preparations and Applications
      Walde, Peter; Cosentino, Katia; Engel, Helen; Stano, Pasquale
      ChemBioChem (2010), 11 (7), 848-865CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. There is considerable interest in prepg. cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biol. cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chem. compds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible prepn. of giant vesicles are available. The key concepts of currently known methods for prepg. giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.
    35. 35
      Lee, W.; Debasitis, J.; Lee, V. Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication. Biomaterials 2009, 30 (8), 1587– 1595,  DOI: 10.1016/j.biomaterials.2008.12.009
      35
      Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication
      Lee, Wonhye; Debasitis, Jason Cushing; Lee, Vivian Kim; Lee, Jong-Hwan; Fischer, Krisztina; Edminster, Karl; Park, Je-Kyun; Yoo, Seung-Schik
      Biomaterials (2009), 30 (8), 1587-1595CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
      The authors present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-contg. collagen was crosslinked by coating the layer with nebulized aq. sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in 2 distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell-hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was obsd. on both planar and non-planar surfaces. The authors' results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing.
    36. 36
      Blaeser, A.; Duarte Campos, D. F.; Puster, U.; Richtering, W.; Stevens, M. M.; Fischer, H. Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity. Adv. Healthc. Mater. 2016, 5 (3), 326– 333,  DOI: 10.1002/adhm.201500677
      There is no corresponding record for this reference.
    37. 37
      Lucas, L.; Aravind, A.; Emma, P.; Marquette, M.; Courtial, C. Rheology, simulation and data analysis toward bioprinting cell viability awareness. Bioprinting 2021, 21, e00119  DOI: 10.1016/j.bprint.2020.e00119
      There is no corresponding record for this reference.
    38. 38
      Bhatia, T.; Husen, P.; Brewer, J. Preparing giant unilamellar vesicles (GUVs) of complex lipid mixtures on demand: Mixing small unilamellar vesicles of compositionally heterogeneous mixtures. Biochim. Biophys. Acta, Biomembr. 2015, 1848 (12), 3175– 3180,  DOI: 10.1016/j.bbamem.2015.09.020
      38
      Preparing giant unilamellar vesicles (GUVs) of complex lipid mixtures on demand: Mixing small unilamellar vesicles of compositionally heterogeneous mixtures
      Bhatia, Tripta; Husen, Peter; Brewer, Jonathan; Bagatolli, Luis A.; Hansen, Per L.; Ipsen, John H.; Mouritsen, Ole G.
      Biochimica et Biophysica Acta, Biomembranes (2015), 1848 (12), 3175-3180CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      Giant unilamellar vesicles (GUVs) are simple model membrane systems of cell-size, which are instrumental to study the function of more complex biol. membranes involving heterogeneities in lipid compn., shape, mech. properties, and chem. properties. The authors have devised a method that makes it possible to prep. a uniform sample of ternary GUVs of a prescribed compn. and heterogeneity by mixing different populations of small unilamellar vesicles (SUVs). The validity of the protocol has been demonstrated by applying it to ternary lipid mixt. of DOPC, DPPC, and cholesterol by mixing small unilamellar vesicles (SUVs) of two different populations and with different lipid compns. The compositional homogeneity among GUVs resulting from SUV mixing is quantified by measuring the area fraction of the liq. ordered-liq. disordered phases in giant vesicles and is comparable to that in GUVs of the prescribed compn. produced from hydration of dried lipids mixed in org. solvent. The authors' method opens up the possibility to quickly increase and manipulate the complexity of GUV membranes in a controlled manner at physiol. buffer and temp. conditions. The new protocol will permit quant. biophys. studies of a whole new class of well-defined model membrane systems of a complexity that resembles biol. membranes with rafts.
    39. 39
      Lira, R. B.; Dimova, R. Fusion assays for model membranes: a critical review. Adv. Biomembr. Lipid Self-Assem. 2019, 30, 229– 270,  DOI: 10.1016/bs.abl.2019.09.003
      There is no corresponding record for this reference.
    40. 40
      Banquy, X.; Kristiansen, K.; Lee, D. W.; Israelachvili, J. N. Adhesion and hemifusion of cytoplasmic myelin lipid membranes are highly dependent on the lipid composition. Biochim. Biophys. Acta, Biomembr. 2012, 1818 (3), 402– 410,  DOI: 10.1016/j.bbamem.2011.10.015
      40
      Adhesion and hemifusion of cytoplasmic myelin lipid membranes are highly dependent on the lipid composition
      Banquy, Xavier; Kristiansen, Kai; Lee, Dong Woog; Israelachvili, Jacob N.
