Protecting Encapsulin Nanoparticles with Cysteine-Knot Miniproteins

Abstract

A big hurdle for the use of protein-based drugs is that they are easily degraded by proteases in the human body. In an attempt to solve this problem, we show the possibility to functionalize TM encapsulin nanoparticles with an mEETI-II knottin miniprotein from the cysteine-stabilized knot class. The resulting particles did not show aggregation and retained part of their protease inhibitive function. This imposes a protection toward protease, in this case, trypsin, degradation of the protein cage. The used chemistry is easy to apply and thus suitable to protect other protein systems from degradation. In addition, this proof of principle opens up the use of other knottins or cysteine-stabilized knots, which can be attached to protein cages to create a heterofunctionalized protein nanocage. This allows specific targeting and tumor suppression among other types of functionalization. Overall, this is a promising strategy to protect a protein of interest which brings oral administration of protein-based drugs one step closer.

Artificial Organelles: Towards Adding or Restoring Intracellular Activity

“…This discovery revealed the potential for targeted sorting of exogenous cargoes inside these nanocompartments, making encapsulins great candidates for a plethora of applications in nanotechnology [167] . A variety of promising biomedical advancements have been made, such as the generation of iron‐sequestering encapsulins that could function as electron microscopy gene reporters, [168] the formation of antimicrobial encapsulins that contain silver nanoparticles [169] or enhance the production of antimicrobial peptides, [170] and the modification of the encapsulin surface with photo‐switchable fluorophores [171] or protection groups against protease degradation [172] . However, the focus below will be on developments that are beneficial for artificial organelle fabrication.…”

Advances in encapsulin nanocompartment biology and engineering

“…Scale bar = 20 nm. Adapted with “ACS Author's Choice” permission from (Klem et al, 2018), which can be found at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.8b00630. Copyright 2018 ACS.…”

“…To address the concern of proteolytic degradation of encapsulins used within the human body, another study aimed to protect the TM encapsulin from trypsin by functionalizing it with cysteine‐knot miniproteins (Klem et al, 2018). As the ecballium elaterium trypsin inhibitor II (EETI‐II) knottins are known to have trypsin inhibitory activity, they were chosen for external functionalization of the TM encapsulin.…”

Cysteine‐Based Biomaterials as Drug Nanocarriers

“…Since the major barrier for oral peptide and protein‐based drugs was the enzymatic obstacle, it was vital to design a drug delivery system that had a good enzyme inhibitory potential besides the other mentioned advantages. In an attempt to protect TM encapsulin from trypsin induced degradation, an mEETI‐II knottin miniprotein from cysteine‐stabilized knot class was used to design a functional TM encapsulin nanoparticle without affecting their activity, which may bring oral administration of protein‐based drugs one step closer . To further improve the ability of polyacrylates to suppress proteolytic enzymes, Perera et al.…”

“…The preparation process is particularly easy, which advances the designs of diverse multifunctional delivery platform for enhanced human disease therapy. Cysteine could be used to design a functional nanoparticle to protect protein‐based formulations from trypsin induced degradation without affecting their activity and biocompatibility, which may bring oral administration of protein‐based drugs one step closer . Moreover, cysteine could also be used to fabricate injectable and in situ formed hydrogels that are promising for their use as therapeutic drug/protein delivery systems under mild conditions .…”