Biology subjects

Care, A.

Publications and source records attributed to Care, A..

8 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↗

bioRxiv2025

Multi-omic data integration and analyses for biomarker discovery of spontaneous preterm birth phenotypes

BackgroundPreterm birth, delivery <37 weeks of gestation, is a global health concern affecting 1.3 million infants annually. A higher morbidity burden is associated with infants born <34 weeks of gestation. A more robust biomarker of spontaneous preterm birth needs to be identified. To our knowledge, this study uniquely integrated three ome-wide datasets prospectively collected from the same individuals within a single, defined UK cohort. The study aimed to integrate genomic, transcriptomic, and metabolomic data collected from the same cohort of women for biomarker discovery. MethodsPregnant women with a history of a previous spontaneous preterm birth (sPTB) <34 weeks were recruited in Liverpool in a subsequent pregnancy at 16 and/or 20 weeks of gestation. Pregnancy outcomes were followed up and categorised into the different clinical subgroups of SPTB <34 weeks, PPROM <34 weeks and term delivery >37 weeks (controls). Blood samples were profiled for genomics, transcriptomics and metabolomics. ANOVA analyses were performed at each gestational timepoint. Network enrichment analysis was performed on significant genes. ResultsAfter multi-omic data integration, 43 women at week 16 and 40 women at week 20 of gestation had all three genomics, transcriptomics and metabolomics profiles available. Multiple significant transcripts were detected (p<0.05) from the ANOVA analyses, though these were mostly in non-coding regions. ConclusionThree different omic data, genomics, transcriptomics and metabolomics, were integrated across the same individuals for biomarker analyses. Molecular signatures were detected that could lead to understanding the pathways involved in the different subgroups of preterm birth: SPTB and PPROM.

systems biology↗

bioRxiv2024

High-fidelity in vitro packaging of diverse synthetic cargo into encapsulin protein cages

Cargo-filled protein cages are powerful tools in biotechnology with demonstrated potential as catalytic nanoreactors and vehicles for targeted drug delivery. While endogenous biomolecules can be packaged into protein cages during their expression and self-assembly inside cells, synthetic cargo molecules are typically incompatible with live cells and must be packaged in vitro. Here we report a fusion-based in vitro assembly method for packaging diverse synthetic cargo into encapsulin protein cages that outperforms standard in cellulo assembly, producing cages with superior uniformity and thermal stability. Fluorescent dyes, proteins and cytotoxic drug molecules can all be selectively packaged with high efficiency via a peptide-mediated targeting process. The exceptional fidelity and broad compatibility of our in vitro assembly platform enables generalisable access to cargo-filled protein cages that host novel synthetic functionality for diverse biotechnological applications.

biochemistry↗

bioRxiv2024

Protein Coronas Derived from Cerebrospinal Fluid Enhance the Interactions Between Nanoparticles and Brain Cells

Neuronanomedicine harnesses nanoparticle technology for the treatment of neurological disorders. An unavoidable consequence of nanoparticle delivery to biological systems is the formation of a protein corona on the nanoparticle surface. Despite the well-established influence of the protein corona on nanoparticle behavior and fate, as well as FDA approval of neuro-targeted nanotherapeutics, the effect of a physiologically relevant protein corona on nanoparticle-brain cell interactions is insufficiently explored. Indeed, less than 1% of protein corona studies have investigated protein coronas formed in cerebrospinal fluid (CSF), the fluid surrounding the brain. Herein, we utilize two clinically relevant polymeric nanoparticles (PLGA and PLGA-PEG) to evaluate the formation of serum and CSF protein coronas. LC-MS analysis revealed distinct protein compositions, with selective enrichment/depletion profiles. Following incubation with brain cells, serum and CSF coronas on PLGA particles showed enhanced associations with all cell types as compared to their corresponding corona on PLGA-PEG particles. CSF-derived protein coronas on PLGA nanoparticles, specifically, showed the greatest nanoparticle-cell interactions, with Pearsons correlation analysis revealing that proteins associated with enhanced nanoparticle-cell interactions were exclusively enriched in this protein corona. This study demonstrates the importance of correct choice of physiologically relevant biological fluids, and its influence on the formation of the protein corona, subsequent nanoparticle-cell interactions.

bioengineering↗

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↗

bioRxiv2020

Bioengineering a light-responsive encapsulin nanoreactor: a potential tool for photodynamic therapy

Encapsulins, a prokaryotic class of self-assembling protein nanocompartments, are being re-engineered to serve as ‘nanoreactors’ for the augmentation or creation of key biochemical reactions. However, approaches that allow encapsulin nanoreactors to be functionally activated with spatial and temporal precision is lacking. We report the construction of a light-responsive encapsulin nanoreactor for “on-demand” production of reactive oxygen species (ROS). Herein, encapsulins were loaded with the fluorescent flavoprotein mini-Singlet Oxygen Generator (miniSOG), a biological photosensitizer that is activated by blue-light to generate ROS, primarily singlet oxygen (1O2). We established that the nanocompartments stably encased miniSOG, and in response to blue-light were able to mediate the photoconversion of molecular oxygen into ROS. Using an in vitro model of lung cancer, ROS generated by the nanoreactor was shown to trigger photosensitized oxidation reactions that exerted a toxic effect on tumour cells, suggesting utility in photodynamic therapy. This encapsulin nanoreactor thus represents a platform for the light-controlled initiation and/or modulation of ROS-driven processes in biomedicine and biotechnology.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