      Biochimica et Biophysica Acta, Biomembranes (2012), 1818 (3), 402-410CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      We report the effects of calcium ions on the adhesion and hemifusion mechanisms of model supported myelin lipid bilayer membranes of differing lipid compn. As in our previous studies the lipid compns. used mimic "healthy" and "diseased-like" (exptl. autoimmune encephalomyelitis, EAE) membranes. Our results show that the interaction forces as a function of membrane sepn. distance are well described by a generic model that also (and in particular) includes the hydrophobic interaction arising from the hydrophobically exposed (interior) parts of the bilayers. The model is able to capture the mech. instability that triggers the onset of the hemifusion event, and highlights the primary role of the hydrophobic interaction in membrane fusion. The effects of lipid compn. on the fusion mechanism, and the adhesion forces between myelin lipid bilayers, can be summarized as follows: in calcium-free buffer, healthy membranes do not present any signs of adhesion or hemifusion, while diseased membranes hemifuse easily. Addn. of 2 mM calcium favors adhesion and hemifusion of the membranes independently of their compn., but the mechanisms involved in the two processes were different: healthy bilayers systematically presented stronger adhesion forces and lower energy barriers to fusion compared to diseased bilayers. These results are of particular relevance for understanding lesion development (demyelination, swelling, vacuolization and/or vesiculation) in myelin assocd. diseases such as multiple sclerosis and its relationship to lipid domain formation in myelin membranes.
    41. 41
      Kowalska, M.; Broniatowski, M.; Płachta, Ł; Wydro, P.; Wydro, P. The effect of the polyethylene glycol chain length of a lipopolymer (DSPE-PEGn) on the properties of DPPC monolayers and bilayers. J. Mol. Liq. 2021, 335, 116529,  DOI: 10.1016/j.molliq.2021.116529
      41
      The effect of the polyethylene glycol chain length of a lipopolymer (DSPE-PEGn) on the properties of DPPC monolayers and bilayers
      Kowalska, Magdalena; Broniatowski, Marcin; Mach, Marzena; Plachta, Lukasz; Wydro, Pawel
      Journal of Molecular Liquids (2021), 335 (), 116529CODEN: JMLIDT; ISSN:0167-7322. (Elsevier B.V.)
      Due to the growing importance of controlled drug delivery systems (DDS), the main task of nanotechnol. is to develop stable, effective and non-toxic nanocarriers in which the drug can be encapsulated and delivered to a specific diseased site in the patient's body. Currently, one of the most popular ways to improve the pharmacokinetic and physicochem. properties of liposomes is introducing into their structure poly(ethylene glycol) chains conjugated with 1,2-disteroil-sn-glycero-3-phosphoethanolamine (DSPE) mols. Because the research so far does not give an unequivocal answer which length of PEG chains is more beneficial for liposomes properties, the aim of this work was to investigate the influence of this parameter (DSPE-PEG350, DSPE-PEG750 and DSPE-PEG2000) on model DPPC membrane. The studies were performed on monolayer and bilayer systems and were related to the surface pressure measurements, Brewster angle microscopy expts., Grazing Incidence X-ray Diffraction studies, dynamic light scattering and zeta potential measurements and the expts. with the calcein release and steady-state fluorescence anisotropy of DPH. The obtained results proved that the mol. organization of the DPPC membrane strongly depends on the length of poly(ethylene glycol) chains conjugated with DSPE. Moreover, the addn. of different lengths of polymer chains changes the properties of formulated liposomes, esp. their stability, permeability, size and surface charge.
    42. 42
      Allen, T. M.; Hansen, C.; Martin, F.; Redemann, C.; Yau-Young, A. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim. Biophys. Acta, Biomembr. 1991, 1066, 29– 36,  DOI: 10.1016/0005-2736(91)90246-5
      42
      Liposomes containing synthetic lipid derivatives of polyethylene glycol show prolonged circulation half-lives in vivo
      Allen, T. M.; Hansen, C.; Martin, F.; Redemann, C.; Yau-Young, A.
      Biochimica et Biophysica Acta, Biomembranes (1991), 1066 (1), 29-36CODEN: BBBMBS; ISSN:0005-2736.
      Novel synthetic lipid derivs. of polyethylene glycol (PEG) were synthesized and tested for their ability to decrease uptake of liposomes into the mononuclear phagocyte system (MPS, reticuloendothelial system) in mice and to prolong circulation half-lives of liposomes. A carbamate deriv. of PEG-1900 with distearoylphosphatidylethanolamine (PEG-DSPE) had the greatest ability to decrease MPS uptake of liposomes, at optimum concns. of 5-7 mol% in liposomes composed of sphingomyelin/egg phosphatidylcholine/cholesterol (SM/PC/Chol, 1:1:1, molar ratio). Results obtained with this compd. were equiv. to results previously obtained with 10 mol% monosialoganglioside GM1 in liposomes of similar compns. (Allen, T. M. and Chonn, A., 1987). Non-derivatized Me PEG or PEG-stearic acid (PEG-SA) were incapable of decreasing MPS uptake of liposomes. PEG-Chol and PEG-dipalmitoylglycerol (PEG-DPG) were intermediate in their effects on MPS uptake. Altering liposome size for liposomes contg. PEG-DSPE resulted in only minor changes in blood levels of liposomes. Half-lives of 0.1 μm liposomes of SM/PC/Chol/PEG-DSPE (1:1:1:0.2, molar ratio) in circulation was in excess of 20 h following either i.v. or i.p. injection. Liver plus spleen liposome levels for these liposomes was below 15% of injected label at 48 h following i.v. liposome injection and below 10% following i.p. injection. The major site of liposome uptake was in carcass tissues, with over 50% of label remaining in vivo at 48 h post-injections, either i.v. or i.p., in the carcass.
