Proposed Implementation

Our project focuses on utilizing encapsulins as a versatile and selective delivery system for cells. The encapsulin structure is very simple and is robust to chemical modifications. This allows for multiple stable derivatives of the original wild-type encapsulin that can be specialized in various functions.

NanoReactors

A family of encapsulins categorized as Family 1 encapsulins act as natural nanoreactors that can compartmentalize specific enzymatic reactions in order to create a stable environment that facilitates the reaction essential to the cell as well as preventing toxicity from intermediate products or competition for the enzyme. This is achieved through a cargo-loading peptide mechanism (CLP) and can further be demonstrated with a modified encapsulin (EncTM) that was loaded with a mini singlet oxygen generator protein (miniSOG) which could produce singlet oxygen when stimulated with blue light. Relative to non-isolated miniSOG, the encapsulated miniSOG could produce approximately 2 fold more singlet oxygen [1].

Figure 1: Overview of Encapsulin Nanoreactor [Diaz et al., 2021]

Targeted Delivery and Controlled Immune Responses

A key advantage with encapsulins is the ability to modify the outer shell of the encapsulin to contain a variety of cell receptors which can make it highly selective. This is a critical feature for drug delivery as it would increase the efficiency of the drug being delivered without excess. Furthermore, this reduces potential side effects and complications with the delivery as the cargo will have limited interaction with other proteins or cells besides the intended target. Experiments have already shown that encapsulins are a viable drug delivery platform specifically for biomolecules. This can be seen with the fusion of SP94, a hepatocellular carcinoma cell-targeting peptide, to the encapsulin (EncTm) [2]. The specific cargo that was encapsulated was aldoxorubicin, an acid-sensitive prodrug. The experiments showed similar cell viability between encapsulated doxorubicin and free doxorubicin.

In addition, the ability to add various proteins to the surface of encapsulins has lead to innovative ways to induce various immune responses from cells. This would prove useful as it would help researchers further understand mechanisms behind immune responses and simulate controlled immune responses in the lab utilizing modified encapsulins. In summary, the versatility of the encapsulin structure gives it the potential to act as various pseudoviruses in research. This occurs in both empty encapsulins and cargo-loaded encapsulins. This is seen specifically when an experiment shows a major glycoprotein, gp350, for the Epstein-Barr virus fused to an Encapsulin. This would induce a strong immune response in both mice and non-human primates [3].

Figure 2: Design of Encapsulin-based Vaccine [Kanekiyo et al., 2015]

These two real-world implementations are what our team aims to focus on when conducting experiments involving the mechanisms and behavior of encapsulins specifically when interacting with yeast cells. MSBT is currently focusing on selectively targeting A-type yeast cells through endocytosis. By achieving endocytosis in only A-type cells, this will help validate the idea that encapsulins can act as a potential selective drug delivery platform as well as be repurposed for other tasks. This will bring MSBT closer to delivering genuine pharmaceuticals or other biomolecules of interest into yeast or other cell lines in the future.

References

1. Diaz, D.; Vidal, X.; Sunna, A.; Care, A. Bioengineering a Light-Responsive Encapsulin Nanoreactor: A Potential Tool for In Vitro Photodynamic Therapy. ACS Appl. Mater. Interfaces 2021, 13, 7977–7986.
2. Lo, A.; Lin, C.T.; Wu, H.C. Hepatocellular carcinoma cell-specific peptide ligand for targeted drug delivery. Mol. Cancer Ther. 2008, 7, 579–589.
3. Kanekiyo, M.; Bu, W.; Joyce, M.G.; Meng, G.; Whittle, J.R.; Baxa, U.; Yamamoto, T.; Narpala, S.; Todd, J.P.; Rao, S.S.; et al. Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site. Cell 2015, 162, 1090–1100.
4. Rodríguez, Javier M., et al. “Nanotechnological Applications Based on Bacterial Encapsulins.” Nanomaterials, vol. 11, no. 6, 2021, p. 1467., https://doi.org/10.3390/nano11061467.