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bioRxiv2026

Incorporation of active cell-free expression lysates in chitosan coated alginate microcapsules

Oral routes of delivery are logistically simple and enables easy administration of therapeutics. However, oral delivery of proteins is still challenging due to the proteolytic environment within the gastrointestinal (GI) tract. To protect protein cargo from degradation, polymer encapsulation is commonly used, and when it is combined with cell-free gene expression (CFE) approaches that enable the rapid and flexible production of proteins, it potentially allows for on-demand production of protein therapeutics. Here, we investigated the suitability of chitosan coated alginate (Alg/Cht) microcapsules for encapsulation of proteins and CFE lysates for oral delivery. We show that CFE lysates can produce functional mCherry, a model fluorescent protein, in the presence of alginate polymers, although direct contact with chitosan did inhibit protein synthesis. We encapsulated CFE lysates or purified mCherry protein into alginate cores before crosslinking them using internal gelation techniques and coating with chitosan to test their protective capacity for oral delivery. Alg/Cht microcapsules protected mCherry protein cargo from degradation in simulated human gastric fluids and mouse gastric extracts and facilitated controlled cargo release upon exposure to conditions that simulate the intestinal environment. None of the individual CFE or encapsulation components induced inflammation in mouse GI tracts when administered via oral gavage. We also observed a delayed release of fluorescent bead cargo from Alg/Cht microcapsules in mouse intestines following oral gavage. Together, our data suggest that CFE lysate-loaded Alg/Cht formulations can be flexibly used to produce proteins and safely deliver them to the GI tract for potential therapeutic applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=40 SRC="FIGDIR/small/730178v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@d3e4c3org.highwire.dtl.DTLVardef@14fbea1org.highwire.dtl.DTLVardef@6c68cforg.highwire.dtl.DTLVardef@15508ec_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LICell-free gene expression lysates are active in chitosan coated alginate (Alg/Cht) microcapsules. C_LIO_LIAlg/Cht microcapsules exhibit controlled release in vitro in simulated intestinal-like conditions. C_LIO_LICell-free and encapsulation components do not induce inflammation in the gastrointestinal tracts of male or female mice. C_LIO_LIAlg/Cht microcapsules show controlled delayed cargo release in vivo when orally gavaged in mice. C_LI

bioengineering↗

bioRxiv2025

Deletion of core septin gene aspB in Aspergillus fumigatus results in fungicidal activity of caspofungin

Septins are a family of GTP-binding proteins found in many eukaryotic lineages. Although highly conserved throughout many eukaryotes, their functions vary across species. In Aspergillus fumigatus, the etiological agent of invasive aspergillosis, septins participate in a variety of processes, including conidiation, septation, and responses to cell wall stress. Previous studies determined that the {Delta}aspB strain had a greater sensitivity to anti-cell wall drugs, especially the echinocandins, yet mechanisms behind this augmented sensitivity are unknown. We performed cell viability staining of the deletion strains after caspofungin exposure and found that the {Delta}aspA, {Delta}aspB, and {Delta}aspC strains had significantly lower cell viability. Concomitant with the reduced viability, deletion strains are more susceptible to caspofungin on solid media. These results indicate that the septin cytoskeleton is important for A. fumigatus survival in the presence of caspofungin. Due to the potential of improved therapeutic outcome, we followed up using a neutropenic murine model of invasive aspergillosis. Animals infected with the {Delta}aspB strain and treated with caspofungin showed improved survival compared to the animals infected with akuBKU80 wild-type or complemented strains. Additionally, histological analysis showed reduced fungal burden and inflammation in the {Delta}aspB infected, caspofungin-treated group. Affinity purification coupled with quantitative proteomics identified proteins involved in the septin-dependent response to caspofungin, including four candidate interactors involved in cell wall stress response. Deletion of these candidate genes resulted in increased susceptibility to caspofungin and moderately reduced viability post-drug exposure. Taken together, these data suggest that septin AspB contributes to the fungistatic response to caspofungin. Author SummaryInvasive aspergillosis is a pulmonary disease caused by the fungus Aspergillus fumigatus that primarily occurs in immunocompromised patients. Invasive aspergillosis has a high mortality rate, ranging from 50-90%. Therapy options are limited due to few available drugs with fungicidal activity and growing global drug resistance. Treatment typically starts with triazoles, which target the fungal cell membrane. If unsuccessful, an echinocandin, which targets the cell wall, is given as a salvage therapy in the U.S. Echinocandins, including caspofungin, are fungistatic against A. fumigatus, slowing growth of the fungus rather than killing it. Due to this, echinocandins have a high therapeutic failure rate. Previous work suggests that deletion of the cytoskeletal septin genes increases sensitivity to caspofungin. Here we describe our finding that the septin genes aspA, aspB, and aspC are involved in the fungal response to caspofungin. Additionally, the deletion of aspB results in fungicidal activity of this otherwise fungistatic drug. These findings show promise for novel therapy options that block the septin-mediated response to caspofungin.

microbiology↗