    43. 43
      Allen, C.; Dos Santos, N.; Gallagher, R.; Chiu, G. N. C.; Shu, Y.; Li, W. M.; Johnstone, S. A.; Janoff, A. S.; Mayer, L. D.; Webb, M. S.; Bally, M. B. Controlling the Physical Behavior and Biological Performance of Liposome Formulations through Use of Surface Grafted Poly(ethylene Glycol). Biosci. Rep. 2002, 22, 225– 250,  DOI: 10.1023/A:1020186505848
      43
      Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol)
      Allen, C.; Dos Santos, N.; Gallagher, R.; Chiu, G. N. C.; Shu, Y.; Li, W. M.; Johnstone, S. A.; Janoff, A. S.; Mayer, L. D.; Webb, M. S.; Bally, M. B.
      Bioscience Reports (2002), 22 (2), 225-250CODEN: BRPTDT; ISSN:0144-8463. (Kluwer Academic/Plenum Publishers)
      A review. The presence of poly(ethylene glycol) (PEG) at the surface of a liposomal carrier has been clearly shown to extend the circulation lifetime of the vehicle. To this point, the extended circulation lifetime that the polymer affords has been attributed to the redn. or prevention of protein adsorption. However, there is little evidence that the presence of PEG at the surface of a vehicle actually reduces total serum protein binding. In this review we examine all aspects of PEG in order to gain a better understanding of how the polymer fulfills its biol. role. The phys. and chem. properties of the polymer are explored and compared to properties of other hydrophilic polymers. An evidence based assessment of several in vitro protein binding studies as well as in vivo pharmacokinetics studies involving PEG is included. The ability of PEG to prevent the self-aggregation of liposomes is considered as a possible means by which it extends circulation longevity. Also, a "dysopsonization" phenomenon where PEG actually promotes binding of certain proteins that then mask the vehicle is discussed.
    44. 44
      Kenworthy, A. K.; Simon, S. A.; McIntosh, T. J. Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 1995, 68 (5), 1903– 1920,  DOI: 10.1016/S0006-3495(95)80368-1
      There is no corresponding record for this reference.
    45. 45
      Staufer, O.; Antona, S.; Zhang, D. Microfluidic production and characterization of biofunctionalized giant unilamellar vesicles for targeted intracellular cargo delivery. Biomaterials 2021, 264, 120203,  DOI: 10.1016/j.biomaterials.2020.120203
      45
      Microfluidic production and characterization of biofunctionalized giant unilamellar vesicles for targeted intracellular cargo delivery
      Staufer, Oskar; Antona, Silvia; Zhang, Dennis; Csatari, Julia; Schroeter, Martin; Janiesch, Jan-Willi; Fabritz, Sebastian; Berger, Imre; Platzman, Ilia; Spatz, Joachim P.
      Biomaterials (2021), 264 (), 120203CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
      Lipid-based vesicles have found widespread applications in the life sciences, allowing for fundamental insights into membrane-based processes in cell biol. and as carrier systems for drug delivery purposes. So far, mostly small unilamellar vesicles (SUVs) with diams. of ∼100 nm have been applied as carrier systems for biomedical applications. Despite this progress, several systematic limitations have arisen due to SUV dimensions, e.g., the size and total amt. of applicable cargo is limited. Giant unilamellar vesicles (GUVs) might offer a pragmatic alternative for efficient cargo delivery. However, due to the lack of reliable high-throughput prodn. technologies for GUV-carrier systems, only little is known about their interaction with cells. Here we present a microfluidic-based mech. droplet-splitting pipeline for the prodn. of carrier-GUVs with diams. of ∼2 μm. The technol. developed allows for highly efficient cargo loading and unprecedented control over the biol. and physicochem. properties of GUV membranes. By generating differently charged (between -31 and + 28 mV), bioligand-conjugated (e.g. with E-cadherin, NrCam and antibodies) and PEG-conjugated GUVs, we performed a detailed investigation of attractive and repulsive GUV-cell interactions. Fine-tuning of these interactions allowed for targeted cellular GUV delivery. Moreover, we evaluated strategies for intracellular GUV cargo release by lysosomal escape mediated by the pH sensitive lipid DOBAQ, enabling cytoplasmic transmission. The presented GUV delivery technol. and the systematic characterization of assocd. GUV-cell interactions could provide a means for more efficient drug administration and will pave the way for hitherto impossible approaches towards a targeted delivery of advanced cargo such as microparticles, viruses or macromol. DNA-robots.
