Smart capsules that recognize targets: Development of antibody-decorated liposomes using a cell-free system
– A new cell-free platform with promising applications in drug delivery and gene therapy –

1. Key Points

• A novel system was developed to synthesize and lipid-modify antibody fragments (VHH antibodies) using a cell-free protein synthesis system, allowing their efficient anchoring on the surface of nanosized liposomes.
• The resulting immunoliposomes, prepared within two days without using living cells, successfully exhibited target-specific binding to cancer cells.
• This technology holds promise as a targeted delivery system, encapsulating drugs or genes inside liposomes for delivery to specific cells.

2. Overview

A research team led by Dr. Yutetsu Kuruma at JAMSTEC/X-star, in collaboration with GeneFrontier Corporation and Institute of Science Tokyo, has developed a cell-free technique to synthesize and lipid-modify antibody proteins for their membrane localization onto liposomes (Fig. 1). This new platform does not rely on living cells and completes the entire process within two days—dramatically shortening timelines that normally require weeks to months.

Not only antibodies but any soluble protein can theoretically be displayed on the liposome surface using this system. The team demonstrated that VHH antibody fragments, once synthesized and fixed on liposomes, bind specifically to cancer cells that express the target-antigen. Since liposomes can encapsulate chemotherapeutic drugs or genes such as mRNA or siRNA, this technique could be applied in drug delivery and gene therapy.

This study will be published in ACS Synthetic Biology (date pending) on June 20(Japan Time). The research was supported by JSPS KAKENHI (Grants: 24K02000, 24H02287, 21H05156, 20K06519), JST CREST (JPMJCR18S6), and the Human Frontier Science Program (RPG0029/2020).

3. Background

New therapeutic modalities using biomolecules such as proteins, antibodies, and mRNA are drawing attention as promising treatments for cancer and other refractory diseases. Especially, liposomes—tiny lipid-based capsules—are widely studied for use in drug delivery systems (DDS). However, enabling these liposomes to specifically target certain cells requires that functional molecules such as antibodies be anchored on their surface, a technically complex and time-consuming process.

The traditional method relies on cultured cells to produce proteins, which slows the process and hinders high-throughput parallel design. To solve this, the research group developed a system to synthesize and lipidate antibodies without any living cells, achieving their efficient immobilization onto nano-sized liposomes.

This approach is based on the PURE system, a reconstituted cell-free protein synthesis method composed of purified translation components from E. coli (Ref. 1). In the previous study (Ref. 2), the research group demonstrated lipid modification of the cell-free synthesized protein by adding myristoyl-CoA and myristoyltransferase in the PURE system. However, the efficiency of membrane localication remained around 20%.

In this study, the researchers significantly improved the efficiency by engineering protein sequences, and further validated that lipidated VHH antibodies could be immobilized on liposomes and bind specifically to HER2-expressing breast cancer cells.

4. Results

The researchers designed fusion protein that include a sequence to enhance translation efficiency and a TEV protease recognition site.

Upon synthesis, the TEV protease cleaves off the upstream sequences, exposing the N-terminal site for lipid modification. As a result, lipid moiety from myristoyl-CoA or palmitoyl-CoA can be enzymatically attached to the N-terminus of protein, achieving lipidation efficiency up to ~100% (Fig. 2).

However, the lipidated proteins alone were insufficient for membrane anchoring on liposome. To address this, polyarginine sequences were inserted upstream of the protein, imparting a positive charge that enhanced interaction with the negatively charged liposome surface. With this strategy, efficient immobilization of the protein was confirmed. Additionally, attaching a more hydrophobic lipid, palmitoyl-CoA (C16:0), further enhanced liposome anchoring.

Applying this method, anti-HER2 VHH antibodies were successfully immobilized on the liposome surface. Analyses showed that over 80% of VHH proteins localized onto the membrane (Fig. 3). By calculation, it was shown that ~2,000 antibody molecules per 100 nm liposome.

When these immunoliposomes were exposed to breast cancer cells, strong binding was observed only in HER2-positive cells, and no binding occurred with HER2-negative controls. These results confirmed that the PURE system can produce functional, lipidated VHH antibodies that maintain their antigen-binding activity after membrane immobilization.
The entire workflow—from protein synthesis to functional immunoliposome production—can be completed in two days.

5. Future Outlook

This newly developed technology for cell-free protein synthesis and lipid modification of liposomes has wide-ranging potential applications.

For instance, by loading liposomes with anticancer drugs, this system can be applied to create next-generation antibody-guided drug carriers with reduced side effects. By encapsulating siRNA or mRNA, it could also contribute to genetic therapies or mRNA vaccine delivery targeting specific cells.

Moreover, as this platform avoids living cell experiments and genetic modification procedures, it reduces regulatory hurdles and supports faster, safer, and purer production of bio-functional materials. This makes it especially valuable for biopharmaceutical manufacturing, drug discovery screening, biosensor development, and synthetic biology.

Although lipidation is limited to N-terminal modifications in the current system, this could be expanded by incorporating other acyltransferases in the future. The concept of producing "smart capsules" entirely in vitro is expected to become a foundational technology across life sciences, medicine, and materials research.

Figure 1. Overview of the cell-free system developed in this study.
VHH antibodies synthesized using the PURE system are first trimmed by TEV protease, and then lipidated with myristoyl-CoA or palmitoyl-CoA by a myristoyltransferase. The resulting lipidated-antibodies are anchored onto the liposome surface through hydrophobic interactions. The antibody-decorated liposomes then bind specifically to target cancer cells.

Figure 2. Membrane anchoring of lipidated proteins onto liposomes membrane.
(A) Schematic of protein construct for lipidation. Polyarginine sequences (PolyR) were tested in three forms: RRR (R3), RRRRRR (R6), and without arginine (R0). Each fragment was analyzed by SDS-PAGE.
(B) Lipidation of the model protein (sfGFP) in the presence of liposomes, and the resulting efficiency of membrane localization onto liposome surfaces.

Figure 3. Binding of VHH antibody-decorated liposomes to breast cancer cells.
(A) Anti-HER2 VHH antibodies were lipidated in the presence of liposomes, then the liposomes were isolated by ultracentrifugation and co-localization with the VHH was assessed by SDS-PAGE analysis. Fractions (Frac.) 1 and 2 correspond to the liposome-containing layers. The gel image below the graph shows the detection of VHH antibody protein. Membrane localization efficiency was calculated as the percentage of VHH protein found in Frac. 1+2 relative to the total. The schematic above the graph illustrates the model of liposome after the reaction.
(B) The liposomes from (A) were exposed to HER2-expressing breast cancer cells and observed under a confocal microscope. Liposomes were stained with rhodamine-lipid.