Biology subjects

Pirolli, N. H.

Publications and source records attributed to Pirolli, N. H..

5 recordsLinked to original sources

bioRxiv2025

Human cell-derived extracellular vesicles exhibit anti-biofilm effects against Pseudomonas aeruginosa

The need for new antimicrobial approaches is one of the most pressing issues in modern medicine. A particular pathogen of concern is multidrug resistant Pseudomonas aeruginosa, which is implicated in over 1 million deaths worldwide yearly. One potential class of novel antimicrobials is extracellular vesicles (EVs), which have been found to have intrinsic antimicrobial and anti-virulence properties. Here, the antimicrobial activity of EVs on P. aeruginosa was explored in the context of biofilms of hyper-virulent strain PA14. We identified the human monocyte cell line THP-1 as a promising source of EVs for this application, inducing reduced PA14 biofilm formation in a dose-dependent manner. THP-1 EVs were not found to affect P. aeruginosa growth planktonically within the biofilm assay or in shaken culture. Additionally, we demonstrate that anti-biofilm effects were conserved with similar efficacy across THP-1 monocyte and differentiated THP-1 macrophage-derived EVs. Further, EVs from induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iMSCs) also reduced PA14 biofilm formation to a similar extent as THP-1 EVs, while EVs from human embryonic kidney cells (HEK293T) had a similar effect to its media control. This work indicates that human cell-derived EVs from several sources possess biological and/or physical properties that reduce PA14 biofilm formation.

microbiology↗

bioRxiv2025

Genetically-programmed Hypervesiculation of Lactiplantibacillus plantarum Increases Production of Bacterial Extracellular Vesicles with Therapeutic Efficacy in a Preclinical Inflammatory Bowel Disease Model

Inflammatory bowel diseases (IBD) affect over 6 million people globally and current treatments achieve only 10-20% rates of durable disease remission. Bacterial extracellular vesicles (BEVs) from probiotic lactic acid bacteria (LAB) are a promising novel therapeutic with mechanisms holding potential to drive increased rates of durable disease remission, including immunomodulation and intestinal epithelial tissue repair. However, translation of these cell-secreted nanovesicles is limited by long standing biomanufacturing hurdles, especially low production yields due to low biogenesis rates from cells. Here, our goal was to identify a candidate probiotic LAB that produces BEVs effective in a preclinical mouse model of IBD, and then genetically engineer the LAB for at least 10-fold increased production yields of BEVs, thereby passing a critical production threshold. We identified Lactiplantibacillus plantarum as a candidate LAB producing BEVs effective in treating acute dextran sulfate sodium (DSS)-induced murine colitis, and with greater efficacy than BEVs from probiotic Escherichia coli Nissle 1917. We then genetically engineered a hypervesiculating L. plantarum strain by inducible expression of a peptidoglycan-modifying enzyme, resulting in a 66-fold increase in BEV productivity. Finally, we confirmed hypervesiculating L. plantarum BEVs were therapeutically effective in the acute DSS mouse model of colitis and found these BEVs were superior in reducing mucosal tissue damage compared to live L. plantarum cells. These findings demonstrate that BEVs from genetically engineered hypervesiculating strain of L. plantarum are a promising preclinical therapeutic candidate for IBD that overcomes historical biomanufacturing limitations of BEV therapeutics.

bioengineering↗

bioRxiv2023

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles (OMVs), have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation co-isolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). E. coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.

bioengineering↗

bioRxiv2023

Induced pluripotent stem cell-derived extracellular vesicles promote wound repair in a diabetic mouse model via an anti-inflammatory immunomodulatory mechanism

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been widely explored in clinical trials for treatment of diseases with complex pathophysiology. However, production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, we initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, we found that their vascularization bioactivity was similar and their anti-inflammatory bioactivity was superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, we employed a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial. In this in vivo model, iPSC EVs more effectively mediated inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.

bioengineering↗

bioRxiv2023

Differentiation state and culture conditions impact neural stem/progenitor cell-derived extracellular vesicle bioactivity

Extracellular vesicles (EVs) derived from neural progenitor/stem cells (NPSCs) have shown promising efficacy in a variety of preclinical models. However, NPSCs lack critical neuroregenerative functionality such as myelinating capacity. Further, culture conditions used in NPSC EV production lack standardization and identification of optimal conditions for NPSC EV neurogenic bioactivity. Here, we assessed whether further differentiated oligodendrocyte precursor cells (OPCs) and immature oligodendrocytes (iOLs) that give rise to mature myelinating oligodendrocytes could yield EVs with neurotherapeutic properties comparable or superior to those from NPSCs as well as mesenchymal stromal cells (MSCs), as MSC EVs are also commonly reported to have neurotherapeutic activity. We additionally examined the effects of four different extracellular matrix (ECM) coating materials (laminin, fibronectin, Matrigel, and collagen IV) and the presence or absence of growth factors (EGF, bFGF, and NGF) in cell culture on the ultimate properties of EVs. The data show that OPC EVs and iOL EVs performed similarly to NPSC EVs in PC-12 proliferation and RAW264.7 mouse macrophage antiinflammatory assays, but NPSC EVs performed better in a PC-12 neurite outgrowth assay. Additionally, the presence of nerve growth factor (NGF) in culture was found to be maximize NPSC EV bioactivity among the conditions tested. NPSC EVs produced under rationally-selected culture conditions (fibronectin + NGF) enhanced axonal regeneration and muscle reinnervation in a rat nerve crush injury model. These results highlight the impact of culture conditions on NPSC EV neuroregenerative bioactivity, thus providing additional rationale for standardization and optimization of culture conditions for NPSC EV production. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=35 SRC="FIGDIR/small/528366v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@447d23org.highwire.dtl.DTLVardef@73a5cborg.highwire.dtl.DTLVardef@89dbb0org.highwire.dtl.DTLVardef@b62d39_HPS_FORMAT_FIGEXP M_FIG C_FIG Extracellular vesicles (EVs) purified from neural progenitor/stem cells (NPSCs) have been investigated for neurotherapeutic activity, however significant variability in culture conditions limits reproducibility and efficacy of this approach. Here, we examined the impact of extracellular matrix (ECM) components and growth factors in NPSC culture on the bioactivity on the bioactivity of their EVs. The results show that EVs from NPSCs cultured with a rationally-selected ECM type (fibronectin) and growth factor (nerve growth factor (NGF)) enhanced nerve regeneration and muscle recovery in a rat sciatic nerve crush injury model.

bioengineering↗