    46. 46
      Mahendra, A.; James, H. P.; Jadhav, S. PEG-grafted phospholipids in vesicles: Effect of PEG chain length and concentration on mechanical properties. Chem. Phys. Lipids 2019, 218, 47– 56,  DOI: 10.1016/j.chemphyslip.2018.12.001
      46
      PEG-grafted phospholipids in vesicles: Effect of PEG chain length and concentration on mechanical properties
      Mahendra, Amit; James, Honey Priya; Jadhav, Sameer
      Chemistry and Physics of Lipids (2019), 218 (), 47-56CODEN: CPLIA4; ISSN:0009-3084. (Elsevier Ireland Ltd.)
      Incorporation of low mol. wt. poly-ethylene glycol (PEG) - grafted phospholipids in vesicle bilayers is known to increase the circulation time of liposomal drug delivery vehicles. Mech. properties of giant unilamellar DPPC vesicles contg. varying concns. of DSPE-PEG (PEG MW: 550, 1000 and 2000) were measured by micropipette aspiration assay or osmotic swelling. While the area compressibility modulus did not change significantly, the bending modulus and water permeability of the bilayer was found to increase with increasing mole fraction of DSPE-PEG. This increase was more pronounced for higher mol. wt. PEG. The measured bending modulus agreed with that predicted by scaling theory only at low mole fractions of DSPE-PEG. The water permeability was also measured as a function of the increase in area per lipid (due to steric repulsion between PEG chains), and for the same area per lipid, the PEG chain with MW 550 provided a greater resistance to water transport across the vesicle membrane compared to PEG 1000 and 2000. Lysis tension of the membrane, detd. by osmotic lysis method at different loading rates showed a decrease in membrane strength on inclusion of the polymer lipid. These results suggest that liposome lifetime in the circulation and the rate of drug delivery are affected by the mol. wt. and concn. of PEG in the bilayer.
    47. 47
      Papaioannou, T. G.; Karatzis, E. N.; Vavuranakis, M.; Lekakis, J. P.; Stefanadis, C. Assessment of vascular wall shear stress and implications for atherosclerotic disease. Int. J. Cardiol. 2006, 113 (1), 12– 18,  DOI: 10.1016/j.ijcard.2006.03.035
      There is no corresponding record for this reference.
    48. 48
      Koutsiaris, A. G.; Tachmitzi, S.; Batis, N.; Kotoula, M. G.; Karabatsas, C. H.; Tsironi, E.; Chatzoulis, D. Z. Volume flow and wall shear stress quantification in the human conjunctival capillaries and post-capillary venules in vivo. Biorheology 2007, 44 (5–6), 375– 386
      48
      Volume flow and wall shear stress quantification in the human conjunctival capillaries and post-capillary venules in vivo
      Koutsiaris Aristotle G; Tachmitzi Sophia V; Batis Nick; Kotoula Maria G; Karabatsas Constantinos H; Tsironi Evagelia; Chatzoulis Dimitrios Z
      Biorheology (2007), 44 (5-6), 375-86 ISSN:0006-355X.
      Understanding the mathematical relationships of volume blood flow and wall shear stress with respect to microvessel diameter is necessary for the study of vascular design. Here, for the first time, volume flow and wall shear stress were quantified from axial red blood cell velocity measurements in 104 conjunctival microvessels of 17 normal human volunteers. Measurements were taken with a slit lamp based imaging system from the post capillary side of the bulbar conjunctiva in microvessel diameters ranging from 4 to 24 micrometers. The variation of the velocity profile with diameter was taken into account by using a profile factor function. Volume flow ranged from 5 to 462 pl/s with a mean value of 102 pl/s and gave a second power law best fitting line (r=0.97) deviating significantly from the third power law relation with diameter. The estimated wall shear stress declined hyperbolically (r=0.93) from a maximum of 9.55 N/m(2) at the smallest capillaries, down to a minimum of 0.28 N/m(2) at the higher diameter post capillary venules. The mean wall shear stress value for all microvessels was 1.54 N/m(2).
    49. 49
      Lorent, J. H.; Levental, K.; Ganesan, L. Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape. Nat. Chem. Biol. 2020, 16 (6), 644– 652,  DOI: 10.1038/s41589-020-0529-6
      49
      Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape
      Lorent, J. H.; Levental, K. R.; Ganesan, L.; Rivera-Longsworth, G.; Sezgin, E.; Doktorova, M. D.; Lyman, E.; Levental, I.
      Nature Chemical Biology (2020), 16 (6), 644-652CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      A fundamental feature of cellular plasma membranes (PMs) is an asym. lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compns. of individual PM leaflets nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophys. properties of both monolayers in living mammalian PMs. Phospholipid unsatn. is dramatically asym., with the cytoplasmic leaflet being approx. twofold more unsatd. than the exoplasmic leaflet. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophys. asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in the asym. structures of protein transmembrane domains. These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.
