Biology subjects

Boyton, I.

Publications and source records attributed to Boyton, I..

4 recordsLinked to original sources

bioRxiv2026

Modular PEGylation Confers Reduced Immune-Cell Uptake and Enhanced Pharmacokinetic Performance to Encapsulin Protein Nanocages

Encapsulins are self-assembling prokaryotic protein nanocages with growing potential as systemic drug delivery systems, but their pharmacokinetic behaviour remains poorly characterised, and rapid immune recognition and clearance may limit delivery to target tissues. Here, we show that modular PEGylation of a SpyCatcher-decorated encapsulin Alkaliphilus metalliredigens (Am-S) markedly reduces macrophage uptake and extends systemic circulation. Site-directed surface PEGylation using SpyTagged PEG achieved 82% conjugation efficiency, corresponding to an estimated average of 49 PEG chains per 60 subunit Am-S nanocage, without compromising nanocage assembly, morphology, or colloidal stability. PEGylated Am-S also retained solubility and protein integrity following freeze-thaw cycling and six months of storage. When interacted with RAW 264.7 macrophages in vitro, PEGylation substantially reduced nanocage association and internalisation relative to non-PEGylated nanocages. Following intravenous administration in BALB/c mice, PEGylated nanocages exhibited markedly prolonged circulation, with >50% of the injected dose remaining after 1 h compared with 2.6% for non-PEGylated Am-S, and a circulatory half-life of 1 h 43 min. To our knowledge, this represents the first pharmacokinetic characterisation of an encapsulin nanocage. Together, these findings demonstrate that controlled PEGylation can substantially reduce macrophage interactions and prolong encapsulin circulation in vivo.

bioengineering↗

bioRxiv2026

Engineering a Novel Bacterial Encapsulin for Programmable Surface Functionalization: From Single-Target to Mosaic Nanovaccines

Encapsulins are prokaryotic self-assembling protein nanocages with promise as nanovaccine scaffolds. Their utility as modular platforms require tolerance to surface engineering, high-yield soluble production, formulation stability, and controlled antigen (co-)display. Herein, a previously uncharacterized encapsulin from Alkaliphilus metalliredigens is engineered into a SpyCatcher-decorated nanoscaffold (Am-S) that enables controlled surface display of SpyTagged antigens. Cryo-EM confirms that the native encapsulin forms a T = 1 icosahedral nanocage, and that C-terminal SpyCatcher fusion yields Am-S without compromising nanocage assembly, symmetry, or structural integrity. Notably, Am-S exhibits high-yield soluble production in Escherichia coli, remains monodisperse after freeze-thaw and extended storage, and supports efficient SpyTagged peptide conjugation for single- and multi-antigen display. As a proof-of-concept, Am-S is functionalized with Alzheimers disease-associated amyloid-{beta} and/or hyperphosphorylated tau epitopes to generate single-target nanocages displaying either antigen and dual-target mosaic nanocages co-displaying both. In mice, Am-S antigen display enhances antigen-specific IgG responses relative to free antigens and induces predominantly IgG1-biased humoral immunity. Mosaic nanocages elicit antibodies against both targets, with immune sera selectively recognizing amyloid-{beta}- and phosphorylated tau-associated pathology in ex vivo brain sections from Alzheimers disease mouse models. These findings position Am-S as a manufacturable scaffold for developing multi-targeting nanovaccines against complex diseases.

synthetic biology↗

bioRxiv2023

In vivo fate of systemically administered encapsulin protein nanocages and implications for their use in targeted drug delivery

Encapsulins, self-assembling protein nanocages derived from prokaryotes, are promising nanoparticle-based drug delivery systems (NDDS). However, the in vivo behavior and fate of encapsulins are poorly understood. In this pre-clinical study, we probe the interactions between the model encapsulin from Thermotoga maritima (TmEnc) and key biological barriers encountered by NDDS. Here, a purified TmEnc formulation that exhibited colloidal stability, storability, and blood compatibility was intravenously injected into BALB/c mice. TmEnc had an excellent nanosafety profile, with no abnormal weight loss or gross pathology observed, and only temporary alterations in toxicity biomarkers detected. Notably, TmEnc demonstrated immunogenic properties, inducing the generation of nanocage-specific IgM and IgG antibodies, but without any prolonged pro-inflammatory effects. An absence of antibody cross-reactivity also suggested immune-orthogonality among encapsulins systems. Moreover, TmEnc formed a serum-derived protein corona on its surface which changed dynamically and appeared to play a role in immune recognition. TmEncs biodistribution profile further revealed its sequestration from the blood circulation by the liver and then biodegraded within Kupffer cells, thus indicating clearance via the mononuclear phagocyte system. Collectively, these findings provide critical insights into how encapsulins behave in vivo, thereby informing their future design, modification, and application in targeted drug delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/549228v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1691ecaorg.highwire.dtl.DTLVardef@647c31org.highwire.dtl.DTLVardef@115a146org.highwire.dtl.DTLVardef@10f2b6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Exploring the Self-Assembly of Encapsulin Protein Nanocages from Different Structural Classes

Encapsulins, self-assembling icosahedral protein nanocages derived from prokaryotes, represent a versatile set of tools for nanobiotechnology. However, a comprehensive understanding of the mechanisms underlying encapsulin self-assembly, disassembly, and reassembly is lacking. Here, we characterise the disassembly/reassembly properties of three encapsulin nanocages that possess different structural architectures: T = 1 (24 nm), T = 3 (32 nm), and T = 4 (42 nm). Using spectroscopic techniques and electron microscopy, encapsulin architectures were found to exhibit varying sensitivities to the denaturant guanidine hydrochloride (GuHCl), extreme pH, and elevated temperature. While all encapsulins showed the capacity to reassemble following GuHCl-induced disassembly (within 75 min), only the smallest T = 1 nanocage reassembled after disassembly in basic pH (within 15 min). Furthermore, atomic force microscopy revealed that all encapsulins showed a significant loss of structural integrity after undergoing sequential disassembly/reassembly steps. These findings provide insights into encapsulins disassembly/reassembly dynamics, thus informing their future design, modification, and application.

biophysics↗