Synthetic cells: happy middle between liposome drug delivery and engineered natural cells

Cells are the fundamental "building blocks" that make up living organisms. However, we don't know exactly how cells were formed in the first place, we also don't know what all the molecules that make up any natural cell do. This causes substantial difficulty in any efforts to re-design natural cells for biomedical applications. Synthetic cells offer an alternative to reprogrammed live cells for responsive biochemical systems engineered as controlled and personalized diagnostics and therapeutics. Synthetic cells are liposomal bioreactors exhibiting some, but not all properties of natural cells. Many natural cell elements have been reconstituted in synthetic cells.1 Fully engineered synthetic cells will have designs that can be radically different from the natural lineages of existing cells. In the biomedical context, synthetic cell technology is uniquely positioned on the intersection of in vitro and live cell therapeutic platforms. The simplicity and programmability of synthetic cells makes them ideal candidates for designing responsive theranostics tailored for particular context: patient and application. Biological complexity of synthetic cells makes it possible to program complex sense and response pathways, delivering therapeutics or performing diagnostic operations using engineered sense and response circuits.

Synthetic cells in biomedical applications

Synthetic cells offer great potential for drug delivery. By changing the combinations of encapsulates and membranes, the synthetic cells control their localization, stabilization, sensingresponding behavior, and their own metabolic reaction. Such flexibility enlarges the strategy for targeted drug delivery. Many aspects of technology towards the synthetic cell in the medicinal application are under development. As one of the successful examples, Schroeder and colleagues opened up the area of synthetic cells in cancer therapy. They designed synthetic cells to produce an anti-cancer protein (Pseudomonas exotoxin A) with in vitro transcription/translation (IVTT) reaction. They showed its anti-cancer effect on both cell culture and in live mice2. Additionally, aiming to deliver the synthetic cells to the targeted tissue, various membrane modifications have been invented. For instance, Wegner and colleagues developed a multi stimulus sensing synthetic cell, which adheres to the specific surface by sensing light, pH, oxidative stress, and metal ions3. The idea of synthetic cells in medicinal use is still new but rapidly growing. Given current technical inventions, synthetic cell therapeutics would become a beneficial tool for drug delivery. However, some issues need to be solved before they can be practical, like further technology development, large-scale production lines, and safety assurance.

The technological roadblocks

The main technical challenge in pathway to designing synthetic cells for biomedical applications is formation of the liposome compartment. Reliable, reproducible formation of unilamellar liposomes, with narrow size distribution and high loading of the payloads in the lumen, remains one of the fast growth of foundational research and pipeline to clinical applications of all liposome technologies4,5.

DFFS: versatile liposome formation

Various protocols for droplet-shooting centrifugal formation of liposomes (termed DSCF) exist whereby liposomes are formed when lumen droplets pass through a lipids-in-oil solution and thence into an aqueous solution via centrifugal force6. Utilizing this general DSCF mechanism it is possible to assemble highly uniform liposomes quickly and easily with tunable lumen and membrane chemistries. See figure 1 for the detailed schematic of the apparatus designed in the Adamala lab7. The biggest advantages of all DFFS methods, and particularly the one using the new apparatus, are the simplicity and reliability of making liposomes. This is crucial for formation of uniform synthetic cells with reproducible payload encapsulation – as is necessary for engineering synthetic cells.

The DSCF liposome formation may be carried out using standard microcentrifuge tubes, commercially available pre-pulled glass capillaries, and 3D-printable capillary holders such as those depicted in figure 2. A prepared DSCF apparatus can be observed in figure 2, prior to centrifugation.

The DFFS apparatus can be produced either by 3D printing, or by injection molding. Detailed protocols and free sample of apparatus will be available on the protocol platform, www.liposome.tech, concurrently with publication of the protocol⁷.

Outlook

There is currently only one type of life known, so all biological studies are performed with a sample size of 1. Learning how to engineer a novel live cell from completely defined parts will provide a leap in our understanding of life, and support the development of novel technologies that will benefit the scientific community and society.

More efficient liposome formation will help to lower the barrier of entry into synthetic cell research, enabling participation of more labs without prior experience in the field. This will, in turn, result in more technologies and application use cases in all areas. Novel, more versatile, programmable drug delivery and diagnostics solutions enabled by synthetic cell platform will change the paradigm in biomedical engineering.

Literature

1. Gaut, N. J. & Adamala, K. P. Reconstituting Natural Cell Elements in Synthetic Cells. Adv. Biol. 5, 1–20 (2021).

2. Krinsky, N. et al. Synthetic Cells Synthesize Therapeutic Proteins inside Tumors. Adv. Healthc. Mater. 7, 1–10 (2018).

3. Xu, D., Kleineberg, C., Vidaković-Koch, T. & Wegner, S. V. Multistimuli Sensing Adhesion Unit for the Self-Positioning of Minimal Synthetic Cells. Small 16, 1–8 (2020).

4. Has, C. & Sunthar, P. A comprehensive review on recent preparation techniques of liposomes. J. Liposome Res. 0, 1–30 (2019).

5. Sharma, D., Ali, A. A. E. & Trivedi, L. R. An Updated Review on: Liposomes as drug delivery system. PharmaTutor 6, 50–62 (2018).

6. Morita, M. et al. Droplet-Shooting and Size-Filtration (DSSF) Method for Synthesis of Cell-Sized Liposomes with Controlled Lipid Compositions. ChemBioChem 16, 2029–2035 (2015).

7. Editors: Karim, A. S. & Jewett, M. C. (Eds. . CELL-FREE GENE EXPRESSION : methods and protocols. (2022).