    50. 50
      Doktorova, M.; LeVine, M. V.; Khelashvili, G.; Weinstein, H. A New Computational Method for Membrane Compressibility: Bilayer Mechanical Thickness Revisited. Biophys. J. 2019, 116 (3), 487– 502,  DOI: 10.1016/j.bpj.2018.12.016
      50
      A New Computational Method for Membrane Compressibility: Bilayer Mechanical Thickness Revisited
      Doktorova, Milka; Le Vine, Michael V.; Khelashvili, George; Weinstein, Harel
      Biophysical Journal (2019), 116 (3), 487-502CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, phys. models of bilayer deformation have utilized mech. consts. such as the moduli for bending rigidity (κC) and area compressibility (KA). However, the use of these models to quantify the energetics of membrane deformation assocd. with protein-membrane interactions, and the membrane response to stress is often hampered by the shortage of exptl. data suitable for the estn. of the mech. consts. of various lipid mixts. Although computational tools such as mol. dynamics simulations can provide alternative means to est. KA values, current approaches suffer significant tech. limitations. Here, we present a novel, to our knowledge, computational framework that allows for a direct estn. of KA values for individual bilayer leaflets. The theory is based on the concept of elasticity and derives KA from real-space anal. of local thickness fluctuations sampled in mol. dynamics simulations. We explore and validate the model on a large set of single and multicomponent bilayers of different lipid compns. and sizes, simulated at different temps. The calcd. bilayer compressibility moduli agree with values estd. previously from expts. and those obtained from a std. computational method based on a series of constrained tension simulations. We further validate our framework in a comparison with an existing polymer brush model and confirm the polymer brush model's predicted linear relationship with proportionality coeff. of 24, using elastic parameters calcd. from the simulation trajectories. The robustness of the results that emerge from the method allows us to revisit the origins of the bilayer mech. (compressible) thickness and in particular its dependence on acyl-chain unsatn. and the presence of cholesterol.
    51. 51
      Karal, M. A. S.; Mokta, N.; Levadny, V. Effects of cholesterol on the size distribution and bending modulus of lipid vesicles. PLoS One 2022, 17 (1), e0263119  DOI: 10.1371/journal.pone.0263119
      51
      Effects of cholesterol on the size distribution and bending modulus of lipid vesicles
      Karal, Mohammad Abu Sayem; Mokta, Nadia Akter; Levadny, Victor; Belaya, Marina; Ahmed, Marzuk; Ahamed, Md. Kabir; Ahammed, Shareef
      PLoS One (2022), 17 (1), e0263119CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      The influence of cholesterol fraction in the membranes of giant unilamellar vesicles (GUVs) on their size distributions and bending moduli has been investigated. The membranes of GUVs were synthesized by a mixt. of two elements: elec. neutral lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol and also a mixt. of three elements: elec. charged lipid 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), DOPC and cholesterol. The size distributions of GUVs have been presented by a set of histograms. The classical lognormal distribution is well fitted to the histograms, from where the av. size of vesicle is obtained. The increase of cholesterol content in the membranes of GUVs increases the av. size of vesicles in the population. Using the framework of Helmholtz free energy of the system, the theory developed by us is extended to explain the exptl. results. The theory dets. the influence of cholesterol on the bending modulus of membranes from the fitting of the proper histograms. The increase of cholesterol in GUVs increases both the av. size of vesicles in population and the bending modulus of membranes.
    52. 52
      Lira, R. B.; Steinkühler, J.; Knorr, R. L.; Dimova, R.; Riske, K. A. Posing for a picture: Vesicle immobilization in agarose gel. Sci. Rep. 2016, 6, 25254,  DOI: 10.1038/srep25254
      52
      Posing for a picture: vesicle immobilization in agarose gel
      Lira, Rafael B.; Steinkuhler, Jan; Knorr, Roland L.; Dimova, Rumiana; Riske, Karin A.
      Scientific Reports (2016), 6 (), 25254CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Taking a photo typically requires the object of interest to stand still. In science, imaging is potentiated by optical and electron microscopy. However, living and soft matter are not still. Thus, biol. prepns. for microscopy usually include a fixation step. Similarly, immobilization strategies are required for or substantially facilitate imaging of cells or lipid vesicles, and even more so for acquiring high-quality data via fluorescence-based techniques. Here, we describe a simple yet efficient method to immobilize objects such as lipid vesicles with sizes between 0.1 and 100 μm using agarose gel. We show that while large and giant unilamellar vesicles (LUVs and GUVs) can be caged in the pockets of the gel meshwork, small mols., proteins and micelles remain free to diffuse through the gel and interact with membranes as in agarose-free solns., and complex biochem. reactions involving several proteins can proceed in the gel. At the same time, immobilization in agarose has no adverse effect on the GUV size and stability. By applying techniques such as FRAP and FCS, we show that the lateral diffusion of lipids is not affected by the gel. Finally, our immobilization strategy allows capturing high-resoln. 3D images of GUVs.
    53. 53
      Sandström, M. C.; Johansson, E.; Edwards, K. Structure of mixed micelles formed in PEG-lipid/lipid dispersions. Langmuir 2007, 23 (8), 4192– 4198,  DOI: 10.1021/la063501s
      53
      Structure of mixed micelles formed in PEG-lipid/lipid dispersions
      Sandstrom Maria C; Johansson Emma; Edwards Katarina
      Langmuir : the ACS journal of surfaces and colloids (2007), 23 (8), 4192-8 ISSN:0743-7463.
