Bioengineering bacterial encapsulin nanocompartments as targeted drug delivery system

Alexander Van De Steen,Rana Khalife,Noelle Colant,Hasan Mustafa Khan,Matas Deveikis,Saverio Charalambous,Clare M Robinson,Rupali Dabas,Sofia Esteban Serna,Diana A Catana,Konstantin Pildish,Vladimir Kalinovskiy,Kenth Gustafsson,Stefanie Frank

doi:10.1016/j.synbio.2021.09.001

Alexander Van De Steen, Rana Khalife + Show 12 more

Open Access

https://doi.org/10.1016/j.synbio.2021.09.001

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Abstract

The development of Drug Delivery Systems (DDS) has led to increasingly efficient therapies for the treatment and detection of various diseases. DDS use a range of nanoscale delivery platforms produced from polymeric of inorganic materials, such as micelles, and metal and polymeric nanoparticles, but their variant chemical composition make alterations to their size, shape, or structures inherently complex. Genetically encoded protein nanocages are highly promising DDS candidates because of their modular composition, ease of recombinant production in a range of hosts, control over assembly and loading of cargo molecules and biodegradability. One example of naturally occurring nanocompartments are encapsulins, recently discovered bacterial organelles that have been shown to be reprogrammable as nanobioreactors and vaccine candidates. Here we report the design and application of a targeted DDS platform based on the Thermotoga maritima encapsulin reprogrammed to display an antibody mimic protein called Designed Ankyrin repeat protein (DARPin) on the outer surface and to encapsulate a cytotoxic payload. The DARPin9.29 chosen in this study specifically binds to human epidermal growth factor receptor 2 (HER2) on breast cancer cells, as demonstrated in an in vitro cell culture model. The encapsulin-based DDS is assembled in one step in vivo by co-expressing the encapsulin-DARPin9.29 fusion protein with an engineered flavin-binding protein mini-singlet oxygen generator (MiniSOG), from a single plasmid in Escherichia coli. Purified encapsulin-DARPin_miniSOG nanocompartments bind specifically to HER2 positive breast cancer cells and trigger apoptosis, indicating that the system is functional and specific. The DDS is modular and has the potential to form the basis of a multi-receptor targeted system by utilising the DARPin screening libraries, allowing use of new DARPins of known specificities, and through the proven flexibility of the encapsulin cargo loading mechanism, allowing selection of cargo proteins of choice.

Highlights

Cytotoxic chemotherapy had been the predominant medical treatment for breast cancer
In this work encapsulins were coupled with the designed ankyrin repeat protein DARPin9.29 which was selected for specific binding to the human epidermal growth factor receptor 2 (HER2) overexpressed by the human breast cancer cell line SK-BR-3 [48]
Both fusion proteins were capable of binding to SK-BR-3 cells, which indicates that DARPin9.29 tolerates fusion to another pro tein without abolishing binding to the receptor

Summary

Introduction

Cytotoxic chemotherapy had been the predominant medical treatment for breast cancer. The Nanomedicine approach to encapsulating cytotoxic therapeutic small molecules provides several benefits to pharmacological properties, most critically, the passive targeting to the tumour site via the associated leaky vasculature, called the Enhanced Permeability and Retention (EPR) effect [5]. Other nanoparticle (NPs)- associated benefits include longer circulation times, slow clearance, greater formulation flexibility [6], tumour penetration and facilitated cellular uptake [7]. All of these factors raise the therapeutic index of the administered chemotherapy drugs [8]

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