      Polyethylene glycol (PEG)-conjugated lipids are commonly employed for steric stabilization of liposomes. When added in high concentrations PEG-lipids induce formation of mixed micelles, and depending on the lipid composition of the sample, these may adapt either a discoidal or a long threadlike shape. The factors governing the type of micellar aggregate formed have so far not been investigated in detail. In this study we have systematically varied the lipid composition in lipid/PEG-lipid mixtures and characterized the aggregate structure by means of cryo-transmission electron microscopy (cryo-TEM). The effects caused by adding sterols, phosphatidylethanolamines, and phospholipids with saturated acyl chains to egg phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000 (EPC/DSPE-PEG2000) mixtures with a fixed amount (25 mol %) of DSPE-PEG2000 was studied. Further, the aggregate structure in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPC/DMPE-PEG2000) samples above and below the gel to liquid crystalline phase transition temperature (TC) was investigated. Our results revealed that lipid components, as well as environmental conditions, that reduce the lipid spontaneous curvature and increase the monolayer bending modulus tend to promote formation of discoidal micelles. At temperatures below the gel-to-liquid crystalline phase transition temperature reduced lipid/PEG-lipid miscibility, furthermore, likely contribute to the observed formation of discoidal rather than threadlike micelles.
    54. 54
      Garbuzenko, O.; Barenholz, Y.; Priev, A. Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer. Chem. Phys. Lipids 2005, 135 (2), 117– 129,  DOI: 10.1016/j.chemphyslip.2005.02.003
      54
      Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer
      Garbuzenko, Olga; Barenholz, Yechezkel; Priev, Aba
      Chemistry and Physics of Lipids (2005), 135 (2), 117-129CODEN: CPLIA4; ISSN:0009-3084. (Elsevier B.V.)
      The aim of this study was to elucidate the effect of various mole percentages (0-25 mol%) of 2000 Da polyethylene glycol-disteroylphosphoethanolamine (PEG-DSPE) in the presence or absence of 40 mol% cholesterol and the effect of degree of satn. of phosphatidylcholine (PC) on the size and the lipid bilayer packing of large unilamellar vesicles (LUV). Egg PC (EPC, unsatd.) LUV and fully hydrogenated soy PC (HSPC, satd.) LUV partial sp. vol., specific compressibility, size, and packing parameter (PP) of lipids were characterized by measurements of d., ultrasonic velocity, specific turbidity, and dynamic light scattering. Liposome size and specific turbidity decreased with increase in temp. and PEG-DSPE mol%, except at 7 ± 2 mol%. At this PEG-DSPE mol%, an anomalous peak in liposome size of 15 ± 5 nm was obsd. We attribute this effect mainly to the change in the spatial structure of the PEG-DSPE mol., depending on whether the grafted PEG is in the mushroom or brush configuration. In the mushroom regime, i.e., when the grafted PEG is up to 4 mol% in LUV, the PEG moiety did not affect the additive PP of the lipids in the bilayer, and the PP value of PEG-DSPE is 1.044; while in the brush regime, i.e., when the grafted PEG is higher than 4 mol%, the PP of PEG-DSPE decreases exponentially, reaching the value of 0.487 at 30 mol% of grafted lipopolymer. The specific compressibility and additive PP values for the mixt. of matrix lipid (EPC or HSPC), cholesterol, and PEG-DSPE for all liposome compns. investigated reached their max. at 7 ± 2 mol% PEG-DSPE, the concn. of PEG-DSPE at which the highest biol. stability of the LUV is achieved.
    55. 55
      Holthuis, J. C. M. Regulating membrane curvature. In Regulatory mechanisms of intracellular membrane transport Springer: 2004, 39 64.  DOI: 10.1007/b98566 .
      There is no corresponding record for this reference.
    56. 56
      Tirosh, O.; Barenholz, Y.; Katzhendler, J.; Priev, A. Hydration of polyethylene glycol-grafted liposomes. Biophys. J. 1998, 74 (3), 1371– 1379,  DOI: 10.1016/S0006-3495(98)77849-X
      56
      Hydration of polyethylene glycol-grafted liposomes
      Tirosh, Oren; Barenholz, Yechezkel; Katzhendler, Jehoshua; Priev, Aba
      Biophysical Journal (1998), 74 (3), 1371-1379CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      This study aimed to characterize the effect of polyethylene glycol of 2000 mol. wt. (PEG2000) attached to a dialkylphosphatidic acid (dihexadecylphosphatidyl (DHP)-PEG2000) on the hydration and thermodn. stability of lipid assemblies. Differential scanning calorimetry, densitometry, and ultrasound velocity and absorption measurements were used for thermodn. and hydrational characterization. Using a differential scanning calorimetry technique we showed that each mol. of PEG2000 binds 136±4 mols. of water. For PEG2000 covalently attached to the lipid mols. organized in micelles, the water binding increases to 210±6 water mols. This demonstrates that the two different structural configurations of the PEG2000, a random coil in the case of the free PEG and a brush in the case of DHP-PEG2000 micelles, differ in their hydration level. Ultrasound absorption changes in liposomes reflect mainly the heterophase fluctuations and packing defects in the lipid bilayer. The PEG-induced excess ultrasound absorption of the lipid bilayer at 7.7 MHz for PEG-lipid concns. over 5 mol % indicates the increase in the relaxation time of the headgroup rotation due to PEG-PEG interactions. The adiabatic compressibility (calcd. from ultrasound velocity and d.) of the lipid bilayer of the liposome increases monotonically with PEG-lipid concn. up to ∼7 mol %, reflecting release of water from the lipid headgroup region. Elimination of this water, induced by grafted PEG, leads to a decrease in bilayer defects and enhanced lateral packing of the phospholipid acyl chains. We assume that the dehydration of the lipid headgroup region in conjunction with the increase of the hydration of the outer layer by grafting PEG in brush configuration are responsible for increasing thermodn. stability of the liposomes at 5-7 mol % of PEG-lipid. At higher PEG-lipid concns., compressibility and partial vol. of the lipid phase of the samples decrease. This reflects the increase in hydration of the lipid headgroup region (up to five addnl. water mols. per lipid mol. for 12 mol % PEG-lipid) and the weakening of the bilayer packing due to the lateral repulsion of PEG chains.
    57. 57
      Pepelanova, I.; Kruppa, K.; Scheper, T.; Lavrentieva, A. Gelatin-methacryloyl (GelMA) hydrogels with defined degree of functionalization as a versatile toolkit for 3D cell culture and extrusion bioprinting. Bioengineering 2018, 5 (3), 55,  DOI: 10.3390/bioengineering5030055
      57
      Gelatin-methacryloyl (GelMA) hydrogels with defined degree of functionalization as a versatile toolkit for 3D cell culture and extrusion bioprinting
      Pepelanova, Iliyana; Kruppa, Katharina; Scheper, Thomas; Lavrentieva, Antonina
      Bioengineering (2018), 5 (3), 55CODEN: BIOEBG; ISSN:2306-5354. (MDPI AG)
      Gelatin-methacryloyl (GelMA) is a semi-synthetic hydrogel which consists of gelatin derivatized with methacrylamide and methacrylate groups. These hydrogels provide cells with an optimal biol. environment (e.g., RGD motifs for adhesion) and can be quickly photo-crosslinked, which provides shape fidelity and stability at physiol. temp. In the present work, we demonstrated how GelMA hydrogels can be synthesized with a specific degree of functionalization (DoF) and adjusted to the intended application as a three-dimensional (3D) cell culture platform. The focus of this work lays on producing hydrogel scaffolds which provide a cell promoting microenvironment for human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) and are conductive to their adhesion, spreading, and proliferation. The control of mech. GelMA properties by variation of concn., DoF, and UV polymn. conditions is described. Moreover, hAD-MSC cell viability and morphol. in GelMA of different stiffness was evaluated and compared. Polymd. hydrogels with and without cells could be digested in order to release encapsulated cells without loss of viability. We also demonstrated how hydrogel viscosity can be increased by the use of biocompatible additives, in order to enable the extrusion bioprinting of these materials. Taken together, we demonstrated how GelMA hydrogels can be used as a versatile tool for 3D cell cultivation.
    58. 58
      Guo, L.; Har, J. Y.; Sankaran, J.; Hong, Y.; Kannan, B.; Wohland, T. Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study. ChemPhyschem 2008, 9 (5), 721– 728,  DOI: 10.1002/cphc.200700611
      58
      Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study
      Guo, Lin; Har, Jia Yi; Sankaran, Jagadish; Hong, Yimian; Kannan, Balakrishnan; Wohland, Thorsten
      ChemPhysChem (2008), 9 (5), 721-728CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Mol. diffusion in biol. membranes is a detg. factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring mol. diffusion in artificial and biol. membranes at very different concn. ranges and spatial resolns. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) can det. abs. diffusion coeffs. at high (> 100 μm-2) and very low surface concns. (single-mol. level), resp. Fluorescence correlation spectroscopy (FCS), on the other hand, is well-suited for the intermediate concn. range of about 0.1-100 μm-2. However, FCS in general requires calibration with a std. dye of known diffusion coeff., and yields only relative measurements with respect to the calibration. A variant of FCS, z-scan FCS, is calibration-free for membrane measurements, but requires several expts. at different well-controlled focusing positions. A recently established FCS method, electron-multiplying charge-coupled-device-based total internal reflection FCS (TIR-FCS), referred to here as imaging TIR-FCS (ITIR-FCS), is also independent of calibration stds., but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR-FCS by measuring the lateral diffusion coeffs. in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.
    59. 59
      Pincet, F.; Adrien, V.; Yang, R. FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments. PLoS One 2016, 11 (7), e0158457  DOI: 10.1371/journal.pone.0158457
      59
      FRAP to characterize molecular diffusion and interaction in various membrane environments
      Pincet, Frederic; Adrien, Vladimir; Yang, Rong; Delacotte, Jerome; Rothman, James E.; Urbach, Wladimir; Tareste, David
      PLoS One (2016), 11 (7), e0158457/1-e0158457/19CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Fluorescence recovery after photobleaching (FRAP) is a std. method used to study the dynamics of lipids and proteins in artificial and cellular membrane systems. The advent of confocal microscopy two decades ago has made quant. FRAP easily available to most labs. Usually, a single bleaching pattern/area is used and the corresponding recovery time is assumed to directly provide a diffusion coeff., although this is only true in the case of unrestricted Brownian motion. Here, we propose some general guidelines to perform FRAP expts. under a confocal microscope with different bleaching patterns and area, allowing the experimentalist to establish whether the mols. undergo Brownian motion (free diffusion) or whether they have restricted or directed movements. Using in silico simulations of FRAP measurements, we further indicate the data acquisition criteria that have to be verified in order to obtain accurate values for the diffusion coeff. and to be able to distinguish between different diffusive species. Using this approach, we compare the behavior of lipids in three different membrane platforms (supported lipid bilayers, giant liposomes and sponge phases), and we demonstrate that FRAP measurements are consistent with results obtained using other techniques such as Fluorescence Correlation Spectroscopy (FCS) or Single Particle Tracking (SPT). Finally, we apply this method to show that the presence of the synaptic protein Munc18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A.
    60. 60
      Hernandez Bücher, J. E.; Staufer, O.; Ostertag, L.; Mersdorf, U.; Platzman, I.; Spatz, J. P. Bottom-up assembly of target-specific cytotoxic synthetic cells. Biomaterials 2022, 285, 121522,  DOI: 10.1016/j.biomaterials.2022.121522
      There is no corresponding record for this reference.
    61. 61
      Bour, A.; Kruglik, S. G.; Chabanon, M.; Rangamani, P.; Puff, N.; Bonneau, S. Lipid Unsaturation Properties Govern the Sensitivity of Membranes to Photoinduced Oxidative Stress. Biophys. J. 2019, 116 (5), 910– 920,  DOI: 10.1016/j.bpj.2019.01.033
      There is no corresponding record for this reference.
    62. 62
      Heuvingh, J.; Bonneau, S. Asymmetric oxidation of giant vesicles triggers curvature-associated shape transition and permeabilization. Biophys. J. 2009, 97 (11), 2904– 2912,  DOI: 10.1016/j.bpj.2009.08.056
      62
      Asymmetric oxidation of giant vesicles triggers curvature-associated shape transition and permeabilization
      Heuvingh, Julien; Bonneau, Stephanie
      Biophysical Journal (2009), 97 (11), 2904-2912CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Oxidn. of unsatd. lipids is a fundamental process involved in cell bioenergetics as in well as in cell death. Using giant unilamellar vesicles and a chlorin photosensitizer, we asym. oxidized the outer or inner monolayers of lipid membranes. We obsd. different shape transitions such as oblate to prolate and budding, which are typical of membrane curvature modifications. The asymmetry of the shape transitions is in accordance with a lowered effective spontaneous curvature of the leaflet being targeted. We interpret this effect as a decrease in the preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidn. (cleaved-lipids). Permeabilization of giant vesicles by light-induced oxidn. is obsd. after a lag and is characterized in relation with the photosensitizer concn. We interpret permeabilization as the opening of pore above a crit. membrane tension, resulting from the budding of vesicles. The evolution of photosensitized giant vesicle lysis tension was measured and yields an estn. of the effective spontaneous curvature at lysis. Addnl. photo-oxidn. was shown to be fusogenic.
    63. 63
      Hermann, E.; Bleicken, S.; Subburaj, Y.; García-Sáez, A. J. Automated analysis of giant unilamellar vesicles using circular Hough transformation. Bioinformatics 2014, 30 (12), 1747– 1754,  DOI: 10.1093/bioinformatics/btu102
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      Jahnke, K.; Weiss, M.; Frey, C.; Antona, S.; Janiesch, J.-W.; Platzman, I.; Göpfrich, K.; Spatz, J. P. Programmable Functionalization of Surfactant-Stabilized Microfluidic Droplets via DNA-Tags. Adv. Funct. Mater. 2019, 29 (23), 1808647,  DOI: 10.1002/adfm.201808647
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.4c00137.

    • Efficiency of the GUV filtering process (Figure S1); GUV images after DOD and extrusion printing (Figure S2); MSC viability postbioprinting (Figure S3); effect of salts on the agglomeration of GUVs (Figure S4); agglomeration of DOPC-GUVs in DMEM (Figure S5); agglomeration of PEG5-GUVs in DMEM (Figure S6); interaction of PEGylated GUVs with cells (Figure S7); fluorescence intensity curves for FRAP measurements on printed PEG5-GUVs (Figure S8) (PDF)


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